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John R. Paul and the Definition of Preventive Medicine | 04557c79-14a9-47b5-a245-b663917d3efd | 2596460 | Preventive Medicine[mh] | |
Measuring vital signs in children with fever at the emergency department: an observational study on adherence to the NICE recommendations in Europe | 01aa30a9-a90c-4577-a15b-1822500a7ce2 | 7314716 | Pediatrics[mh] | Fever is the most common reason for children to be brought to an emergency department (ED) , with causes ranging from self-limiting illnesses of childhood to serious bacterial infections (SBIs) that can prove fatal . Vital signs can help clinicians identify children at risk of serious illness. Even though the level of evidence for the diagnostic accuracy of vital signs is varying, their importance is widely acknowledged . Vital signs form the basis of paediatric early warning scores (PEWS) that are widely used to monitor disease severity of children in the inpatient setting . Moreover, they are included in several prediction models for serious infections and in disease-specific guidelines for the ED setting . The NICE guideline for the assessment and initial management of fever in children under five recommends a routine measurement of temperature, heart rate, capillary refill and respiratory rate in all children presenting to the ED with a fever . These recommendations have been adopted throughout a large number of European hospitals. Not measuring vital signs may pose the patient at risk of underestimating the severity of illness and may delay appropriate treatment . From adult research and single-country studies, we know that incomplete and inaccurate recording of vital signs is common . This problem may be even larger in Europe, given the diversity of the countries, cultures and healthcare systems. However, international data on recording of vital signs across Europe in children are lacking . Information on the measurement of vital signs is crucial in order to fuel research on serious illness and to target quality improvement initiatives in paediatric emergency medicine. This research aims to evaluate the current practice of measuring vital signs in febrile children in European EDs and, more specifically, the level of adherence to the NICE guideline recommendation to routinely measure four distinct vital signs.
Study design and population We performed a prospective observational study in 28 EDs in 11 European countries, including patients under the age of 16 and with a fever as their presenting complaint. Children were excluded if they presented to the ED repeatedly for the same problem within 7 days, if they were treated with antibiotics in the 7 days before the ED visit, or if they had a documented allergy to antibiotics. For the current study, children with comorbidities were also excluded, as disease-specific characteristics may influence their management. In the whole population, we evaluated the measurement of vital signs. In children under five, we assessed the adherence to the recommendations to measure four distinct vital signs from the NICE guideline ‘Fever in under 5s: assessment and initial management’ . Data collection Data collection took place between October 2014 and February 2016 within the network of Research in European Pediatric Emergency Medicine (REPEM). Detailed methods have been published earlier . In short, all participating 28 EDs recorded medical information for all attending children with fever for one random day each month. We recorded general characteristics of patients (age, sex, weight, height, comorbidities), vital signs (heart rate, respiratory rate, temperature, oxygen saturation, capillary refill time) and information regarding diagnosis and management. Data were extracted from routine patient records, and filled in on an electronic study case report form (CRF) by the local investigator after the sampling day (Electronic Supplementary Material ). Comorbidities and diagnoses were recorded according to pre-specified categories. We neither used ICD-codes for the recording of diagnoses nor had we access to data after the ED visit. Consequently, ‘diagnosis’ in this manuscript refers to a presumed diagnosis at ED discharge. All items in the CRF were mandatory to fill in, with the option to choose ‘unknown’. Unknown values on vital signs were seen as ‘not measured’, and were therefore considered to be outcomes rather than omissions. Local investigators were aware of the sampling days and the general scope of the study as a registry of febrile children, but vital sign measurement was not known as a specific point of interest. Hospital information was collected using a survey, including questions on guideline use. We collected data on hospital setting (inner city/rural/mixed), hospital type (academic/teaching/non-teaching), triage system and number of annual paediatric ED visits, similar to other studies on the organization of care (Electronic Supplementary Material ) . Setting reflects the population in the catchment area of the hospital. Academic hospitals are connected to a university, teaching hospitals non-university hospitals that provide training for paediatrics residents; non-teaching hospitals do not provide training of residents. Definitions Not every study hospital used the same triage system, but they all classified children according to a five-point scale, ranging from ‘non-urgent’ to ‘immediate’, making comparisons possible. Owing to the small number of cases, patients in the ‘immediate’ and ‘very urgent’ categories were grouped together. Tachycardia and tachypnoea were defined according to the advanced pediatric life support (APLS) guideline . Fever was defined as temperature ≥ 38 °C, hypoxia as peripheral oxygen saturation level of ≤ 94%. Crowding of the ED was defined for each hospital according to their number of total paediatric ED visits on the sampling day (less than usual/as usual/more than usual). We defined a usual number of total visits as the interquartile range of the number of total visits across the different sampling days per hospital. If on a sampling day the number of total visits was lower than the 25th percentile for that hospital, crowding was less than usual, if the number was higher than the 75th percentile, the ED was more crowded than usual. Adherence to the NICE guideline was based on the following indicator: ‘ Measure and record temperature, heart rate, respiratory rate and capillary refill time as part of the routine assessment of a child with fever. ’ . Adherence to the NICE guideline was defined as the complete measurements of those four vital signs in children under 5 years old. Statistical analysis We used descriptive analyses to evaluate the frequency of measurement for all of the available vital signs in the study population. We examined practice variations between countries, age groups, triage levels and diagnoses, visualizing the measurement of vital signs by heat maps. We used diagnosis in these analyses as a proxy of presenting complaint (next to the fever) and suspicion of severity, assuming that children with RTIs would present with respiratory symptoms, enteric infections with vomiting or diarrhoea and that children with fever without source, urinary tract infections and sepsis/meningitis mostly present without specific symptoms but with a higher suspicion of invasive infections. We compared the frequency of detecting abnormal vital signs between countries that frequently measured vital signs and countries that measured them less often. In assessing adherence to the NICE guideline, we measured the frequency of complete measurements in children under five from all hospitals that used the NICE recommendations. We tested the influence of age, triage level, diagnosis and crowding of the ED on adherence using a multilevel logistic regression model that included hospital as a random variable. Analyses were performed using SPSS (IBM, version 24) and R (version 3.5.2).
We performed a prospective observational study in 28 EDs in 11 European countries, including patients under the age of 16 and with a fever as their presenting complaint. Children were excluded if they presented to the ED repeatedly for the same problem within 7 days, if they were treated with antibiotics in the 7 days before the ED visit, or if they had a documented allergy to antibiotics. For the current study, children with comorbidities were also excluded, as disease-specific characteristics may influence their management. In the whole population, we evaluated the measurement of vital signs. In children under five, we assessed the adherence to the recommendations to measure four distinct vital signs from the NICE guideline ‘Fever in under 5s: assessment and initial management’ .
Data collection took place between October 2014 and February 2016 within the network of Research in European Pediatric Emergency Medicine (REPEM). Detailed methods have been published earlier . In short, all participating 28 EDs recorded medical information for all attending children with fever for one random day each month. We recorded general characteristics of patients (age, sex, weight, height, comorbidities), vital signs (heart rate, respiratory rate, temperature, oxygen saturation, capillary refill time) and information regarding diagnosis and management. Data were extracted from routine patient records, and filled in on an electronic study case report form (CRF) by the local investigator after the sampling day (Electronic Supplementary Material ). Comorbidities and diagnoses were recorded according to pre-specified categories. We neither used ICD-codes for the recording of diagnoses nor had we access to data after the ED visit. Consequently, ‘diagnosis’ in this manuscript refers to a presumed diagnosis at ED discharge. All items in the CRF were mandatory to fill in, with the option to choose ‘unknown’. Unknown values on vital signs were seen as ‘not measured’, and were therefore considered to be outcomes rather than omissions. Local investigators were aware of the sampling days and the general scope of the study as a registry of febrile children, but vital sign measurement was not known as a specific point of interest. Hospital information was collected using a survey, including questions on guideline use. We collected data on hospital setting (inner city/rural/mixed), hospital type (academic/teaching/non-teaching), triage system and number of annual paediatric ED visits, similar to other studies on the organization of care (Electronic Supplementary Material ) . Setting reflects the population in the catchment area of the hospital. Academic hospitals are connected to a university, teaching hospitals non-university hospitals that provide training for paediatrics residents; non-teaching hospitals do not provide training of residents.
Not every study hospital used the same triage system, but they all classified children according to a five-point scale, ranging from ‘non-urgent’ to ‘immediate’, making comparisons possible. Owing to the small number of cases, patients in the ‘immediate’ and ‘very urgent’ categories were grouped together. Tachycardia and tachypnoea were defined according to the advanced pediatric life support (APLS) guideline . Fever was defined as temperature ≥ 38 °C, hypoxia as peripheral oxygen saturation level of ≤ 94%. Crowding of the ED was defined for each hospital according to their number of total paediatric ED visits on the sampling day (less than usual/as usual/more than usual). We defined a usual number of total visits as the interquartile range of the number of total visits across the different sampling days per hospital. If on a sampling day the number of total visits was lower than the 25th percentile for that hospital, crowding was less than usual, if the number was higher than the 75th percentile, the ED was more crowded than usual. Adherence to the NICE guideline was based on the following indicator: ‘ Measure and record temperature, heart rate, respiratory rate and capillary refill time as part of the routine assessment of a child with fever. ’ . Adherence to the NICE guideline was defined as the complete measurements of those four vital signs in children under 5 years old.
We used descriptive analyses to evaluate the frequency of measurement for all of the available vital signs in the study population. We examined practice variations between countries, age groups, triage levels and diagnoses, visualizing the measurement of vital signs by heat maps. We used diagnosis in these analyses as a proxy of presenting complaint (next to the fever) and suspicion of severity, assuming that children with RTIs would present with respiratory symptoms, enteric infections with vomiting or diarrhoea and that children with fever without source, urinary tract infections and sepsis/meningitis mostly present without specific symptoms but with a higher suspicion of invasive infections. We compared the frequency of detecting abnormal vital signs between countries that frequently measured vital signs and countries that measured them less often. In assessing adherence to the NICE guideline, we measured the frequency of complete measurements in children under five from all hospitals that used the NICE recommendations. We tested the influence of age, triage level, diagnosis and crowding of the ED on adherence using a multilevel logistic regression model that included hospital as a random variable. Analyses were performed using SPSS (IBM, version 24) and R (version 3.5.2).
Population characteristics In total, 5255 children were included in the complete cohort, all presenting with fever and without prior antibiotic treatment or repeated ED visits. In the current study, we included 4560 children. Exclusion was mostly because of comorbidities (Fig. ). Of the included children, 53.8% were male and the median age was 2.4 years (interquartile range 1.1–4.7). Table shows their baseline characteristics and provides information regarding patients’ way of referral and follow-up. Baseline characteristics of children with comorbidities have been published earlier . In general, these children were more ill and older than children without comorbidities. Of the 28 participating hospitals, 17 were academic hospitals, 10 teaching hospitals and one non-teaching hospital (Table ). They varied from inner city hospitals ( n = 17) to regional ( n = 2) and mixed hospitals ( n = 9), and their number of annual paediatric ED visits ranged from 2700 to 88,000. Most hospitals used a local triage system ( n = 8) or the Manchester triage system ( n = 7, Table ). All except the Spanish hospitals used the recommendation to routinely measure vital signs as mentioned in the NICE guideline. Overall measurement of vital signs and per country The measurement of vital signs occurred in varying degrees, both when comparing the different vital signs with each other and across participating countries. Overall, temperature was measured most frequently (97%, 4435/4560, 95% confidence interval 97–98%), ranging between countries from 70% (78/111) in Hungary to 100% in Denmark and England ( n = 24 and n = 145 respectively). Capillary refill was next (86%, 85–89%), followed by heart rate (73%, 72–75%), saturation (56%, 55–58%) and respiratory rate (51%, 50–53%), although the latter two had much wider ranges between countries. Figure a contains a heat map visualizing the frequency of vital sign measurements in participating countries. Variability between countries is apparent throughout all of the different vital signs and is most striking for respiratory rates. Temperature was the most consistent, as it was measured in more than 90% of cases in all countries but one. Measurement of vital signs per triage level, age group and diagnosis In the hospital in Turkey, no routine triage was performed. In the remaining hospitals, 99% (3825/3852) of children were triaged upon their arrival to the hospital. Children requiring ‘very urgent’ or ‘immediate’ care had their vital signs measured most frequently (Fig. ). Differences across triage levels were greatest for heart rate, saturation and respiratory rate and amounted to about 30% points between the ‘very urgent/immediate’ and ‘standard’ categories (heart rate: 93% vs. 64%, saturation: 90% vs. 59%, respiratory rate: 65% vs. 37%). Differences in measurement across age groups were smaller (Fig. ). Only heart rate and saturation were more frequently measured in infants than in children > 5 years of age (heart rate: 83% vs. 71%; saturation 78% vs. 47%). We observed an association between the measurement of vital signs and diagnosis (Fig. ). Most children (3307/4461, 74%) had respiratory tract infections (RTIs); only 15 children had sepsis or meningitis. Temperature and capillary refill were measured quite consistently across the different infectious foci (97% and 86% of cases respectively), but the remaining vital signs exhibited a considerable variability. Saturation was measured substantially more often in lower RTIs and in patients with sepsis/meningitis than in other cases. Heart rate was recorded in all patients with sepsis/meningitis (15/15) and in 86% (416/486) of those with lower RTIs. For fevers of unknown origin, on the other hand, heart rate measurements were included in the work-up of only 61% (174/284) of cases. Respiratory rates were measured in less than half of patients for four out of seven infectious foci and were done most frequently in patients with lower RTIs, amounting to 64% (310/486) of cases. Frequency of abnormal findings The incidence of abnormal vital signs when measured was generally low. Of all patients with a measured temperature, 2403 (54.2%) had a fever at the time of evaluation in the ED (Table ). Out of these children, 889 (37%) had a temperature of 39 °C or more. Other than that, heart rate was most often abnormal, in 34.1% of cases. Twenty-nine percent of children were found to be tachypnoeic, hypoxia was found in 3.3% of cases and prolonged capillary refill in 1.5%. We observed no correlation between the frequency of measurement of a vital sign per country and the proportion of abnormal values (out of all values measured in that country). So, less frequent measurement of a vital sign was not related to a higher proportion of abnormal values detected. Adherence to guideline recommendation From all hospitals using the NICE recommendations, 1450/3014 (48%) of children under five underwent a complete measurement of these vital signs (95% CI 46 to 50%). A complete measurement was most frequent in children with lower RTIs and sepsis, although at a moderate compliance of 55% and 46% respectively (193/350 for lower RTIs and 5/11 for sepsis; Table ). Multivariable analysis showed that children with RTIs had complete measurements significantly more often than children with fever without focus (odds ratio for upper RTI 1.75 (1.10–2.77), for lower RTI 3.75 (2.21–6.37); Table ). Also, younger children were more likely to have all recommended vital signs measured than children over 1 year of age. Last, children with high triage urgency had full measurements slightly more often than non-urgent children (immediate/very urgent OR 1.62 (0.95–2.76), urgent level OR 1.36 (0.96–1.95)). Crowding of the ED had no significant effect on the frequency of complete measurement of vital signs. After adjusting for diagnosis, age and triage urgency, a substantial variability between hospitals remained (data not shown).
In total, 5255 children were included in the complete cohort, all presenting with fever and without prior antibiotic treatment or repeated ED visits. In the current study, we included 4560 children. Exclusion was mostly because of comorbidities (Fig. ). Of the included children, 53.8% were male and the median age was 2.4 years (interquartile range 1.1–4.7). Table shows their baseline characteristics and provides information regarding patients’ way of referral and follow-up. Baseline characteristics of children with comorbidities have been published earlier . In general, these children were more ill and older than children without comorbidities. Of the 28 participating hospitals, 17 were academic hospitals, 10 teaching hospitals and one non-teaching hospital (Table ). They varied from inner city hospitals ( n = 17) to regional ( n = 2) and mixed hospitals ( n = 9), and their number of annual paediatric ED visits ranged from 2700 to 88,000. Most hospitals used a local triage system ( n = 8) or the Manchester triage system ( n = 7, Table ). All except the Spanish hospitals used the recommendation to routinely measure vital signs as mentioned in the NICE guideline.
The measurement of vital signs occurred in varying degrees, both when comparing the different vital signs with each other and across participating countries. Overall, temperature was measured most frequently (97%, 4435/4560, 95% confidence interval 97–98%), ranging between countries from 70% (78/111) in Hungary to 100% in Denmark and England ( n = 24 and n = 145 respectively). Capillary refill was next (86%, 85–89%), followed by heart rate (73%, 72–75%), saturation (56%, 55–58%) and respiratory rate (51%, 50–53%), although the latter two had much wider ranges between countries. Figure a contains a heat map visualizing the frequency of vital sign measurements in participating countries. Variability between countries is apparent throughout all of the different vital signs and is most striking for respiratory rates. Temperature was the most consistent, as it was measured in more than 90% of cases in all countries but one.
In the hospital in Turkey, no routine triage was performed. In the remaining hospitals, 99% (3825/3852) of children were triaged upon their arrival to the hospital. Children requiring ‘very urgent’ or ‘immediate’ care had their vital signs measured most frequently (Fig. ). Differences across triage levels were greatest for heart rate, saturation and respiratory rate and amounted to about 30% points between the ‘very urgent/immediate’ and ‘standard’ categories (heart rate: 93% vs. 64%, saturation: 90% vs. 59%, respiratory rate: 65% vs. 37%). Differences in measurement across age groups were smaller (Fig. ). Only heart rate and saturation were more frequently measured in infants than in children > 5 years of age (heart rate: 83% vs. 71%; saturation 78% vs. 47%). We observed an association between the measurement of vital signs and diagnosis (Fig. ). Most children (3307/4461, 74%) had respiratory tract infections (RTIs); only 15 children had sepsis or meningitis. Temperature and capillary refill were measured quite consistently across the different infectious foci (97% and 86% of cases respectively), but the remaining vital signs exhibited a considerable variability. Saturation was measured substantially more often in lower RTIs and in patients with sepsis/meningitis than in other cases. Heart rate was recorded in all patients with sepsis/meningitis (15/15) and in 86% (416/486) of those with lower RTIs. For fevers of unknown origin, on the other hand, heart rate measurements were included in the work-up of only 61% (174/284) of cases. Respiratory rates were measured in less than half of patients for four out of seven infectious foci and were done most frequently in patients with lower RTIs, amounting to 64% (310/486) of cases.
The incidence of abnormal vital signs when measured was generally low. Of all patients with a measured temperature, 2403 (54.2%) had a fever at the time of evaluation in the ED (Table ). Out of these children, 889 (37%) had a temperature of 39 °C or more. Other than that, heart rate was most often abnormal, in 34.1% of cases. Twenty-nine percent of children were found to be tachypnoeic, hypoxia was found in 3.3% of cases and prolonged capillary refill in 1.5%. We observed no correlation between the frequency of measurement of a vital sign per country and the proportion of abnormal values (out of all values measured in that country). So, less frequent measurement of a vital sign was not related to a higher proportion of abnormal values detected.
From all hospitals using the NICE recommendations, 1450/3014 (48%) of children under five underwent a complete measurement of these vital signs (95% CI 46 to 50%). A complete measurement was most frequent in children with lower RTIs and sepsis, although at a moderate compliance of 55% and 46% respectively (193/350 for lower RTIs and 5/11 for sepsis; Table ). Multivariable analysis showed that children with RTIs had complete measurements significantly more often than children with fever without focus (odds ratio for upper RTI 1.75 (1.10–2.77), for lower RTI 3.75 (2.21–6.37); Table ). Also, younger children were more likely to have all recommended vital signs measured than children over 1 year of age. Last, children with high triage urgency had full measurements slightly more often than non-urgent children (immediate/very urgent OR 1.62 (0.95–2.76), urgent level OR 1.36 (0.96–1.95)). Crowding of the ED had no significant effect on the frequency of complete measurement of vital signs. After adjusting for diagnosis, age and triage urgency, a substantial variability between hospitals remained (data not shown).
Main findings In this study of febrile children at 28 European EDs, we observed that of all vital signs, temperature is most frequently measured and respiratory rate least frequently, but with a high degree of variability between countries. Most centres have adopted the recommendation of the NICE guideline ‘ Fever in under 5s: assessment and initial management ’ to always measure temperature, heart rate, respiratory rate and capillary refill, but compliance to this recommendation was moderate. Febrile children that are under 1 year of age, with high triage urgency and those with RTIs were more likely to have a full set of vital signs measured. Interpretation and comparison to existing literature Fever was an inclusion criteria for our study, which explains the high frequency of completed temperature in our database and the high proportion of abnormal temperatures. The relatively high proportion with abnormal heart rate can be explained by the physiological relationship between temperature and heart rate . Respiratory rate was least frequently measured and with large variation across subgroups. Other studies have suggested reasons for such variability, like crying or distress of a child, or limitations in the counting technique . We had no information on the child’s well-being or the devices used for measurement of respiratory rate, but these factors may have contributed to the observed low frequency of measurement of this vital sign. Although ED crowding has been associated with decreased quality of care , we found no association between ED crowding and adherence to the vital signs measurement recommendation in our study. We observed an overall adherence of 48% to the NICE recommendation to measure four vital signs in all children under five, in our study in 2014–2016. This is lower than reported by a previous audit study in primary care in the UK (62%) after educational sessions and introduction of a template to record vital signs in the electronic health record . An audit among paediatric EDs in the UK found that temperature was similarly measured as in our study (94%), but reports lower numbers for capillary refill time (53%) and higher rates for heart rate (94%) and respiratory rate (89%) measurements . It may be striking that full measurement of vital signs children under five was most frequently done in children suspected of RTIs, rather than in those with suspected urinary tract infections and fever without focus. Even though the discharge diagnosis is often unknown at the moment of vital sign measurement, it is likely to assume that children with these last two diagnoses might present without specific symptoms. These children may have more diagnostic uncertainty and be at higher risk of complicated disease. Less than half of the children with suspected sepsis—although represented by a small number in our study—received the full set of vital sign measurements needed for compliance with the NICE guidelines. Patient characteristics can only partly explain the observed practice variations. Professional adherence to guideline recommendations can also be influenced by local policy or professional experience. Even though most participating centres mentioned that their guidelines were based on the NICE guideline, in the process of translation from the UK to another setting, the evidence probably is weighed according to the local setting and practice. This may induce further practice variation across centres . Strengths and limitations This study had the advantage of a sizeable, prospectively generated database containing large amounts of high quality patient information from 28 hospitals of various sizes and hospital types, from 11 different countries in Europe. Compared to the available literature in European paediatric emergency medicine, this number of included hospitals and countries is large, supporting the generalizability of our findings. However, some countries and hospitals included more patients than others, which might have influenced results. Furthermore, countries were represented by different numbers of hospitals (some countries only by one hospital), which adds uncertainty to whether measurements are a reflection of national or local policies. The study was performed in hospitals of the REPEM research collaboration, ensuring high-quality data . Their interest in research indicates that they are likely to uphold a high standard of care. The staff of participating hospitals were only aware of the general study design as a registry of febrile children, so a special focus on vital sign measurement during the study period is unlikely. Lastly, because this research treated missing variables as decisions not to perform certain measurements, some room remains for human error in data collection. However, all items in the data collection form were mandatory, with the option to fill in ‘missing’. During the preparation of this manuscript, the local investigators confirmed that ‘missing’ values were indeed ‘not recorded’. Clinical and research implications Our numbers on compliance to the NICE recommendation obtained from 28 European EDs calls for better recording of vital signs in children. Not measuring vital signs may pose children at risk of underestimating the severity of their illness or delaying necessary treatments . Even though almost all included centres had adopted the NICE recommendation to measure vital signs in all febrile children, compliance in less than half of cases is striking. Even in children with sepsis, fever without source or urinary tract infections in less than 50% of cases the full set was measured. Therefore, special attention should be given to children presenting with fever without specific symptoms, since vital sign measurements may contribute most to the identification of severe infections in this patient group. Although measurement is influenced by age and triage, it might be questioned whether triage appropriately selects children with severe disease . Future research should focus on identifying reasons for non-compliance, including cultural and healthcare factors at the individual, organizational and national level . Qualitative research could provide more in-depth information on the reasons for the observed discrepancies in vital sign measurements across Europe. At the same time, more evidence is needed on the diagnostic value of vital signs in different settings and patient groups and their impact on health outcomes. Such research could provide evidence for targeted measuring of vital signs in children that benefit most from complete measurements.
In this study of febrile children at 28 European EDs, we observed that of all vital signs, temperature is most frequently measured and respiratory rate least frequently, but with a high degree of variability between countries. Most centres have adopted the recommendation of the NICE guideline ‘ Fever in under 5s: assessment and initial management ’ to always measure temperature, heart rate, respiratory rate and capillary refill, but compliance to this recommendation was moderate. Febrile children that are under 1 year of age, with high triage urgency and those with RTIs were more likely to have a full set of vital signs measured.
Fever was an inclusion criteria for our study, which explains the high frequency of completed temperature in our database and the high proportion of abnormal temperatures. The relatively high proportion with abnormal heart rate can be explained by the physiological relationship between temperature and heart rate . Respiratory rate was least frequently measured and with large variation across subgroups. Other studies have suggested reasons for such variability, like crying or distress of a child, or limitations in the counting technique . We had no information on the child’s well-being or the devices used for measurement of respiratory rate, but these factors may have contributed to the observed low frequency of measurement of this vital sign. Although ED crowding has been associated with decreased quality of care , we found no association between ED crowding and adherence to the vital signs measurement recommendation in our study. We observed an overall adherence of 48% to the NICE recommendation to measure four vital signs in all children under five, in our study in 2014–2016. This is lower than reported by a previous audit study in primary care in the UK (62%) after educational sessions and introduction of a template to record vital signs in the electronic health record . An audit among paediatric EDs in the UK found that temperature was similarly measured as in our study (94%), but reports lower numbers for capillary refill time (53%) and higher rates for heart rate (94%) and respiratory rate (89%) measurements . It may be striking that full measurement of vital signs children under five was most frequently done in children suspected of RTIs, rather than in those with suspected urinary tract infections and fever without focus. Even though the discharge diagnosis is often unknown at the moment of vital sign measurement, it is likely to assume that children with these last two diagnoses might present without specific symptoms. These children may have more diagnostic uncertainty and be at higher risk of complicated disease. Less than half of the children with suspected sepsis—although represented by a small number in our study—received the full set of vital sign measurements needed for compliance with the NICE guidelines. Patient characteristics can only partly explain the observed practice variations. Professional adherence to guideline recommendations can also be influenced by local policy or professional experience. Even though most participating centres mentioned that their guidelines were based on the NICE guideline, in the process of translation from the UK to another setting, the evidence probably is weighed according to the local setting and practice. This may induce further practice variation across centres .
This study had the advantage of a sizeable, prospectively generated database containing large amounts of high quality patient information from 28 hospitals of various sizes and hospital types, from 11 different countries in Europe. Compared to the available literature in European paediatric emergency medicine, this number of included hospitals and countries is large, supporting the generalizability of our findings. However, some countries and hospitals included more patients than others, which might have influenced results. Furthermore, countries were represented by different numbers of hospitals (some countries only by one hospital), which adds uncertainty to whether measurements are a reflection of national or local policies. The study was performed in hospitals of the REPEM research collaboration, ensuring high-quality data . Their interest in research indicates that they are likely to uphold a high standard of care. The staff of participating hospitals were only aware of the general study design as a registry of febrile children, so a special focus on vital sign measurement during the study period is unlikely. Lastly, because this research treated missing variables as decisions not to perform certain measurements, some room remains for human error in data collection. However, all items in the data collection form were mandatory, with the option to fill in ‘missing’. During the preparation of this manuscript, the local investigators confirmed that ‘missing’ values were indeed ‘not recorded’.
Our numbers on compliance to the NICE recommendation obtained from 28 European EDs calls for better recording of vital signs in children. Not measuring vital signs may pose children at risk of underestimating the severity of their illness or delaying necessary treatments . Even though almost all included centres had adopted the NICE recommendation to measure vital signs in all febrile children, compliance in less than half of cases is striking. Even in children with sepsis, fever without source or urinary tract infections in less than 50% of cases the full set was measured. Therefore, special attention should be given to children presenting with fever without specific symptoms, since vital sign measurements may contribute most to the identification of severe infections in this patient group. Although measurement is influenced by age and triage, it might be questioned whether triage appropriately selects children with severe disease . Future research should focus on identifying reasons for non-compliance, including cultural and healthcare factors at the individual, organizational and national level . Qualitative research could provide more in-depth information on the reasons for the observed discrepancies in vital sign measurements across Europe. At the same time, more evidence is needed on the diagnostic value of vital signs in different settings and patient groups and their impact on health outcomes. Such research could provide evidence for targeted measuring of vital signs in children that benefit most from complete measurements.
Measuring vital signs in children with fever in the emergency department occurs with a high degree of practice variation between different European hospitals and is done more often in younger children, those with a higher triage urgency or who have respiratory tract infections. The overall adherence to the NICE recommendation to measure four vital signs in all febrile children under five is moderate. Our practice variation study is essential as a benchmark for current clinical practice. It can guide future research into the drivers and consequences of the observed under-recording of vital signs. Moreover, it can be used to tailor implementation strategies of the NICE recommendation to different European settings.
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Stem cell pathology: histogenesis of malignant fibrous histiocytoma and characterization of myofibroblasts appearing in fibrotic lesions | 8c52f2af-80f1-44f6-b720-84e195918ae3 | 10539815 | Anatomy[mh] | What are MFHs (recently named “undifferentiated pleomorphic sarcomas”)? Malignant mesenchymal tumors, such as fibrosarcoma, osteosarcoma, liposarcoma, leiomyosarcoma, and rhabdomyosarcoma, as well as malignant peripheral nerve sheath tumors, are relatively
easily diagnosed based on their own cellular and histological characteristics . MFH was introduced as a new entity of malignant mesenchymal tumor in
1963 . After that, the diagnostic term of MFH has been used, but the histogenesis of MFH remained to be determined, albeit MFH is the most common
neoplasm of the soft-tissue in the elderly . In veterinary medicine, spontaneously occurring MFHs have rarely been detected in domestic and
laboratory animals . Experimentally induced MFH-like sarcomas
by using chemicals such as 4-(hydroxyamino) quinoline-1-oxide and nickel sulfide have been reported in rats and mice . Histopathologically, MFHs are characterized by a mixture of fibroblastic, histiocytic and undifferentiated mesenchymal cells arranged in a storiform growth pattern . Besides, human MFHs sometimes exhibit various features of mesenchymal tumors such as osteosarcoma, liposarcoma and leiomyosarcoma in parts of tumor tissues . Such histological heterogeneity of MFHs drew a hypothesis that the tumor may contain cells with mesenchymal
multipotency. Characteristics of a rat transplantable tumor line (MFH-MT) and MFH-MT-derived cultured cell line (MT-P) In order to explore the cell properties of MFHs, the author established a transplantable tumor line (MFH-MT) from a spontaneous MFH developed in subcutaneous tissue of F344 rat. Cultured
MT-P cell line was induced from MFH-MT. The original and serially transplanted MFH-MT tumors, as well as tumors induced in syngeneic rats by injection with MT-P cells, were composed of an
admixture of fibroblastic, histiocytic cells and immature mesenchymal cells arranged in a storiform growth pattern, of which histology was similar to that of human MFHs . Characterization of cloned cell lines (MT-8 and MT-9) derived from MT-P cells Two cloned cell lines, named MT-8 and MT-9, were induced from MT-P cells by using the limiting dilution technique. MT-8 and MT-9 cells possessed mesenchymal nature with different morphology
to each other . MT-8 cells had less developed organelles and the induced tumors represented histological characteristics of undifferentiated
sarcoma , whereas MT-9 cells had relatively well-developed intracytoplasmic organelles such as endoplasmic reticulum, mitochondria and lysosomes and the induced tumors showed a storiform
growth pattern typical of human MFHs . Although MT-9
cells, besides immunoreactivity for vimentin, reacted to stem cell markers such as nestin, CD90, and CD34; more interestingly, MT-9 cells also reacted in parts to α-smooth muscle actin
(α-SMA) for myofibroblasts/smooth muscle cells , S-100 /glial fibrillary acidic protein
(GFAP) for neurogenic cells and bone morphogenetic protein (BMP)-6 for osteogenic cells, as well as CD68/CD163 for histiocytic
cells/macrophages, whereas MT-8 cells reacted faintly and negatively to these marker antigens .
Interestingly, the reactivity to CD68 and CD163 were also seen in cultured MT-9 cells ( , respectively), indicating that cells reacting to
CD68/CD163 seen in MT-9 tumors were not infiltrated macrophages but neoplastic cells. The gene analyses revealed that genes relating to “cell differentiation” were more activated in MT-9
than MT-8 tumors, whereas those involved in “cell cycle” were greater in MT-8 than MT-9 tumors. These findings indicated that MT-8 cells are mesenchymal stem cells that have not yet been
decided for cellular differentiation, whereas MT-9 cells are the stem cells having somewhat capacity of mesenchymal differentiations (that is, mesenchymal differentiation capacity of MT-9
cells is greater than that of MT-8 cells). MT-8 tumors are diagnosed as sarcoma not otherwise specified (sarcoma NOS) , whereas MT-9 tumors
showed a histology typical of MFH . In fact, in in vitro studies, adipogenic supplement-added MT-9 cells showed increased accumulation of cytoplasmic lipid droplets stained red with the oil red staining. Addition of BMP-2 (a factor for osteogenesis) to MT-9 enhanced osteoblastic differentiation (as shown by increases in alkaline phosphatase
activity, osteocalcin mRNA expression and calcification . TGF-β1-treated MT-9 cells revealed increased numbers of α-SMA-immunopositive
cells, and enhanced protein levels of α-SMA and fibronectin, indicating myofibrogenesis . TGF-β1 is the most powerful factor for myofibroblast
induction . More interestingly, when injected in syngeneic rats, MT-9 cells, that had been incubated under some conditions in
vitro , developed tumors partly including leiomyosarcoma , osteosarcoma , fibrosarcoma , liposarcoma , and granular cell tumors , in addition to MFH-like storiform growth
. MT-9 cells show greater multipotency presumably under microenvironmental conditions . According to “the cancer stem cells theory” , MT-8 cells may be so-called cancer stem cells (less differentiated cells and greater mitotic
activity), whereas MT-9 cells may be precursor cells (more differentiated cells with less mitotic capacity) of tumors . Based on characteristics of
MFH-constituting cells, therefore, the new term “undifferentiated pleomorphic sarcoma” is suitable for MFHs . The possible histogenesis of MFH is shown in , on the basis of findings mentioned above. Generation of rat MFH-specific antibody (A3), and distribution of A3-labeled cells in the rat body An antibody (named A3) was generated using whole cell antigens prepared from MT-8 cells .
Immunohistochemically, A3 reacted specifically to rat MFH cells, but did not react to other types of tumors in rats . A3 is a conformation-specific antibody recognizing both N -glycan and peptide . The carbohydrate chain is
changed during cellular development from ES cells as a landmark for cell differentiation . Taken these findings together, Rat MFH cells
immunopositive to A3 are considered to have pluripotential differentiations, like ES cells. In normal rat tissues of fetuses, A3 labeled primitive mesenchymal cells in visceral organs . In adult rats, A3 labeled mesenchymal cells in the
bone marrow ; these bone marrow cells co-expressed mesenchymal stem cell markers such as vimentin, CD90 (Thy-1) and CD105 , as well as rat endothelial cell antigen-1 (RECA-1) . Human ES cells and iPS cells show direct differentiation to
endothelial cells, indicating that immature endothelial cells at the early stages have stem cell nature . In addition, A3 labeled perivascular cells (so-called pericytes) which are regarded as mesenchymal somatic stem cells in the
connective tissues . Bone marrow-derived stem cells have the capacity to differentiate not only into various
types of mesenchymal cells, but also into cells with visceral mesoderm, neuroectoderm and endoderm characteristics . The pericytes undergo osteogenic or chondrogenic differentiation in vitro . A3-labeled pericytes are considered to
be in the lineage of bone marrow stem cells. In hair follicles, the hair shaft is veiled by the epidermic hair follicle which are surrounded by the connective tissue sheath (CTS) as the outermost component of hair follicle (dermic
hair follicle). The bulge of hair follicle contains epithelial stem cells which can be activated at the anagen to generate a new hair follicle and the mesenchymal cells in the CTS possess the nature of stromal stem cells . Immunohistochemically, A3 labeled both epithelial cells of the bulge and mesenchymal cells in the CTS . The cells in the
bulge are regarded as somatic stem cells with potential epithelial differentiation . A3-labeled cells in the CTS co-expressed partly CD90, CD34,
and nestin. Antibodies to CD90, CD34 and nestin have been used as immature mesenchymal cell markers . Mesenchymal nestin-positive cells are regarded as stem cells
with neural and mesoderm differentiation potentials . In addition, A3-labeled cells contribute to the tissue regeneration in rat colonic lesions,
indicating that they may take part in the stem cell niche for the formation of mucosal epithelial cells . A3-labeled cells seem to act as rescue
cells after injury in colon ulcer . The rescue cells have nature of both mesenchymal and epithelial cells to maintain self-renewal, keeping the
barrier from external environment under progress of re-epithelialization after injury. The recue cells are considered to be derived from the bone-marrow stem cells . Collectively, the progenitor of MFHs is considered to be A3-labeled pericytes which may be in the lineage of bone marrow stem cells capable of differentiating into both mesenchymal and
epithelial cells . A probable relationship between MFH cells
(MT-8 and MT-9) and the well-defined mesenchymal cells is shown in Possible stem cell derivation of subcutaneous mesenchymal tumors in cats and dogs Feline injection site sarcomas (FISSs; also known as “vaccine-associated sarcomas”) are characterized by subcutaneous abnormal proliferation of atypical mesenchymal cells and often by
infiltration of macrophages/multinucleated foreign body type giant cells. Histologically, the majority are fibrosarcomas, and other histologic variants such as MFH, poorly differentiated
sarcoma, rhabdomyosarcoma, myxosarcoma, liposarcoma, nerve sheath tumor, histiocytic sarcoma, and extraskeletal osteosarcoma and chondrosarcoma have been recognized . FISSs may be derived from somatic stem cells with multipotential mesenchymal differentiations. It is speculated that
inflammation at the injection site induced by vaccines or other injections likely plays a critical role in tumor development, presumably through genetic damage in stem cells due to oxidative
stress which may be excessively generated by exudative macrophages and giant cells. Perivascular wall tumors (PWTs; previously classified “canine hemangiopericytomas”) are relatively common tumor arising in the subcutis of dogs, and considered to be nonendothelial vascular
neoplasms with a histological characteristic of whirling pattern . In PWTs, however, there are a variety
of histological features, occasionally including histology of peripheral nerve sheath tumors. Therefore, the histogenesis is still under debate. The PWTs may be derived from undifferentiated
stem cells which may differentiate into perivascular fibroblasts, pericytes, myopericytes, smooth muscle cells, myofibroblasts, or transitional cells with overlapping features . The hypothesis that a subgroup of PWTs could arise from a pluripotent perivascular cell (presumably, pericyte) would explain the diverse
differentiation of PWTs . FISSs and PWTs are considered to be originated from somatic stem cells in the subcutis, like pericytes in the lineage of stem cells as a possible origin in MFHs.
Malignant mesenchymal tumors, such as fibrosarcoma, osteosarcoma, liposarcoma, leiomyosarcoma, and rhabdomyosarcoma, as well as malignant peripheral nerve sheath tumors, are relatively
easily diagnosed based on their own cellular and histological characteristics . MFH was introduced as a new entity of malignant mesenchymal tumor in
1963 . After that, the diagnostic term of MFH has been used, but the histogenesis of MFH remained to be determined, albeit MFH is the most common
neoplasm of the soft-tissue in the elderly . In veterinary medicine, spontaneously occurring MFHs have rarely been detected in domestic and
laboratory animals . Experimentally induced MFH-like sarcomas
by using chemicals such as 4-(hydroxyamino) quinoline-1-oxide and nickel sulfide have been reported in rats and mice . Histopathologically, MFHs are characterized by a mixture of fibroblastic, histiocytic and undifferentiated mesenchymal cells arranged in a storiform growth pattern . Besides, human MFHs sometimes exhibit various features of mesenchymal tumors such as osteosarcoma, liposarcoma and leiomyosarcoma in parts of tumor tissues . Such histological heterogeneity of MFHs drew a hypothesis that the tumor may contain cells with mesenchymal
multipotency.
In order to explore the cell properties of MFHs, the author established a transplantable tumor line (MFH-MT) from a spontaneous MFH developed in subcutaneous tissue of F344 rat. Cultured
MT-P cell line was induced from MFH-MT. The original and serially transplanted MFH-MT tumors, as well as tumors induced in syngeneic rats by injection with MT-P cells, were composed of an
admixture of fibroblastic, histiocytic cells and immature mesenchymal cells arranged in a storiform growth pattern, of which histology was similar to that of human MFHs .
Two cloned cell lines, named MT-8 and MT-9, were induced from MT-P cells by using the limiting dilution technique. MT-8 and MT-9 cells possessed mesenchymal nature with different morphology
to each other . MT-8 cells had less developed organelles and the induced tumors represented histological characteristics of undifferentiated
sarcoma , whereas MT-9 cells had relatively well-developed intracytoplasmic organelles such as endoplasmic reticulum, mitochondria and lysosomes and the induced tumors showed a storiform
growth pattern typical of human MFHs . Although MT-9
cells, besides immunoreactivity for vimentin, reacted to stem cell markers such as nestin, CD90, and CD34; more interestingly, MT-9 cells also reacted in parts to α-smooth muscle actin
(α-SMA) for myofibroblasts/smooth muscle cells , S-100 /glial fibrillary acidic protein
(GFAP) for neurogenic cells and bone morphogenetic protein (BMP)-6 for osteogenic cells, as well as CD68/CD163 for histiocytic
cells/macrophages, whereas MT-8 cells reacted faintly and negatively to these marker antigens .
Interestingly, the reactivity to CD68 and CD163 were also seen in cultured MT-9 cells ( , respectively), indicating that cells reacting to
CD68/CD163 seen in MT-9 tumors were not infiltrated macrophages but neoplastic cells. The gene analyses revealed that genes relating to “cell differentiation” were more activated in MT-9
than MT-8 tumors, whereas those involved in “cell cycle” were greater in MT-8 than MT-9 tumors. These findings indicated that MT-8 cells are mesenchymal stem cells that have not yet been
decided for cellular differentiation, whereas MT-9 cells are the stem cells having somewhat capacity of mesenchymal differentiations (that is, mesenchymal differentiation capacity of MT-9
cells is greater than that of MT-8 cells). MT-8 tumors are diagnosed as sarcoma not otherwise specified (sarcoma NOS) , whereas MT-9 tumors
showed a histology typical of MFH . In fact, in in vitro studies, adipogenic supplement-added MT-9 cells showed increased accumulation of cytoplasmic lipid droplets stained red with the oil red staining. Addition of BMP-2 (a factor for osteogenesis) to MT-9 enhanced osteoblastic differentiation (as shown by increases in alkaline phosphatase
activity, osteocalcin mRNA expression and calcification . TGF-β1-treated MT-9 cells revealed increased numbers of α-SMA-immunopositive
cells, and enhanced protein levels of α-SMA and fibronectin, indicating myofibrogenesis . TGF-β1 is the most powerful factor for myofibroblast
induction . More interestingly, when injected in syngeneic rats, MT-9 cells, that had been incubated under some conditions in
vitro , developed tumors partly including leiomyosarcoma , osteosarcoma , fibrosarcoma , liposarcoma , and granular cell tumors , in addition to MFH-like storiform growth
. MT-9 cells show greater multipotency presumably under microenvironmental conditions . According to “the cancer stem cells theory” , MT-8 cells may be so-called cancer stem cells (less differentiated cells and greater mitotic
activity), whereas MT-9 cells may be precursor cells (more differentiated cells with less mitotic capacity) of tumors . Based on characteristics of
MFH-constituting cells, therefore, the new term “undifferentiated pleomorphic sarcoma” is suitable for MFHs . The possible histogenesis of MFH is shown in , on the basis of findings mentioned above.
An antibody (named A3) was generated using whole cell antigens prepared from MT-8 cells .
Immunohistochemically, A3 reacted specifically to rat MFH cells, but did not react to other types of tumors in rats . A3 is a conformation-specific antibody recognizing both N -glycan and peptide . The carbohydrate chain is
changed during cellular development from ES cells as a landmark for cell differentiation . Taken these findings together, Rat MFH cells
immunopositive to A3 are considered to have pluripotential differentiations, like ES cells. In normal rat tissues of fetuses, A3 labeled primitive mesenchymal cells in visceral organs . In adult rats, A3 labeled mesenchymal cells in the
bone marrow ; these bone marrow cells co-expressed mesenchymal stem cell markers such as vimentin, CD90 (Thy-1) and CD105 , as well as rat endothelial cell antigen-1 (RECA-1) . Human ES cells and iPS cells show direct differentiation to
endothelial cells, indicating that immature endothelial cells at the early stages have stem cell nature . In addition, A3 labeled perivascular cells (so-called pericytes) which are regarded as mesenchymal somatic stem cells in the
connective tissues . Bone marrow-derived stem cells have the capacity to differentiate not only into various
types of mesenchymal cells, but also into cells with visceral mesoderm, neuroectoderm and endoderm characteristics . The pericytes undergo osteogenic or chondrogenic differentiation in vitro . A3-labeled pericytes are considered to
be in the lineage of bone marrow stem cells. In hair follicles, the hair shaft is veiled by the epidermic hair follicle which are surrounded by the connective tissue sheath (CTS) as the outermost component of hair follicle (dermic
hair follicle). The bulge of hair follicle contains epithelial stem cells which can be activated at the anagen to generate a new hair follicle and the mesenchymal cells in the CTS possess the nature of stromal stem cells . Immunohistochemically, A3 labeled both epithelial cells of the bulge and mesenchymal cells in the CTS . The cells in the
bulge are regarded as somatic stem cells with potential epithelial differentiation . A3-labeled cells in the CTS co-expressed partly CD90, CD34,
and nestin. Antibodies to CD90, CD34 and nestin have been used as immature mesenchymal cell markers . Mesenchymal nestin-positive cells are regarded as stem cells
with neural and mesoderm differentiation potentials . In addition, A3-labeled cells contribute to the tissue regeneration in rat colonic lesions,
indicating that they may take part in the stem cell niche for the formation of mucosal epithelial cells . A3-labeled cells seem to act as rescue
cells after injury in colon ulcer . The rescue cells have nature of both mesenchymal and epithelial cells to maintain self-renewal, keeping the
barrier from external environment under progress of re-epithelialization after injury. The recue cells are considered to be derived from the bone-marrow stem cells . Collectively, the progenitor of MFHs is considered to be A3-labeled pericytes which may be in the lineage of bone marrow stem cells capable of differentiating into both mesenchymal and
epithelial cells . A probable relationship between MFH cells
(MT-8 and MT-9) and the well-defined mesenchymal cells is shown in
Feline injection site sarcomas (FISSs; also known as “vaccine-associated sarcomas”) are characterized by subcutaneous abnormal proliferation of atypical mesenchymal cells and often by
infiltration of macrophages/multinucleated foreign body type giant cells. Histologically, the majority are fibrosarcomas, and other histologic variants such as MFH, poorly differentiated
sarcoma, rhabdomyosarcoma, myxosarcoma, liposarcoma, nerve sheath tumor, histiocytic sarcoma, and extraskeletal osteosarcoma and chondrosarcoma have been recognized . FISSs may be derived from somatic stem cells with multipotential mesenchymal differentiations. It is speculated that
inflammation at the injection site induced by vaccines or other injections likely plays a critical role in tumor development, presumably through genetic damage in stem cells due to oxidative
stress which may be excessively generated by exudative macrophages and giant cells. Perivascular wall tumors (PWTs; previously classified “canine hemangiopericytomas”) are relatively common tumor arising in the subcutis of dogs, and considered to be nonendothelial vascular
neoplasms with a histological characteristic of whirling pattern . In PWTs, however, there are a variety
of histological features, occasionally including histology of peripheral nerve sheath tumors. Therefore, the histogenesis is still under debate. The PWTs may be derived from undifferentiated
stem cells which may differentiate into perivascular fibroblasts, pericytes, myopericytes, smooth muscle cells, myofibroblasts, or transitional cells with overlapping features . The hypothesis that a subgroup of PWTs could arise from a pluripotent perivascular cell (presumably, pericyte) would explain the diverse
differentiation of PWTs . FISSs and PWTs are considered to be originated from somatic stem cells in the subcutis, like pericytes in the lineage of stem cells as a possible origin in MFHs.
General concept of fibrosis, and myofibroblasts in hepatic, renal and pancreatic fibrosis of dogs and cats After tissue injury, inflammation occurs, and subsequently fibrosis is developed via deposition of collagen fibers, in order to repair the injured area. The phenomenon is considered as
appropriate mechanisms equipped in multicellular organisms. Infiltrated macrophages that respond to injury and myofibroblasts that produce collagen fibers play a crucial role in the fibrosis
. If these cells work together and function effectively, the injured tissue recovers completely, whereas, if their coordination is disturbed,
organ and tissue dysfunction occurs due to abnormal deposition of collagen fibers . Cirrhosis and atrophied kidney are representative lesions of
intractable fibrosis. Generally, myofibroblasts are divided into following types: cells expressing only vimentin, those expressing vimentin and desmin, those expressing vimentin and α-SMA, and those expressing
vimentin, α-SMA, and desmin . In spontaneous pathological lesions, myofibroblasts are identified
exclusively by immunohistochemistry with α-SMA antibody . Using fibrotic lesions in liver, kidneys, and pancreas obtained from dogs and
cats, the appearance of α-SMA-immunopositive myofibroblasts was investigated . With the grade of
fibrosis, the appearance of myofibroblasts increased. In addition, macrophages also increased in relation to advancing grade of fibrosis. Macrophages and myofibroblasts are confirmed to be
key cells playing a central role in spontaneously occurring fibrotic lesions in the liver, kidneys and pancreas. It is considered that myofibroblasts may be induced by factors (particularly,
TGF-β1) produced by infiltrated macrophages . Myofibroblasts in chemically-induced hepatic fibrosis Characterization of myofibroblasts in hepatic reparative fibrosis : Hepatic fibrosis, regardless of the etiology, is a dynamic and highly integrated molecular and cellular
process, leading to cirrhosis at the advanced stage . It is considered that hepatic stellate cells (HSCs), which control blood stream in the sinusoid in normal livers, may be a contributor in hepatic
fibrosis through the conversion into myofibroblasts . The HSCs express GFAP, a type III intermediate
filament protein, which is originally an astrocyte marker in the brain . Cellular properties and possible derivation of GFAP-expressing
myofibroblasts were investigated in livers injured in rats by thioacetamide (TAA; a hepatotoxicant) and subsequent reparative fibrosis . After hepatocyte injury in the perivenular areas, the fibrotic lesion consisting of myofibroblasts developed with increased macrophage infiltration.
Immunohistochemically, the myofibroblasts gathering in the hepatic reparative fibrosis expressed much stronger GFAP than those in controls, and came to show co-expressions such cytoskeletons as vimentin, desmin and α-SMA in varying degrees . Furthermore,
some GFAP-expressing myofibroblasts reacted simultaneously to nestin or A3, and there were also myofibroblasts co-expressing nestin/A3. Nestin is a neural and mesenchymal stem cell marker
, and A3 is a somatic stem cell marker in rats . GFAP-expressing myofibroblasts showed the
proliferating activity, but they did not undergo apoptosis. On the other hand, some α-SMA positive myofibroblasts underwent apoptosis. HSCs can proliferate and then convert into
myofibroblasts with co-expressing cytoskeletal proteins for myofibroblast markers (vimentin, desmin, and α-SMA) . Likely, hepatic myofibroblasts
are derived partly from immature HSCs reacting to nestin and A3 which may be in the stem cell lineage . Additionally, well-differentiated
myofibroblasts expressing α-SMA disappear by apoptosis in reparative fibrosis, a healing process . D-galactosamine (D-GalN)-induced hepatic lesions
consist of hepatocyte necrosis/apoptosis and reparative fibrosis present diffusely or focally within the hepatic lobules, of which findings resemble those of human acute viral hepatitis
. The lesions are different from those of TAA-induced centrilobular coagulation necrosis. Immunohistochemically, myofibroblasts seen in
D-GalN-induced liver lesions reacted simultaneously to vimentin/α-SMA, desmin/α-SMA, and desmin/vimentin; furthermore, some myofibroblasts reacting to vimentin, desmin, and α-SMA also
co-expressed GFAP. Additionally, GFAP-expressing myofibroblasts reacted to nestin and A3. Cells immunopositive to CD90 also appeared in fibrotic lesions . These characteristics of myofibroblasts were similar to each other in D-GalN- and TAA-induced hepatotoxicity. Characterization of myofibroblasts and hepatic progenitor cells (HPCs) in cirrhosis : Prolonged and repeated exposures of TAA to rats cause cirrhosis with histological
findings similar partly to those seen in human chronic viral hepatitis . Immunohistochemically, GFAP-expressing myofibroblasts appeared in fibrotic
bridges, separating pseudolobules by increasing collagen bundles. The majority of GFAP-expressing myofibroblasts co-expressed vimentin, desmin and α-SMA in varying degrees . Some GFAP-positive myofibroblasts co-expressed stem cell markers such as nestin, A3 and CD90. Similar to reparative fibrosis, there is a dynamic
participation of GFAP-expressing myofibroblasts in cirrhosis at advanced stage of fibrosis; it is considered that they are also derived mainly from HSCs, and partly from immature HSCs
labeled by A3 and CD90 antibody in the stem cell lineage . GFAP-expressing HSC-converted myofibroblasts have nature of stem cells, demonstrable with immunoreactions to nestin, A3 and CD90. It is reported that GFAP is also expressed in hepatic
progenitor cells (HPCs), which are also called “oval cells” . The HPCs have been considered to be the somatic stem cells capable of differentiating
into both hepatocytes and biliary epithelial cells after liver injury . The ductular reaction (called “pseudo-bile ducts”), a pattern of
proliferation of the most terminal branches of the biliary tree, are characteristically observed in progressive cirrhosis . Immunohistochemically,
ductular cells observed in rat cirrhosis were stained with CK19 (for an epithelial marker) antibody, and some CK19 expressing cells reacted to GFAP, indicating that GFAP-positive HPCs
participate in the ductular reaction ; in fact, the canals of Hering which represent the smaller branches of intrahepatic biliary trees contain a
progenitor cell niche formed by HPCs . As another characteristic of HPCs in rat cirrhosis, there were proliferating ductular cells immunostained
with antibody to α-fetoprotein or β-catenin; the ductular reaction was related to increased expression of hepatocarcinogenesis-related factors such as Wnt2, Wnt4 and glypican-3 . Of interest, the development of myofibroblasts by
epithelial-mesenchymal transition (EMT) from bile ductular cells has been proposed in some animal models and human cases of biliary cirrhosis . It
is shown in a mouse model of bile duct ligation that periductal myofibroblasts were generated from bile duct epithelia through the EMT . In rat
TAA-induced cirrhosis, on the contrary, there is no direct evidence for the EMT in bile ducts of ductular reaction, because there was no co-expression between α-SMA-positive
HSCs/myofibroblasts and CK19-positive ductural epithelial cells in the fibrotic lesions . In rat cirrhosis, however, GFAP-positive HPCs give rise
to the ductular reaction and GFAP-positive HSCs are thought to be the major contributor in the development of myofibroblasts through the conversion . There is a close proximity between HSCs and HPCs
in terms of GFAP expression. The EMT could be a retroactive phenomenon of mesenchymal-epithelial transition (MET) in hepatogenesis . The evidence that HPCs from fetal livers undergo MET in hepatogenesis points out a possible formation of myofibroblasts via the EMT in HPCs relating
to ductular reaction . Significance of microRNAs (miRNAs) in hepatic myofibroblasts : MicroRNAs (miRNAs) are a class of small noncoding RNAs (~25 nucleotides), which play a significant role in
regulating a wide variety of cellular and biological processes, including differentiation, proliferation, cell cycle control, and cell death . miR-214, a microRNA, has recently attracted attention as a fibrosis-related factor . miR-214 was highly
upregulated in the fibrotic area in parallel with the cirrhosis progression . Interestingly, miR-214 overexpression in MT-9 cells (a rat
mesenchymal cell line with stem cell nature ) under TGF-β1 stimulation resulted in decreased cell number with declined expression of α-SMA, whereas
cleaved caspase 3 was increased; these findings suggest that miR-214 induces apoptosis in stem cell-derived myofibroblasts and inhibits myofibroblast differentiation under stimulation of
fibrogenic factors . miR-214 has an anti-fibrotic role in chemically-induced liver fibrosis/cirrhosis. Characterization of myofibroblasts appearing in chemically-induced renal fibrosis Cisplatin (CDDP)-induced renal fibrosis and myofibroblasts : Irrespective of its etiology for kidney injury, renal fibrosis is regarded as the common pathway leading to
chronic kidney disease (CKD) at the advanced stage . Different from hepatic fibrosis to which pre-existing HSCs contribute through the conversion
into myofibroblasts, there are few mesenchymal cells in renal interstitium. However, a lot of myofibroblasts expressing α-SMA appear in renal fibrosis, plying an important role in the
development of fibrotic lesions by producing collagens . Cisplatin (CDDP; cis -diamminedichloroplatinum), an anti-cancer drug with nephrotoxicity as a side-effect, has been used to induce progressive renal fibrosis in rats . CDDP-induced renal lesions consist of degeneration/necrosis/desquamation of epithelial cells of the renal
tubules mainly in the cortico-medullary junction at the early stage, and then the affected tubules are variously dilated or atrophied, being lined by flattened or cuboidal epithelial cells
with basophilic cytoplasm indicating regeneration of epithelial cells; around the affected tubules, interstitial fibrosis is developed gradually with macrophage infiltration . The progressive interstitial fibrosis results in collapse of
renal structures with scar formation. The commencement of interstitial fibrosis corresponds to increased level of TGF-β1 which might be produced by infiltrated macrophages . In addition to CD68-expressing M1 macrophages (associated with tissue injury and inflammation induction) and CD163-expressing M2 macrophages (associated with anti-inflammatory and fibrosis)
, in CDDP-induced renal lesions, MHC class II-expressing macrophages with the polarity toward M1 macrophages as well as CD4 helper T cells and
CD8 cytotoxic T cells are characteristically increased with advancing fibrotic lesions . Because MHC class II macrophages induce T cells in the
immune system , continuous renal injury, in particular by CD8 cytotoxic T cells, may be occurring; the injury also increases exudative macrophages
producing TGF-β1. The progressive renal interstitial fibrosis in CDDP-injected rats is formed by the induction/development of myofibroblasts via the interaction of macrophages and T cells
. In CDDP-induced renal fibrosis, immunohistochemically, there are myofibroblasts reacting to vimentin, desmin and α-SMA, and the expression degrees are increased with advancing fibrosis
. There are myofibroblasts reacting to α-SMA/vimentin, α-SMA/desmin or desmin/vimentin, indicating that renal myofibroblasts can express
simultaneously different cytoskeletal proteins , of which findings are similar to those in
hepatic myofibroblasts. CD90 expression in renal myofibroblasts : To explore the possible origin of renal myofibroblasts, the participation of mesenchymal stem cells expressing CD90 was examined in
CDDP-induced renal fibrosis, . CD90, a 25–37 kDa GPI-anchored cell surface protein, is expressed in immature mesenchymal cells such as bone
marrow stem cells and pericytes . Immunohistochemically, some vimentin- or desmin-positive
myofibroblasts in renal fibrosis reacted to CD90, whereas myofibroblasts reacting to both CD90 and α-SMA is very few . Because the myofibroblasts
expressing α-SMA are considered to be well-differentiated type , vimentin- and desmin-reacting to
myofibroblasts, which co-express CD90, may be the precursor of myofibroblasts in the renal fibrosis . In normal adult rats, immunohistochemically, tubulointerstitial cells in the medulla and mesangial cells in the glomeruli express CD90, whereas the cortical interstitial cells are negative
to CD90 . In developing kidneys of rat fetuses and neonates, renal blastemal cell-derived mesenchymal cells react to CD90 and A3 . The interstitial cells reacting to CD90 in the medulla of adults look similar in nature to
CD90-expressing mesenchymal blastemal cells with immature nature in developing kidneys. In addition, the pericytes express CD90 and A3 . The pericytes have been considered to be stromal stem cells in
connective tissues . In in vitro studies, immature mesenchymal cell line (MT-9;
established from rat MFH cells as mentioned above) originally express CD90, but MT-9 cells incubated with TGF-β1 (the major fibrogenic factor) decreased CD90 expression and conversely
increased α-SMA expression , indicating enhancement of myofibroblastic nature. MT-9 cells have nature of pericytes (mesenchymal somatic stem cells), because MT-9 cells are labeled with A3 and the pericytes in
normal rat connective tissues also show a positive reaction to A3 and CD90 . Additionally, NRK-49F, a rat kidney-derived interstitial fibroblastic cell line, also showed the increased
expression of α-SMA under the incubation with TGF-β1 . Taking these findings together, myofibroblasts in
renal fibrosis are considered to be originated from CD90-expressing interstitial cells in the medulla and pericytes (showing immune reactions to both A3 and CD90) around blood vessels in the
kidney . During the development, renal myofibroblasts
come to show cytoskeletal markers such as vimentin, desmin and α-SMA in varying degrees by reducing CD90 expression . Myofibroblasts formed via the epithelial-mesenchymal transition (EMT) in abnormally regenerating tubular epithelial cells in renal fibrosis : In in vitro studies, a rat kidney-derived epithelial cell line (NRK-52E) incubated with TGF-β1 increased the expression of α-SMA dramatically, with decreased expression of E-cadherin (an epithelial
marker) . Cultured LLC-PK1 cells, a different renal epithelial cell line established from porcine kidney, showed myofibroblastic differentiation
under incubation with TGF-β1 . The EMT has been demonstrated mainly by such in vitro studies. β-catenin, an intracellular molecule involved in the developmental, physiological, and pathological processes in the kidney , binds to E-cadherin
at the cell membrane, which can then stabilize the cell adhesion junction. Additionally, β-catenin mediates Wnt signaling, which regulates the gene expression associated with cell
differentiation and proliferation . Immunohistochemically, β-catenin expression is not seen in the
proximal renal tubules of normal rats, whereas, in CDDP-induced renal injury, the regenerating renal tubular epithelial cells, which reacted strongly to Ki67 (a marker for
proliferating/regenerating cells), expressed membranous or cytoplasmic β-catenin, but did not show a positive reaction to α-SMA . Interestingly,
epithelial cells of abnormally dilated or atrophied renal tubules at the advanced stage of renal fibrosis do not react to β-catenin or Ki67, indicating that regeneration of renal epithelial
cells is incomplete/abnormal; such renal tubules may be at G 0 /G 1 arrest in cell cycle . Interestingly, some of renal tubules
showing incomplete regeneration reacted to α-SMA , but not to E-cadherin ; this phenomenon is
similar to TGF-β1-induced EMT in in vitro studies as mentioned above. Because α-SMA expression is a marker for development of myofibroblasts , it is likely that EMT occurs in abnormally regenerating epithelial cells after renal injury in vivo , particularly
at the advanced stage , thereby resulting in progressive renal fibrosis. It is known that incomplete
regeneration takes place in cases of renal tubules with disrupted basal lamina due to injury . The presence or absence of β-catenin expression
would become a marker for the EMT phenomenon in progressive renal fibrosis in vivo . During nephrogenesis, renal blastemal cell-derived mesenchymal cells exhibit variously vimentin, CD90 and α-SMA , of which immunohistochemical
findings are generally similar to those in myofibroblasts seen in renal fibrosis. Because renal blastemal cells have the capacity to differentiate into both renal epithelial cells and
interstitial mesenchymal cells , myofibroblasts formed via the EMT in abnormally regenerating epithelial cells of the injured tubules may be
regarded as so-called “retroactive phenomenon” into blastemal cells ; this phenomenon seems to be
similar to that seen in bile ductual reaction by HPCs in cirrhosis . In short, myofibroblasts in renal fibrosis are considered to be derived from the pre-existing medullary interstitial cells and pericytes, and also to be formed via the EMT phenomenon in
abnormally regenerating renal epithelial cells . Presumably, these cells in fibrosis and regeneration could be immature cells in the stem cell lineage. Renal fibrosis-related factors : In addition to TGF-β1 which is known to be the most powerful fibrogenic factor , increased
expressions of BMP-6 and osteopontin (OPN) are seen consistently with advancing CDDP-induced renal fibrosis . BMP-6 deficiency mice applied with
unilateral ureteral obstruction aggravates tubular damage and enhances interstitial fibrosis . OPN is used as a biomarker for renal injury; its
expression is related to the extent of renal damage . Immunohistochemically, BMP-6 is expressed both in
the damaged renal epithelial cells and spindle-shaped myofibroblasts (co-expressing α-SMA) in the fibrotic areas; OPN expression is seen exclusively in the injured renal epithelial cells. In in vitro studies, incubation with BMP-6 or OPN increased α-SMA in MT-9 cells, similar to effects of TGF-β1 on MT-9 and NRK-49F cells. Interestingly, TGF-β1 addition
increased BMP-6 and OPN in NRK-52E cells in vitro . Collectively, BMP-6 and OPN considerably participate in progressive renal fibrosis through development of myofibroblasts,
in cooperation with TGF-β1 . Platelet-derived growth factor BB exhibits an additive effect on TGF-β1-induced α-SMA expression in LLC-PK1, NRK-49F
and MT-9 cells . On the other hand, it has been reported that hepatocyte growth factor (HGF) and BMP-7 have inhibitory effects on renal
myofibroblast development . Cyclooxygenase (COX)-1 and COX-2 are thought to be responsible for basal prostaglandin (PG) production in homeostasis or inflammation . The
expression patterns of PGE 2 synthesis enzymes such as COX-1, COX-2 and microsomal PGE synthase (mPGES)-1, and PGE 2 receptors (EP2 and EP4) were examined in CDDP-induced
rat renal lesions . Immunohistochemically, the expressions of COX-1, mPGES-1 and EP4 receptor were increased exclusively in the affected renal
tubules, but those of COX-2 and EP2 receptor were not detected. The addition of PGE 2 or 11-deoxy-PGE 1 (EP4 receptor agonist) to NRK-52E (a rat renal epithelial cell
line) increased the cell number, indicating the regenerating effects of PGE 2 via EP4 receptor. Furthermore, 11-deoxy-PGE 1 -treated NRK-52E cells underwent the
G 0 /G 1 arrest and inhibited apoptosis, suggestive of favorable regeneration of renal epithelial cells. NRK-52E cells incubated with TGF-β1, an inducer of EMT, in the
presence of 11-deoxy-PGE1 decreased the α-SMA expression (a marker of myofibroblasts) . EP4-selective agonist reduced renal lesions in a mercury
chloride-induced rat renal failure model . PGE 2 , which may be produced by COX-1 rather than COX-2 in CDDP-induced renal fibrosis, can
regulate renal epithelial regeneration via EP4 receptor through inhibition of epithelial apoptosis and EMT . It is important to investigate the
factors which can influence the development of renal myofibroblasts. Myofibroblasts appearing in fibrosis in other organs Apart from the liver and kidneys, mainly with immunohistochemical methods, characteristics of myofibroblasts in fibrotic lesions in the skin, pancreas, lungs, myocardium, and colon have
been analyzed by using rat models. Myofibroblasts in cutaneous excisional wound healing reacted exclusively to vimentin and α-SMA, and they also show a positive reaction to CD90 . In the cutaneous fibrosis, connective tissue stem cells, such as the pericytes (in particular, of neovascularization) and perifollicular dermal sheath
mesenchymal cells in the wound periphery, participate in the lesion development. Interestingly, there is considerable immunophenotypical similarity between myofibroblasts (expressing
vimentin, α-SMA and CD90), pericytes (reacting to vimentin, α-SMA, CD90 and A3), and perifollicular dermal sheath mesenchymal stem cells (reacting to vimentin, CD90 and A3). In
bleomycin-induced scleroderma with greater fibrosis, the reactivity of vimentin, α-SMA, and CD90 in the pericytes and perifollicular dermal sheath mesenchymal stem cells were more prominent
. Apoptosis in the hair follicle induced by bleomycin might have mediated the perifollicular fibrosis by inducing macrophages capable of
fibrogenic factors, resulting in extensive scleroderma . In addition to the pre-existing cutaneous fibroblasts, myofibroblasts developed in
cutaneous fibrosis seem to be generated from the pericytes particularly in newly-developed blood vessels or perifollicular dermal sheath mesenchymal stem cells. The concept of
endothelial-mesenchymal transition (EnMT) has been proposed as a novel mechanism for generating fibrotic lesions through induction of collagen-producing myofibroblasts . Because there is a close relationship of endothelial cells and
pericytes (both showing a positive reaction to A3) in neovascularization and they are considered to be in the stem cell lineage relating to the regeneration and repair , the EnMT could occur in various tissues after injury, thereby contributing to fibrosis. Pancreatic stellate cells (PSCs) expressing GFAP, similar in nature to HSCs, are present around the acinar cells in the pancreas . In pancreatic fibrosis induced by dibutyltin dichloride, immunohistochemically, myofibroblasts showed co-expression to vimentin/α-SMA, desmin/α-SMA,
and GFAP/α-SMA; further, CD90-expressing immature mesenchymal cells also increased in fibrotic lesions. Pancreatic myofibroblasts may be originated from GFAP-expressing PSCs and CD90-expressing immature mesenchymal cells . In the bleomycin-induced rat pulmonary fibrosis, a small number of α-SMA-expressing myofibroblasts were present in the fibrotic lesion. In the fibrotic lesions, RECA-1-expressing
endothelial cells co-expressed A3; some of A3-positive cells reacted to CD34. A3 and CD34 are expressed in somatic stem cells . According to the concept of EnMT , immature endothelial cells
expressing A3 and CD34 in the pulmonary septa may have been involved in the pulmonary fibrosis as a possible progenitor of myofibroblasts. Myofibroblasts appearing in isoproterenol-induced myocardial fibrosis expressed vimentin and α-SMA, and many CD90-expressing cells are present in the fibrotic areas . Additionally, there are newly-developed blood vessels reacting to A3 in and around the myocardial fibrosis. Myofibroblasts appearing in rat myocardial fibrosis may
have been derived from immature mesenchymal cells immunopositive for CD90 or A3, including the pericytes and endothelial cells relating to EnMT . In dextran sulfate sodium (DSS)-induced colonic ulcerated lesions with fibrosis, A3-positive cells aggregated beneath the desquamated mucosa where the re-epithelialization is occurring
nearby; the cells reacted simultaneously to CD90, vimentin or CK19 (an epithelial cell marker). Likely, the aggregated A3-positive cells may be rescue cells with nature of both mesenchymal
and epithelial cells to maintain self-renewal after injury in the colon . In addition, there were myofibroblasts reacting to vimentin, α-SMA and
CD90 in the fibrotic lesions, and newly-formed capillaries were labeled by A3. The mesenchymal stem cells (including pericytes and endothelial cells) that they reacted to CD90 and A3 may be
a precursor of myofibroblasts in fibrotic colon lesions . The derivation of myofibroblasts in the fibrotic lesions As introduced above, myofibroblasts are characterized by immune-expressions of vimentin, desmin and α-SMA in varying degrees, although desmin-expression are rarely seen in the cutaneous,
myocardial and pulmonary fibrosis. Interestingly, HSCs and PSCs, both reacting to GFAP, participate in the hepatic and pancreatic fibrosis, respectively; these cells are the pre-existing
mesenchymal cells with immature nature which may be derived partly from the somatic stem cells. More interestingly, immunohistochemically, CD90-expressing immature mesenchymal cells appear
in various fibrotic lesions, co-expressing vimentin and α-SMA; in addition, A3-labeled cells are seen in the newly-developed blood vessels in and around the fibrotic lesions; the pericytes
and endothelial cells in neovascularization express A3 antigen. The endothelial cells could contribute to the fibrosis via the EnMT. The mesenchymal stem cells, which may be involved in
pathological lesions, are speculated to be derived from bone marrow stem cells as a possible contributor of fibrotic lesion development . The bone marrow-derived stem cells and connective tissue stem cells such as pericytes are called “Muse cells” (multilineage-differentiating
stress-enduring cells) . In fact, immunohistochemically, the bone marrow-stem cells and pericytes react to CD90 and A3. Immature mesenchymal cells
reacting to A3, CD90, CD34 or nestin are considered to be progenitor cells of myofibroblasts, partly which are considered to be in the bone marrow-stem cell lineage. Further, the development
of myofibroblasts via the EMT phenomenon occurs in abnormally regenerating renal epithelial cells in the fibrotic kidneys and in bile ductular reaction in cirrhosis; these cells may be also
in the stem cell lineage relating to the regeneration and repair. Although myofibroblasts appearing in the fibrotic lesions may be heterogeneous in the derivation, the somatic stem cells
seem to contribute largely to the development of myofibroblasts as the basic line . Therefore, the somatic stem cells equipped in the body
would be a useful tool for regenerative therapy .
After tissue injury, inflammation occurs, and subsequently fibrosis is developed via deposition of collagen fibers, in order to repair the injured area. The phenomenon is considered as
appropriate mechanisms equipped in multicellular organisms. Infiltrated macrophages that respond to injury and myofibroblasts that produce collagen fibers play a crucial role in the fibrosis
. If these cells work together and function effectively, the injured tissue recovers completely, whereas, if their coordination is disturbed,
organ and tissue dysfunction occurs due to abnormal deposition of collagen fibers . Cirrhosis and atrophied kidney are representative lesions of
intractable fibrosis. Generally, myofibroblasts are divided into following types: cells expressing only vimentin, those expressing vimentin and desmin, those expressing vimentin and α-SMA, and those expressing
vimentin, α-SMA, and desmin . In spontaneous pathological lesions, myofibroblasts are identified
exclusively by immunohistochemistry with α-SMA antibody . Using fibrotic lesions in liver, kidneys, and pancreas obtained from dogs and
cats, the appearance of α-SMA-immunopositive myofibroblasts was investigated . With the grade of
fibrosis, the appearance of myofibroblasts increased. In addition, macrophages also increased in relation to advancing grade of fibrosis. Macrophages and myofibroblasts are confirmed to be
key cells playing a central role in spontaneously occurring fibrotic lesions in the liver, kidneys and pancreas. It is considered that myofibroblasts may be induced by factors (particularly,
TGF-β1) produced by infiltrated macrophages .
Characterization of myofibroblasts in hepatic reparative fibrosis : Hepatic fibrosis, regardless of the etiology, is a dynamic and highly integrated molecular and cellular
process, leading to cirrhosis at the advanced stage . It is considered that hepatic stellate cells (HSCs), which control blood stream in the sinusoid in normal livers, may be a contributor in hepatic
fibrosis through the conversion into myofibroblasts . The HSCs express GFAP, a type III intermediate
filament protein, which is originally an astrocyte marker in the brain . Cellular properties and possible derivation of GFAP-expressing
myofibroblasts were investigated in livers injured in rats by thioacetamide (TAA; a hepatotoxicant) and subsequent reparative fibrosis . After hepatocyte injury in the perivenular areas, the fibrotic lesion consisting of myofibroblasts developed with increased macrophage infiltration.
Immunohistochemically, the myofibroblasts gathering in the hepatic reparative fibrosis expressed much stronger GFAP than those in controls, and came to show co-expressions such cytoskeletons as vimentin, desmin and α-SMA in varying degrees . Furthermore,
some GFAP-expressing myofibroblasts reacted simultaneously to nestin or A3, and there were also myofibroblasts co-expressing nestin/A3. Nestin is a neural and mesenchymal stem cell marker
, and A3 is a somatic stem cell marker in rats . GFAP-expressing myofibroblasts showed the
proliferating activity, but they did not undergo apoptosis. On the other hand, some α-SMA positive myofibroblasts underwent apoptosis. HSCs can proliferate and then convert into
myofibroblasts with co-expressing cytoskeletal proteins for myofibroblast markers (vimentin, desmin, and α-SMA) . Likely, hepatic myofibroblasts
are derived partly from immature HSCs reacting to nestin and A3 which may be in the stem cell lineage . Additionally, well-differentiated
myofibroblasts expressing α-SMA disappear by apoptosis in reparative fibrosis, a healing process . D-galactosamine (D-GalN)-induced hepatic lesions
consist of hepatocyte necrosis/apoptosis and reparative fibrosis present diffusely or focally within the hepatic lobules, of which findings resemble those of human acute viral hepatitis
. The lesions are different from those of TAA-induced centrilobular coagulation necrosis. Immunohistochemically, myofibroblasts seen in
D-GalN-induced liver lesions reacted simultaneously to vimentin/α-SMA, desmin/α-SMA, and desmin/vimentin; furthermore, some myofibroblasts reacting to vimentin, desmin, and α-SMA also
co-expressed GFAP. Additionally, GFAP-expressing myofibroblasts reacted to nestin and A3. Cells immunopositive to CD90 also appeared in fibrotic lesions . These characteristics of myofibroblasts were similar to each other in D-GalN- and TAA-induced hepatotoxicity. Characterization of myofibroblasts and hepatic progenitor cells (HPCs) in cirrhosis : Prolonged and repeated exposures of TAA to rats cause cirrhosis with histological
findings similar partly to those seen in human chronic viral hepatitis . Immunohistochemically, GFAP-expressing myofibroblasts appeared in fibrotic
bridges, separating pseudolobules by increasing collagen bundles. The majority of GFAP-expressing myofibroblasts co-expressed vimentin, desmin and α-SMA in varying degrees . Some GFAP-positive myofibroblasts co-expressed stem cell markers such as nestin, A3 and CD90. Similar to reparative fibrosis, there is a dynamic
participation of GFAP-expressing myofibroblasts in cirrhosis at advanced stage of fibrosis; it is considered that they are also derived mainly from HSCs, and partly from immature HSCs
labeled by A3 and CD90 antibody in the stem cell lineage . GFAP-expressing HSC-converted myofibroblasts have nature of stem cells, demonstrable with immunoreactions to nestin, A3 and CD90. It is reported that GFAP is also expressed in hepatic
progenitor cells (HPCs), which are also called “oval cells” . The HPCs have been considered to be the somatic stem cells capable of differentiating
into both hepatocytes and biliary epithelial cells after liver injury . The ductular reaction (called “pseudo-bile ducts”), a pattern of
proliferation of the most terminal branches of the biliary tree, are characteristically observed in progressive cirrhosis . Immunohistochemically,
ductular cells observed in rat cirrhosis were stained with CK19 (for an epithelial marker) antibody, and some CK19 expressing cells reacted to GFAP, indicating that GFAP-positive HPCs
participate in the ductular reaction ; in fact, the canals of Hering which represent the smaller branches of intrahepatic biliary trees contain a
progenitor cell niche formed by HPCs . As another characteristic of HPCs in rat cirrhosis, there were proliferating ductular cells immunostained
with antibody to α-fetoprotein or β-catenin; the ductular reaction was related to increased expression of hepatocarcinogenesis-related factors such as Wnt2, Wnt4 and glypican-3 . Of interest, the development of myofibroblasts by
epithelial-mesenchymal transition (EMT) from bile ductular cells has been proposed in some animal models and human cases of biliary cirrhosis . It
is shown in a mouse model of bile duct ligation that periductal myofibroblasts were generated from bile duct epithelia through the EMT . In rat
TAA-induced cirrhosis, on the contrary, there is no direct evidence for the EMT in bile ducts of ductular reaction, because there was no co-expression between α-SMA-positive
HSCs/myofibroblasts and CK19-positive ductural epithelial cells in the fibrotic lesions . In rat cirrhosis, however, GFAP-positive HPCs give rise
to the ductular reaction and GFAP-positive HSCs are thought to be the major contributor in the development of myofibroblasts through the conversion . There is a close proximity between HSCs and HPCs
in terms of GFAP expression. The EMT could be a retroactive phenomenon of mesenchymal-epithelial transition (MET) in hepatogenesis . The evidence that HPCs from fetal livers undergo MET in hepatogenesis points out a possible formation of myofibroblasts via the EMT in HPCs relating
to ductular reaction . Significance of microRNAs (miRNAs) in hepatic myofibroblasts : MicroRNAs (miRNAs) are a class of small noncoding RNAs (~25 nucleotides), which play a significant role in
regulating a wide variety of cellular and biological processes, including differentiation, proliferation, cell cycle control, and cell death . miR-214, a microRNA, has recently attracted attention as a fibrosis-related factor . miR-214 was highly
upregulated in the fibrotic area in parallel with the cirrhosis progression . Interestingly, miR-214 overexpression in MT-9 cells (a rat
mesenchymal cell line with stem cell nature ) under TGF-β1 stimulation resulted in decreased cell number with declined expression of α-SMA, whereas
cleaved caspase 3 was increased; these findings suggest that miR-214 induces apoptosis in stem cell-derived myofibroblasts and inhibits myofibroblast differentiation under stimulation of
fibrogenic factors . miR-214 has an anti-fibrotic role in chemically-induced liver fibrosis/cirrhosis.
Cisplatin (CDDP)-induced renal fibrosis and myofibroblasts : Irrespective of its etiology for kidney injury, renal fibrosis is regarded as the common pathway leading to
chronic kidney disease (CKD) at the advanced stage . Different from hepatic fibrosis to which pre-existing HSCs contribute through the conversion
into myofibroblasts, there are few mesenchymal cells in renal interstitium. However, a lot of myofibroblasts expressing α-SMA appear in renal fibrosis, plying an important role in the
development of fibrotic lesions by producing collagens . Cisplatin (CDDP; cis -diamminedichloroplatinum), an anti-cancer drug with nephrotoxicity as a side-effect, has been used to induce progressive renal fibrosis in rats . CDDP-induced renal lesions consist of degeneration/necrosis/desquamation of epithelial cells of the renal
tubules mainly in the cortico-medullary junction at the early stage, and then the affected tubules are variously dilated or atrophied, being lined by flattened or cuboidal epithelial cells
with basophilic cytoplasm indicating regeneration of epithelial cells; around the affected tubules, interstitial fibrosis is developed gradually with macrophage infiltration . The progressive interstitial fibrosis results in collapse of
renal structures with scar formation. The commencement of interstitial fibrosis corresponds to increased level of TGF-β1 which might be produced by infiltrated macrophages . In addition to CD68-expressing M1 macrophages (associated with tissue injury and inflammation induction) and CD163-expressing M2 macrophages (associated with anti-inflammatory and fibrosis)
, in CDDP-induced renal lesions, MHC class II-expressing macrophages with the polarity toward M1 macrophages as well as CD4 helper T cells and
CD8 cytotoxic T cells are characteristically increased with advancing fibrotic lesions . Because MHC class II macrophages induce T cells in the
immune system , continuous renal injury, in particular by CD8 cytotoxic T cells, may be occurring; the injury also increases exudative macrophages
producing TGF-β1. The progressive renal interstitial fibrosis in CDDP-injected rats is formed by the induction/development of myofibroblasts via the interaction of macrophages and T cells
. In CDDP-induced renal fibrosis, immunohistochemically, there are myofibroblasts reacting to vimentin, desmin and α-SMA, and the expression degrees are increased with advancing fibrosis
. There are myofibroblasts reacting to α-SMA/vimentin, α-SMA/desmin or desmin/vimentin, indicating that renal myofibroblasts can express
simultaneously different cytoskeletal proteins , of which findings are similar to those in
hepatic myofibroblasts. CD90 expression in renal myofibroblasts : To explore the possible origin of renal myofibroblasts, the participation of mesenchymal stem cells expressing CD90 was examined in
CDDP-induced renal fibrosis, . CD90, a 25–37 kDa GPI-anchored cell surface protein, is expressed in immature mesenchymal cells such as bone
marrow stem cells and pericytes . Immunohistochemically, some vimentin- or desmin-positive
myofibroblasts in renal fibrosis reacted to CD90, whereas myofibroblasts reacting to both CD90 and α-SMA is very few . Because the myofibroblasts
expressing α-SMA are considered to be well-differentiated type , vimentin- and desmin-reacting to
myofibroblasts, which co-express CD90, may be the precursor of myofibroblasts in the renal fibrosis . In normal adult rats, immunohistochemically, tubulointerstitial cells in the medulla and mesangial cells in the glomeruli express CD90, whereas the cortical interstitial cells are negative
to CD90 . In developing kidneys of rat fetuses and neonates, renal blastemal cell-derived mesenchymal cells react to CD90 and A3 . The interstitial cells reacting to CD90 in the medulla of adults look similar in nature to
CD90-expressing mesenchymal blastemal cells with immature nature in developing kidneys. In addition, the pericytes express CD90 and A3 . The pericytes have been considered to be stromal stem cells in
connective tissues . In in vitro studies, immature mesenchymal cell line (MT-9;
established from rat MFH cells as mentioned above) originally express CD90, but MT-9 cells incubated with TGF-β1 (the major fibrogenic factor) decreased CD90 expression and conversely
increased α-SMA expression , indicating enhancement of myofibroblastic nature. MT-9 cells have nature of pericytes (mesenchymal somatic stem cells), because MT-9 cells are labeled with A3 and the pericytes in
normal rat connective tissues also show a positive reaction to A3 and CD90 . Additionally, NRK-49F, a rat kidney-derived interstitial fibroblastic cell line, also showed the increased
expression of α-SMA under the incubation with TGF-β1 . Taking these findings together, myofibroblasts in
renal fibrosis are considered to be originated from CD90-expressing interstitial cells in the medulla and pericytes (showing immune reactions to both A3 and CD90) around blood vessels in the
kidney . During the development, renal myofibroblasts
come to show cytoskeletal markers such as vimentin, desmin and α-SMA in varying degrees by reducing CD90 expression . Myofibroblasts formed via the epithelial-mesenchymal transition (EMT) in abnormally regenerating tubular epithelial cells in renal fibrosis : In in vitro studies, a rat kidney-derived epithelial cell line (NRK-52E) incubated with TGF-β1 increased the expression of α-SMA dramatically, with decreased expression of E-cadherin (an epithelial
marker) . Cultured LLC-PK1 cells, a different renal epithelial cell line established from porcine kidney, showed myofibroblastic differentiation
under incubation with TGF-β1 . The EMT has been demonstrated mainly by such in vitro studies. β-catenin, an intracellular molecule involved in the developmental, physiological, and pathological processes in the kidney , binds to E-cadherin
at the cell membrane, which can then stabilize the cell adhesion junction. Additionally, β-catenin mediates Wnt signaling, which regulates the gene expression associated with cell
differentiation and proliferation . Immunohistochemically, β-catenin expression is not seen in the
proximal renal tubules of normal rats, whereas, in CDDP-induced renal injury, the regenerating renal tubular epithelial cells, which reacted strongly to Ki67 (a marker for
proliferating/regenerating cells), expressed membranous or cytoplasmic β-catenin, but did not show a positive reaction to α-SMA . Interestingly,
epithelial cells of abnormally dilated or atrophied renal tubules at the advanced stage of renal fibrosis do not react to β-catenin or Ki67, indicating that regeneration of renal epithelial
cells is incomplete/abnormal; such renal tubules may be at G 0 /G 1 arrest in cell cycle . Interestingly, some of renal tubules
showing incomplete regeneration reacted to α-SMA , but not to E-cadherin ; this phenomenon is
similar to TGF-β1-induced EMT in in vitro studies as mentioned above. Because α-SMA expression is a marker for development of myofibroblasts , it is likely that EMT occurs in abnormally regenerating epithelial cells after renal injury in vivo , particularly
at the advanced stage , thereby resulting in progressive renal fibrosis. It is known that incomplete
regeneration takes place in cases of renal tubules with disrupted basal lamina due to injury . The presence or absence of β-catenin expression
would become a marker for the EMT phenomenon in progressive renal fibrosis in vivo . During nephrogenesis, renal blastemal cell-derived mesenchymal cells exhibit variously vimentin, CD90 and α-SMA , of which immunohistochemical
findings are generally similar to those in myofibroblasts seen in renal fibrosis. Because renal blastemal cells have the capacity to differentiate into both renal epithelial cells and
interstitial mesenchymal cells , myofibroblasts formed via the EMT in abnormally regenerating epithelial cells of the injured tubules may be
regarded as so-called “retroactive phenomenon” into blastemal cells ; this phenomenon seems to be
similar to that seen in bile ductual reaction by HPCs in cirrhosis . In short, myofibroblasts in renal fibrosis are considered to be derived from the pre-existing medullary interstitial cells and pericytes, and also to be formed via the EMT phenomenon in
abnormally regenerating renal epithelial cells . Presumably, these cells in fibrosis and regeneration could be immature cells in the stem cell lineage. Renal fibrosis-related factors : In addition to TGF-β1 which is known to be the most powerful fibrogenic factor , increased
expressions of BMP-6 and osteopontin (OPN) are seen consistently with advancing CDDP-induced renal fibrosis . BMP-6 deficiency mice applied with
unilateral ureteral obstruction aggravates tubular damage and enhances interstitial fibrosis . OPN is used as a biomarker for renal injury; its
expression is related to the extent of renal damage . Immunohistochemically, BMP-6 is expressed both in
the damaged renal epithelial cells and spindle-shaped myofibroblasts (co-expressing α-SMA) in the fibrotic areas; OPN expression is seen exclusively in the injured renal epithelial cells. In in vitro studies, incubation with BMP-6 or OPN increased α-SMA in MT-9 cells, similar to effects of TGF-β1 on MT-9 and NRK-49F cells. Interestingly, TGF-β1 addition
increased BMP-6 and OPN in NRK-52E cells in vitro . Collectively, BMP-6 and OPN considerably participate in progressive renal fibrosis through development of myofibroblasts,
in cooperation with TGF-β1 . Platelet-derived growth factor BB exhibits an additive effect on TGF-β1-induced α-SMA expression in LLC-PK1, NRK-49F
and MT-9 cells . On the other hand, it has been reported that hepatocyte growth factor (HGF) and BMP-7 have inhibitory effects on renal
myofibroblast development . Cyclooxygenase (COX)-1 and COX-2 are thought to be responsible for basal prostaglandin (PG) production in homeostasis or inflammation . The
expression patterns of PGE 2 synthesis enzymes such as COX-1, COX-2 and microsomal PGE synthase (mPGES)-1, and PGE 2 receptors (EP2 and EP4) were examined in CDDP-induced
rat renal lesions . Immunohistochemically, the expressions of COX-1, mPGES-1 and EP4 receptor were increased exclusively in the affected renal
tubules, but those of COX-2 and EP2 receptor were not detected. The addition of PGE 2 or 11-deoxy-PGE 1 (EP4 receptor agonist) to NRK-52E (a rat renal epithelial cell
line) increased the cell number, indicating the regenerating effects of PGE 2 via EP4 receptor. Furthermore, 11-deoxy-PGE 1 -treated NRK-52E cells underwent the
G 0 /G 1 arrest and inhibited apoptosis, suggestive of favorable regeneration of renal epithelial cells. NRK-52E cells incubated with TGF-β1, an inducer of EMT, in the
presence of 11-deoxy-PGE1 decreased the α-SMA expression (a marker of myofibroblasts) . EP4-selective agonist reduced renal lesions in a mercury
chloride-induced rat renal failure model . PGE 2 , which may be produced by COX-1 rather than COX-2 in CDDP-induced renal fibrosis, can
regulate renal epithelial regeneration via EP4 receptor through inhibition of epithelial apoptosis and EMT . It is important to investigate the
factors which can influence the development of renal myofibroblasts.
Apart from the liver and kidneys, mainly with immunohistochemical methods, characteristics of myofibroblasts in fibrotic lesions in the skin, pancreas, lungs, myocardium, and colon have
been analyzed by using rat models. Myofibroblasts in cutaneous excisional wound healing reacted exclusively to vimentin and α-SMA, and they also show a positive reaction to CD90 . In the cutaneous fibrosis, connective tissue stem cells, such as the pericytes (in particular, of neovascularization) and perifollicular dermal sheath
mesenchymal cells in the wound periphery, participate in the lesion development. Interestingly, there is considerable immunophenotypical similarity between myofibroblasts (expressing
vimentin, α-SMA and CD90), pericytes (reacting to vimentin, α-SMA, CD90 and A3), and perifollicular dermal sheath mesenchymal stem cells (reacting to vimentin, CD90 and A3). In
bleomycin-induced scleroderma with greater fibrosis, the reactivity of vimentin, α-SMA, and CD90 in the pericytes and perifollicular dermal sheath mesenchymal stem cells were more prominent
. Apoptosis in the hair follicle induced by bleomycin might have mediated the perifollicular fibrosis by inducing macrophages capable of
fibrogenic factors, resulting in extensive scleroderma . In addition to the pre-existing cutaneous fibroblasts, myofibroblasts developed in
cutaneous fibrosis seem to be generated from the pericytes particularly in newly-developed blood vessels or perifollicular dermal sheath mesenchymal stem cells. The concept of
endothelial-mesenchymal transition (EnMT) has been proposed as a novel mechanism for generating fibrotic lesions through induction of collagen-producing myofibroblasts . Because there is a close relationship of endothelial cells and
pericytes (both showing a positive reaction to A3) in neovascularization and they are considered to be in the stem cell lineage relating to the regeneration and repair , the EnMT could occur in various tissues after injury, thereby contributing to fibrosis. Pancreatic stellate cells (PSCs) expressing GFAP, similar in nature to HSCs, are present around the acinar cells in the pancreas . In pancreatic fibrosis induced by dibutyltin dichloride, immunohistochemically, myofibroblasts showed co-expression to vimentin/α-SMA, desmin/α-SMA,
and GFAP/α-SMA; further, CD90-expressing immature mesenchymal cells also increased in fibrotic lesions. Pancreatic myofibroblasts may be originated from GFAP-expressing PSCs and CD90-expressing immature mesenchymal cells . In the bleomycin-induced rat pulmonary fibrosis, a small number of α-SMA-expressing myofibroblasts were present in the fibrotic lesion. In the fibrotic lesions, RECA-1-expressing
endothelial cells co-expressed A3; some of A3-positive cells reacted to CD34. A3 and CD34 are expressed in somatic stem cells . According to the concept of EnMT , immature endothelial cells
expressing A3 and CD34 in the pulmonary septa may have been involved in the pulmonary fibrosis as a possible progenitor of myofibroblasts. Myofibroblasts appearing in isoproterenol-induced myocardial fibrosis expressed vimentin and α-SMA, and many CD90-expressing cells are present in the fibrotic areas . Additionally, there are newly-developed blood vessels reacting to A3 in and around the myocardial fibrosis. Myofibroblasts appearing in rat myocardial fibrosis may
have been derived from immature mesenchymal cells immunopositive for CD90 or A3, including the pericytes and endothelial cells relating to EnMT . In dextran sulfate sodium (DSS)-induced colonic ulcerated lesions with fibrosis, A3-positive cells aggregated beneath the desquamated mucosa where the re-epithelialization is occurring
nearby; the cells reacted simultaneously to CD90, vimentin or CK19 (an epithelial cell marker). Likely, the aggregated A3-positive cells may be rescue cells with nature of both mesenchymal
and epithelial cells to maintain self-renewal after injury in the colon . In addition, there were myofibroblasts reacting to vimentin, α-SMA and
CD90 in the fibrotic lesions, and newly-formed capillaries were labeled by A3. The mesenchymal stem cells (including pericytes and endothelial cells) that they reacted to CD90 and A3 may be
a precursor of myofibroblasts in fibrotic colon lesions .
As introduced above, myofibroblasts are characterized by immune-expressions of vimentin, desmin and α-SMA in varying degrees, although desmin-expression are rarely seen in the cutaneous,
myocardial and pulmonary fibrosis. Interestingly, HSCs and PSCs, both reacting to GFAP, participate in the hepatic and pancreatic fibrosis, respectively; these cells are the pre-existing
mesenchymal cells with immature nature which may be derived partly from the somatic stem cells. More interestingly, immunohistochemically, CD90-expressing immature mesenchymal cells appear
in various fibrotic lesions, co-expressing vimentin and α-SMA; in addition, A3-labeled cells are seen in the newly-developed blood vessels in and around the fibrotic lesions; the pericytes
and endothelial cells in neovascularization express A3 antigen. The endothelial cells could contribute to the fibrosis via the EnMT. The mesenchymal stem cells, which may be involved in
pathological lesions, are speculated to be derived from bone marrow stem cells as a possible contributor of fibrotic lesion development . The bone marrow-derived stem cells and connective tissue stem cells such as pericytes are called “Muse cells” (multilineage-differentiating
stress-enduring cells) . In fact, immunohistochemically, the bone marrow-stem cells and pericytes react to CD90 and A3. Immature mesenchymal cells
reacting to A3, CD90, CD34 or nestin are considered to be progenitor cells of myofibroblasts, partly which are considered to be in the bone marrow-stem cell lineage. Further, the development
of myofibroblasts via the EMT phenomenon occurs in abnormally regenerating renal epithelial cells in the fibrotic kidneys and in bile ductular reaction in cirrhosis; these cells may be also
in the stem cell lineage relating to the regeneration and repair. Although myofibroblasts appearing in the fibrotic lesions may be heterogeneous in the derivation, the somatic stem cells
seem to contribute largely to the development of myofibroblasts as the basic line . Therefore, the somatic stem cells equipped in the body
would be a useful tool for regenerative therapy .
Stem cells are considered to be a group of cells that have pluripotency and maintain their differentiation capacity even after repeated self-renewal. Pathological studies based on stem cells
existing in the body has been flourishing last decades. The author has attempted to elucidate the histogenesis of undifferentiated mesenchymal cell-derived tumors (so-called MFH) including
stem cells with multi-differentiations, and to investigate the characteristics of myofibroblasts appearing in fibrosis by using a newly/originally-developed antibody (A3) which recognizes rat
somatic stem cells. The myofibroblasts may be derived partly from progenitors in the lineage of stem cells as the baseline. This review introduced the data and their related discussion on
myofibroblasts based on stem cell pathology. Exploring characteristics of stem cells would contribute to elucidation of the pathogenesis of “regeneration, repair and fibrosis”,
“epithelial-mesenchymal transition (EMT)”, and “cancer stem cells”, which are described in “General Pathology”, finally thereby leading to new development and application of “stem cell
pathology”.
The authors declare no conflict of interest.
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Professionelle Gesundheitskompetenz. Berufs- und geschlechtsspezifische Aspekte | ab8bbfcf-2160-4321-a731-84ff4d1aad7f | 11868220 | Health Literacy[mh] | Mit dem Wandel zur Informations- und Wissensgesellschaft wächst die Herausforderung, sich in einer immer größeren Menge von Gesundheitsinformationen zurechtzufinden, in der zuverlässige Informationen und qualitativ fragwürdige Informationen nebeneinanderstehen . Die Nutzung von Gesundheitsinformationen verlangt hohe Gesundheitskompetenz (GK) – verstanden als die Fähigkeit, die richtige Information, egal in welcher Form, zu finden, zu verstehen, ihre Vertrauenswürdigkeit einzuschätzen und sie für die Entscheidungsfindung oder das eigene Gesundheitsverhalten zu nutzen . GK wird in allen Lebensbereichen benötigt – im Bildungs‑, Ernährungs‑, Konsum- und Freizeitbereich und ganz besonders, wenn es um konkrete Gesundheits- und Krankheitsfragen geht. Hier sind insbesondere die Gesundheitsprofessionen (GP) gefordert. Doch auch für sie geht der Informations- und Wissenszuwachs mit neuen Anforderungen einher. Zugleich wird die Förderung von GK für sie zu einer immer wichtigeren Aufgabe, da der Anstieg an Gesundheitsinformationen weite Teile der Bevölkerung vor Schwierigkeiten stellt . Während über die GK der Bevölkerung mittlerweile valide Daten vorliegen, existieren über die GK der GP bislang so gut wie keine empirischen Erkenntnisse. Das gilt speziell dann, wenn das Interesse nicht auf ihre persönliche, sondern die professionelle GK zielt, also auf die Aufgaben abgehoben wird, die sie als GP bei der professionellen Förderung der GK ihrer Patient:innen bewältigen müssen. Um die professionelle GK zu untersuchen, wurden von einem Dreiländerkonsortium, bestehend aus Forscher:innen aus Deutschland (Hertie School, Universität Bielefeld), Österreich (Gesundheit Österreich GmbH) und der Schweiz (Careum), ein neues Konzept und Messinstrument entwickelt und erstmals eingesetzt . In dem vorliegenden Artikel werden ausgewählte, damit für Deutschland gewonnene Daten dargestellt und diskutiert. Ziel ist es, zu analysieren: welche Aufgaben die befragten GP (Pflegefachpersonen und Ärzt:innen) bei der Förderung von GK als besonders schwierig einschätzen, welche berufs- und geschlechtsspezifischen Unterschiede sich dabei identifizieren lassen und inwieweit ausgewählte organisatorische und qualifikatorische Rahmenbedingungen mit der Aufgabenrealisierung in Beziehung stehen. Zunächst erfolgt eine konzeptionelle Verortung der Studie. Konzeptionelle Rahmenüberlegungen Für die Bevölkerung stellen die GP – trotz der Zunahme der Bedeutung des Internets und digitaler Medien – nach wie vor wichtigste Informationsinstanz bei Gesundheits- und Krankheitsfragen dar . Auch bei der Informationssuche, bei unverstandenen, schwer oder nicht einschätzbaren Gesundheitsinformationen sind sie eine wichtige Anlaufstelle, von der Verständnis, Klarstellung, ausreichend Zeit für Rückfragen und kompetente Unterstützung bei der Verarbeitung von Informationen erwartet wird . Denn oft rufen gefundene Informationen eher Verwirrung als Erkenntnisgewinn hervor. Mit der Digitalisierung hat sich das verstärkt: Durch sie hat sich zwar der Zugang zu Informationen vereinfacht, aber zugleich ist die Menge an Informationen stark gewachsen. Auch die Qualität ist fragwürdiger geworden, weil auch Fehl- und Falschinformationen deutlich zugenommen haben. Die daraus erwachsenen Irritationen (etwa bei der Beurteilung von Gesundheitsinformationen) werden ebenfalls oft an die GP herangetragen, sodass die Förderung der GK für sie zu einer bedeutenden Aufgabe geworden ist. Doch ist unklar, ob die GP sich in der Lage sehen, diese Aufgabe auch angemessen zu erfüllen, zumal sie, je nach Setting, vielfach nicht mit den gegebenen Rahmenbedingungen in Übereinstimmung zu bringen ist. Seit vielen Jahren wird beklagt, dass die Zeit für Informationsvermittlung und Kommunikation zu knapp ist, sie nicht ausreichend refinanziert wird, geeignete räumliche Bedingungen fehlen und ebenso, dass die Kommunikation herausfordernd und aufwendig geworden ist – nicht zuletzt, weil Patient:innen immer höhere Ansprüche und Erwartungen an die GP herantragen . Verstärkt wird dies durch den Wandel der Patientenrolle, der die GP speziell im Bereich Informationsvermittlung (und Kommunikation) mit Anforderungen konfrontiert, auf die sie nicht ausreichend vorbereitet und eingestellt sind, wie die jahrelange Diskussion über Shared Decision Making (SDM) exemplarisch andeutet – ein Thema, das als Kernelement der veränderten Patientenrolle gilt, dessen Implementation aber sehr zäh verläuft . Ähnlich ist es mit Veränderungen, die die Digitalisierung mit sich bringt. Gerade sie stößt im Gesundheitssystem und bei vielen GP auf ein schleppendes Anpassungsverhalten . Angesichts des Deutschland seit Langem attestierten digitalen Entwicklungsrückstands verwundert dies nicht, beschränkt aber ebenfalls die Möglichkeiten der Unterstützung von Patient:innen im Umgang mit den mittlerweile meist digitalen Gesundheitsinformationen. Die angeführten Aspekte unterstreichen, wie wichtig es für eine gelingende Stärkung der GK der Bevölkerung ist, die professionelle GK genauer zu erforschen. Bislang wurde das Thema kaum untersucht, sodass zunächst zu klären ist, wie professionelle GK zu definieren und konzeptualisieren ist. Damit wurde im Rahmen des Dreiländerkonsortiums begonnen und folgende Definition erarbeitet: „Professionelle Gesundheitskompetenz umfasst die Motivation, das Wissen und die Fähigkeiten, professionell relevantes Wissen und Informationen in unterschiedlicher – auch digitaler – Form finden, verstehen, beurteilen und nutzen zu können, um im Berufsalltag professionell auf dem State of the Art agieren zu können und gesundheits- und krankheitsrelevantes Wissen und ebensolche Informationen so aufbereiten, vermitteln und kommunizieren zu können, dass sie von Patientinnen und Patienten verstanden, (kritisch) beurteilt und zur Entscheidungsfindung über Gesundheitsfragen genutzt werden können.“ [ , S. 17] Wichtig zu betonen ist, dass diese Definition als Teil einer ganzen „Familie“ von GK-Definitionen zu verstehen ist . Sie nimmt explizit auf die in Europa weitverbreitete Definition von Sørensen et al. Bezug, nach der der Umgang mit den 4 Schritten der Informationsverarbeitung (gesundheitsrelevante Informationen finden, verstehen, beurteilen/einschätzen und anwenden) den Kern von GK bilden. Hervorzuheben ist außerdem, dass die professionelle GK, ebenso wie die generelle GK, als relational zu verstehen ist, d. h., sie ist sowohl das Ergebnis der persönlichen Fähigkeiten der GP als auch der gegebenen situativen und strukturellen Bedingungen. Übereinstimmend mit dieser Definition lassen sich 4 Aufgabenbereiche identifizieren, die bei der professionellen Förderung von GK durch die GP zu bewältigen sind: Eine wichtige Aufgabe im Rahmen professioneller GK stellt ein systematisches, umfassendes professionelles Informations- und Wissensmanagement dar. Denn auch die GP sind mit einem enormen, rasant voranschreitenden (globalen) Informations- und Wissenszuwachs konfrontiert. Daher sind sie ihrerseits gefordert, sich ständig mit neuen (Fach‑)Informationen auseinanderzusetzen, um auf dem aktuellen professionellen Informations- und Wissensstand zu bleiben und in ihrer täglichen Arbeit nach dem jeweiligen State of the Art und der vorliegenden Evidenz handeln zu können und ebenfalls, um Patient:innen auf dem neuesten Wissensstand informieren und beraten zu können. Nicht weniger bedeutsam ist es für sie, sich mit Fragen der Informations- und Wissensvermittlung auseinanderzusetzen, um in der Lage zu sein, professionelles Wissen und Informationen so zu vermitteln und zu erklären, dass Patient:innen diese Informationen auch verstehen, einschätzen und so nutzen können, dass ihre Gesundheit und ihre GK davon profitieren. Eng damit verbunden ist die patientenzentrierte Kommunikation – eine Aufgabe, die zunehmend an Stellenwert gewonnen hat, um der veränderten Patientenrolle entsprechen zu können: nicht mehr über Patient:innen, sondern mit ihnen zu entscheiden, gemeinsam Behandlungs- und Versorgungsziele und -strategien zu beraten und diese auszuhandeln, Informationsasymmetrien abzubauen und der Kommunikation sowie der Informations- und Wissensvermittlung mehr Raum zu geben. Mit der professionellen digitalen GK ist die gesellschaftlich zunehmend bedeutende, aber im Alltag der GP keineswegs einfach leistbare Aufgabe angesprochen, Patient:innen bei den vielfältigen Herausforderungen im Umgang mit digitalen Informationsmedien und -informationen zu unterstützen und ihnen vor allem bei der Beurteilung krankheits- und gesundheitsbezogener Informationen beratend zur Seite zu stehen. Diese 4 Aufgaben stehen im Mittelpunkt des neu erarbeiteten Erhebungsinstruments (PROF-HL-Q). Für die Bevölkerung stellen die GP – trotz der Zunahme der Bedeutung des Internets und digitaler Medien – nach wie vor wichtigste Informationsinstanz bei Gesundheits- und Krankheitsfragen dar . Auch bei der Informationssuche, bei unverstandenen, schwer oder nicht einschätzbaren Gesundheitsinformationen sind sie eine wichtige Anlaufstelle, von der Verständnis, Klarstellung, ausreichend Zeit für Rückfragen und kompetente Unterstützung bei der Verarbeitung von Informationen erwartet wird . Denn oft rufen gefundene Informationen eher Verwirrung als Erkenntnisgewinn hervor. Mit der Digitalisierung hat sich das verstärkt: Durch sie hat sich zwar der Zugang zu Informationen vereinfacht, aber zugleich ist die Menge an Informationen stark gewachsen. Auch die Qualität ist fragwürdiger geworden, weil auch Fehl- und Falschinformationen deutlich zugenommen haben. Die daraus erwachsenen Irritationen (etwa bei der Beurteilung von Gesundheitsinformationen) werden ebenfalls oft an die GP herangetragen, sodass die Förderung der GK für sie zu einer bedeutenden Aufgabe geworden ist. Doch ist unklar, ob die GP sich in der Lage sehen, diese Aufgabe auch angemessen zu erfüllen, zumal sie, je nach Setting, vielfach nicht mit den gegebenen Rahmenbedingungen in Übereinstimmung zu bringen ist. Seit vielen Jahren wird beklagt, dass die Zeit für Informationsvermittlung und Kommunikation zu knapp ist, sie nicht ausreichend refinanziert wird, geeignete räumliche Bedingungen fehlen und ebenso, dass die Kommunikation herausfordernd und aufwendig geworden ist – nicht zuletzt, weil Patient:innen immer höhere Ansprüche und Erwartungen an die GP herantragen . Verstärkt wird dies durch den Wandel der Patientenrolle, der die GP speziell im Bereich Informationsvermittlung (und Kommunikation) mit Anforderungen konfrontiert, auf die sie nicht ausreichend vorbereitet und eingestellt sind, wie die jahrelange Diskussion über Shared Decision Making (SDM) exemplarisch andeutet – ein Thema, das als Kernelement der veränderten Patientenrolle gilt, dessen Implementation aber sehr zäh verläuft . Ähnlich ist es mit Veränderungen, die die Digitalisierung mit sich bringt. Gerade sie stößt im Gesundheitssystem und bei vielen GP auf ein schleppendes Anpassungsverhalten . Angesichts des Deutschland seit Langem attestierten digitalen Entwicklungsrückstands verwundert dies nicht, beschränkt aber ebenfalls die Möglichkeiten der Unterstützung von Patient:innen im Umgang mit den mittlerweile meist digitalen Gesundheitsinformationen. Die angeführten Aspekte unterstreichen, wie wichtig es für eine gelingende Stärkung der GK der Bevölkerung ist, die professionelle GK genauer zu erforschen. Bislang wurde das Thema kaum untersucht, sodass zunächst zu klären ist, wie professionelle GK zu definieren und konzeptualisieren ist. Damit wurde im Rahmen des Dreiländerkonsortiums begonnen und folgende Definition erarbeitet: „Professionelle Gesundheitskompetenz umfasst die Motivation, das Wissen und die Fähigkeiten, professionell relevantes Wissen und Informationen in unterschiedlicher – auch digitaler – Form finden, verstehen, beurteilen und nutzen zu können, um im Berufsalltag professionell auf dem State of the Art agieren zu können und gesundheits- und krankheitsrelevantes Wissen und ebensolche Informationen so aufbereiten, vermitteln und kommunizieren zu können, dass sie von Patientinnen und Patienten verstanden, (kritisch) beurteilt und zur Entscheidungsfindung über Gesundheitsfragen genutzt werden können.“ [ , S. 17] Wichtig zu betonen ist, dass diese Definition als Teil einer ganzen „Familie“ von GK-Definitionen zu verstehen ist . Sie nimmt explizit auf die in Europa weitverbreitete Definition von Sørensen et al. Bezug, nach der der Umgang mit den 4 Schritten der Informationsverarbeitung (gesundheitsrelevante Informationen finden, verstehen, beurteilen/einschätzen und anwenden) den Kern von GK bilden. Hervorzuheben ist außerdem, dass die professionelle GK, ebenso wie die generelle GK, als relational zu verstehen ist, d. h., sie ist sowohl das Ergebnis der persönlichen Fähigkeiten der GP als auch der gegebenen situativen und strukturellen Bedingungen. Übereinstimmend mit dieser Definition lassen sich 4 Aufgabenbereiche identifizieren, die bei der professionellen Förderung von GK durch die GP zu bewältigen sind: Eine wichtige Aufgabe im Rahmen professioneller GK stellt ein systematisches, umfassendes professionelles Informations- und Wissensmanagement dar. Denn auch die GP sind mit einem enormen, rasant voranschreitenden (globalen) Informations- und Wissenszuwachs konfrontiert. Daher sind sie ihrerseits gefordert, sich ständig mit neuen (Fach‑)Informationen auseinanderzusetzen, um auf dem aktuellen professionellen Informations- und Wissensstand zu bleiben und in ihrer täglichen Arbeit nach dem jeweiligen State of the Art und der vorliegenden Evidenz handeln zu können und ebenfalls, um Patient:innen auf dem neuesten Wissensstand informieren und beraten zu können. Nicht weniger bedeutsam ist es für sie, sich mit Fragen der Informations- und Wissensvermittlung auseinanderzusetzen, um in der Lage zu sein, professionelles Wissen und Informationen so zu vermitteln und zu erklären, dass Patient:innen diese Informationen auch verstehen, einschätzen und so nutzen können, dass ihre Gesundheit und ihre GK davon profitieren. Eng damit verbunden ist die patientenzentrierte Kommunikation – eine Aufgabe, die zunehmend an Stellenwert gewonnen hat, um der veränderten Patientenrolle entsprechen zu können: nicht mehr über Patient:innen, sondern mit ihnen zu entscheiden, gemeinsam Behandlungs- und Versorgungsziele und -strategien zu beraten und diese auszuhandeln, Informationsasymmetrien abzubauen und der Kommunikation sowie der Informations- und Wissensvermittlung mehr Raum zu geben. Mit der professionellen digitalen GK ist die gesellschaftlich zunehmend bedeutende, aber im Alltag der GP keineswegs einfach leistbare Aufgabe angesprochen, Patient:innen bei den vielfältigen Herausforderungen im Umgang mit digitalen Informationsmedien und -informationen zu unterstützen und ihnen vor allem bei der Beurteilung krankheits- und gesundheitsbezogener Informationen beratend zur Seite zu stehen. Diese 4 Aufgaben stehen im Mittelpunkt des neu erarbeiteten Erhebungsinstruments (PROF-HL-Q). Messung professioneller Gesundheitskompetenz Der PROF-HL‑Q misst die selbsteingeschätzten Schwierigkeiten bei der Erfüllung von insgesamt 34 Aufgaben in den zuvor dargelegten 4 Bereichen professioneller GK: Informations- und Wissensmanagement (7 Items), Informations- und Wissensvermittlung (17 Items), patientenzentrierte Kommunikation (6 Items) und professionelle digitale GK (4 Items). Es wird gefragt, „wie einfach oder schwierig“ die Aufgaben für die teilnehmenden GP jeweils sind. Zu beantworten sind die Items auf einer 5‑stufigen Antwortskala („sehr schwierig“, „eher schwierig“, „weder einfach noch schwierig“, „eher einfach“, „sehr einfach“). Für die Auswertung wurden die Antworten für jeden Aufgabenbereich summiert und in einen gemeinsamen Score von 0 bis 100 Punkten skaliert, wobei 0 den geringsten und 100 den höchsten Wert darstellt. Ein höherer Score lässt somit auf geringere Schwierigkeiten bei der Aufgabenrealisierung schließen. Ein Score wurde nur dann berechnet, wenn alle Fragen der jeweiligen Itembatterie vollständig beantwortet wurden. Das Erhebungsinstrument wurde im Rahmen der HLS-PROF-Studie auf seine psychometrischen Eigenschaften geprüft. Die Ergebnisse deuten auf zufriedenstellende Eigenschaften des Instruments . (Mit einem Cronbachs Alpha von 0,82–0,90 für Pflegefachpersonen und 0,80–0,91 für Ärzt:innen weisen die Skalen zudem eine zufriedenstellende interne Konsistenz auf.) In die Analyse gingen neben der professionellen GK 5 ausgewählte organisatorische Rahmenbedingungen ein. Dazu wurde gefragt, inwieweit ausreichend Zeit und geeignete Räumlichkeiten für Patientengespräche zur Verfügung stehen, die Möglichkeit besteht, ungestört Gespräche zu führen, zusätzliche Gespräche zur Klärung weiterführender Fragen angeboten werden können und auf Dolmetschdienste oder digitale Übersetzungsmöglichkeiten zurückzugriffen werden kann. Außerdem wurde die Beurteilung der Ausbildung einbezogen: Dazu wurde gefragt, wie gut die Befragten durch ihre Ausbildung auf die bei der Förderung von GK anfallenden Aufgaben vorbereitet worden sind. Zusätzlich wurde danach gefragt, wie vertraut sie mit dem GK-Konzept sind. Eine Übersicht über die Verteilung der Antworten zu den Rahmenbedingungen, der Einschätzung der Ausbildung und der Vertrautheit mit dem GK-Konzept ist im Onlinematerial zu finden (Tab. Z1). Studiendesign und Studienpopulation Bei der Untersuchung (HLS-PROF-GER) handelt es sich um eine quantitative Querschnittserhebung, in der Allgemeinärzt:innen bzw. hausärztlich tätige Internist:innen und Pflegefachpersonen der Gesundheits- und Krankenpflege mit regelmäßigem Patientenkontakt via computerassistierter Web-Interviews (CAWI) zu ihrer professionellen GK befragt wurden. Die Befragung wurde von Mai bis Juni 2022 durchgeführt. Für die Rekrutierung der Ärzt:innen und Pflegefachpersonen wurde jeweils auf ein Online-Panel zurückgegriffen. Über das „Sermo-Panel“, das auf den Austausch zwischen Mediziner:innen zielt, wurden 4468 Ärzt:innen per E‑Mail zur Befragung und zu einem initialen Screening (3 Fragen zur Sicherstellung des passenden Berufsabschlusses und des regelmäßigen Patientenkontakts) eingeladen. Von den 352 Personen, die sich am Screening beteiligten, erfüllten 326 die Einschlusskriterien und wurden zur Hauptbefragung weitergeleitet. Für die Ärzt:innen konnten 297 verwertbare Datensätze generiert werden. Die Rekrutierung der Pflegefachpersonen erfolgte in ähnlicher Weise über das „Payback Access-Panel“ und die Versendung des initialen Screening-Fragebogens. Von den 2644 eingeladenen Personen, wurden 1131 aus nicht bekannten Gründen nicht erreicht, 841 Personen erfüllten nicht die Einschlusskriterien. Letztendlich gingen 624 verwertbare Datensätze für die Zielgruppe der Pflegenden in die Analyse ein. Datenauswertung Mit dem vorliegenden Artikel erfolgte eine Spezialauswertung des HLS-PROF-GER . Dazu wurden in einem ersten Schritt die Scores professioneller GK berechnet und nach Berufsgruppen und dem Geschlecht stratifiziert. Unterschiede zwischen den Geschlechtern wurden mittels bivariater Analyse (t-Test, Mann-Whitney-U-Test) geprüft. In einem zweiten Schritt wurden die 8 von den Befragten als besonders schwierig eingeschätzten Aufgaben zur professionellen GK identifiziert. Dazu wurden die Antwortkategorien „eher schwierig“ und „sehr schwierig“ zusammengefasst und die Items nach Schwierigkeitsgrad geordnet. Potenzielle Unterschiede nach Geschlecht wurden mittels Z‑Tests untersucht. Für die Überprüfung des Zusammenhangs zwischen den 8 schwierigsten Aufgaben und den Rahmenbedingungen, der Bewertung der Ausbildung sowie der Vertrautheit mit dem GK-Konzept wurden Korrelationskoeffizienten nach Spearman berechnet. Alle Analysen wurden mit der Statistiksoftware SPSS Version 28 durchgeführt. Für die Berechnungen wurde ein für das Geschlecht gewichteter Datensatz genutzt, um den vorhandenen Abweichungen zwischen der realisierten Stichprobe und der Grundgesamtheit der Ärzt:innen und Pflegefachpersonen besser zu entsprechen . Für die Berechnung der Korrelationskoeffizienten wurde der ungewichtete Datensatz genutzt. Der PROF-HL‑Q misst die selbsteingeschätzten Schwierigkeiten bei der Erfüllung von insgesamt 34 Aufgaben in den zuvor dargelegten 4 Bereichen professioneller GK: Informations- und Wissensmanagement (7 Items), Informations- und Wissensvermittlung (17 Items), patientenzentrierte Kommunikation (6 Items) und professionelle digitale GK (4 Items). Es wird gefragt, „wie einfach oder schwierig“ die Aufgaben für die teilnehmenden GP jeweils sind. Zu beantworten sind die Items auf einer 5‑stufigen Antwortskala („sehr schwierig“, „eher schwierig“, „weder einfach noch schwierig“, „eher einfach“, „sehr einfach“). Für die Auswertung wurden die Antworten für jeden Aufgabenbereich summiert und in einen gemeinsamen Score von 0 bis 100 Punkten skaliert, wobei 0 den geringsten und 100 den höchsten Wert darstellt. Ein höherer Score lässt somit auf geringere Schwierigkeiten bei der Aufgabenrealisierung schließen. Ein Score wurde nur dann berechnet, wenn alle Fragen der jeweiligen Itembatterie vollständig beantwortet wurden. Das Erhebungsinstrument wurde im Rahmen der HLS-PROF-Studie auf seine psychometrischen Eigenschaften geprüft. Die Ergebnisse deuten auf zufriedenstellende Eigenschaften des Instruments . (Mit einem Cronbachs Alpha von 0,82–0,90 für Pflegefachpersonen und 0,80–0,91 für Ärzt:innen weisen die Skalen zudem eine zufriedenstellende interne Konsistenz auf.) In die Analyse gingen neben der professionellen GK 5 ausgewählte organisatorische Rahmenbedingungen ein. Dazu wurde gefragt, inwieweit ausreichend Zeit und geeignete Räumlichkeiten für Patientengespräche zur Verfügung stehen, die Möglichkeit besteht, ungestört Gespräche zu führen, zusätzliche Gespräche zur Klärung weiterführender Fragen angeboten werden können und auf Dolmetschdienste oder digitale Übersetzungsmöglichkeiten zurückzugriffen werden kann. Außerdem wurde die Beurteilung der Ausbildung einbezogen: Dazu wurde gefragt, wie gut die Befragten durch ihre Ausbildung auf die bei der Förderung von GK anfallenden Aufgaben vorbereitet worden sind. Zusätzlich wurde danach gefragt, wie vertraut sie mit dem GK-Konzept sind. Eine Übersicht über die Verteilung der Antworten zu den Rahmenbedingungen, der Einschätzung der Ausbildung und der Vertrautheit mit dem GK-Konzept ist im Onlinematerial zu finden (Tab. Z1). Bei der Untersuchung (HLS-PROF-GER) handelt es sich um eine quantitative Querschnittserhebung, in der Allgemeinärzt:innen bzw. hausärztlich tätige Internist:innen und Pflegefachpersonen der Gesundheits- und Krankenpflege mit regelmäßigem Patientenkontakt via computerassistierter Web-Interviews (CAWI) zu ihrer professionellen GK befragt wurden. Die Befragung wurde von Mai bis Juni 2022 durchgeführt. Für die Rekrutierung der Ärzt:innen und Pflegefachpersonen wurde jeweils auf ein Online-Panel zurückgegriffen. Über das „Sermo-Panel“, das auf den Austausch zwischen Mediziner:innen zielt, wurden 4468 Ärzt:innen per E‑Mail zur Befragung und zu einem initialen Screening (3 Fragen zur Sicherstellung des passenden Berufsabschlusses und des regelmäßigen Patientenkontakts) eingeladen. Von den 352 Personen, die sich am Screening beteiligten, erfüllten 326 die Einschlusskriterien und wurden zur Hauptbefragung weitergeleitet. Für die Ärzt:innen konnten 297 verwertbare Datensätze generiert werden. Die Rekrutierung der Pflegefachpersonen erfolgte in ähnlicher Weise über das „Payback Access-Panel“ und die Versendung des initialen Screening-Fragebogens. Von den 2644 eingeladenen Personen, wurden 1131 aus nicht bekannten Gründen nicht erreicht, 841 Personen erfüllten nicht die Einschlusskriterien. Letztendlich gingen 624 verwertbare Datensätze für die Zielgruppe der Pflegenden in die Analyse ein. Mit dem vorliegenden Artikel erfolgte eine Spezialauswertung des HLS-PROF-GER . Dazu wurden in einem ersten Schritt die Scores professioneller GK berechnet und nach Berufsgruppen und dem Geschlecht stratifiziert. Unterschiede zwischen den Geschlechtern wurden mittels bivariater Analyse (t-Test, Mann-Whitney-U-Test) geprüft. In einem zweiten Schritt wurden die 8 von den Befragten als besonders schwierig eingeschätzten Aufgaben zur professionellen GK identifiziert. Dazu wurden die Antwortkategorien „eher schwierig“ und „sehr schwierig“ zusammengefasst und die Items nach Schwierigkeitsgrad geordnet. Potenzielle Unterschiede nach Geschlecht wurden mittels Z‑Tests untersucht. Für die Überprüfung des Zusammenhangs zwischen den 8 schwierigsten Aufgaben und den Rahmenbedingungen, der Bewertung der Ausbildung sowie der Vertrautheit mit dem GK-Konzept wurden Korrelationskoeffizienten nach Spearman berechnet. Alle Analysen wurden mit der Statistiksoftware SPSS Version 28 durchgeführt. Für die Berechnungen wurde ein für das Geschlecht gewichteter Datensatz genutzt, um den vorhandenen Abweichungen zwischen der realisierten Stichprobe und der Grundgesamtheit der Ärzt:innen und Pflegefachpersonen besser zu entsprechen . Für die Berechnung der Korrelationskoeffizienten wurde der ungewichtete Datensatz genutzt. In der für das Geschlecht gewichteten Stichprobe sind insgesamt 930 Fälle (304 für Ärzt:innen und 626 für Pflegefachpersonen) enthalten. Dabei überwiegt der Anteil der Frauen deutlich gegenüber dem der Männer (Tab. ). Im Mittel sind die Befragten 46 Jahre alt, wobei die befragten Ärzt:innen rund 11,5 Jahre älter sind als die befragten Pflegefachpersonen. Mit durchschnittlich 18,2 bzw. 19,6 Jahren weisen beide Berufsgruppen eine ähnlich lange Verweildauer im Beruf auf. Der Großteil der Befragten ist in Deutschland geboren. Gut zwei Drittel der Pflegenden sind in einer stationären Einrichtung tätig, rund 90 % der Ärzt:innen praktizieren in einem ambulanten Setting. Scores professioneller Gesundheitskompetenz Beide GP erreichen im Mittel zwischen 54,0 bis 73,8 Punkte in den 4 Aufgabenbereichen professioneller GK. Die niedrigsten Scores zeigen sich bei der professionellen digitalen GK, gefolgt von der Informations- und Wissensvermittlung mit durchschnittlich 61,9 Punkten. Etwas mehr Punkte werden im Bereich des Informations- und Wissensmanagements erzielt (65,0). Deutlich höher fällt die Punktzahl bei der patientenzentrierten Kommunikation aus (73,8), sie wird mit Abstand als am leichtesten eingeschätzt (Abb. ). Betrachtet man die beiden Berufsgruppen getrennt voneinander (Abb. ), zeigt sich ein ähnliches Muster: Sowohl die Pflegefachpersonen als auch die Ärzt:innen erreichen bei der professionellen digitalen GK die geringsten (54,5 bzw. 53,1), bei der patientenzentrierten Kommunikation die höchsten Scores (74,3 bzw. 72,7). Die Informations- und Wissensvermittlung (62,5 bzw. 60,5) und Informations- und Wissensmanagement (64,7 bzw. 65,8) nehmen die Plätze 2 und 3 in der Rangfolge der schwierigsten Aufgabenbereiche ein. Unter geschlechtsspezifischen Gesichtspunkten zeigen sich nur geringfügige Unterschiede. Einzig im Bereich der patientenzentrierten Kommunikation erreichen weibliche Pflegefachpersonen statistisch signifikant höhere Scores als männliche Befragte (MW: 75,2 vs. 70,0 Punkte, p < 0,01). Eine Betrachtung der 8 am häufigsten als „eher schwierig“ oder „sehr schwierig“ beurteilten Aufgaben (Abb. ) zeigt für die Gesamtstichprobe, dass die Befragten am schwierigsten einschätzen, „mit fehl- oder falschinformierten Patient:innen umzugehen“ (PGK21: 39,5 %), gefolgt davon, Patient:innen bei der Beurteilung der Vertrauenswürdigkeit digitaler Informationen zu unterstützen (PGK33: 35,0 %) und „einzuschätzen, inwieweit kulturelle Unterschiede das gegenseitige Verständnis erschweren“ (PGK11: 33,8 %). An 4. und 5. Stelle stehen die Aufgaben, „Patient:innen dabei zu unterstützen, die für sie relevanten digitalen Gesundheitsinformationen zu finden“ (PGK31: 32,1 %) sowie diese „zur Verbesserung ihres Gesundheitsproblems oder ihrer Gesundheit zu nutzen“ (PGK34: 26 %). „Einzuschätzen, inwieweit Patient:innen in der Lage sind, mit Informationen über Krankheits- und Gesundheitsthemen umzugehen“ (PGK10), wird von 24,7 % der Befragten als eher/sehr schwierig eingeschätzt und steht damit an 6. Stelle. Das Item, „Patient:innen dabei zu unterstützen, die gefundenen digitalen Informationen zu verstehen“ (PGK32: 24,5 %), steht an vorletzter Stelle in der Liste der als am schwersten eingeschätzten Aufgaben, noch vor der Aufgabe „bei der Informationsvermittlung mit unsicherer Evidenz umgehen“ (PGK18: 23,4 %). Bei der Untersuchung der geschlechtsspezifischen Unterschiede bei den 8 als am schwierigsten beurteilten Aufgaben (Abb. ) wird deutlich, dass die Differenzen innerhalb der Gruppe der Pflegenden sehr gering sind. Bei den Ärzt:innen zeigen sich größere Unterschiede. Das Item PGK21, „mit fehl- oder falschinformierten Patient:innen umzugehen“, wird von den befragten Ärztinnen statistisch signifikant als schwieriger eingeschätzt als von den Ärzten (Kategorie „eher schwierig“: 42,9 % vs. 31,8 %, p < 0,049). Dies gilt auch für Item PGK31, „Patient:innen dabei zu unterstützen, die für sie relevanten digitalen Gesundheitsinformationen zu finden“ (Kategorie „eher schwierig“: 37,2 % vs. 22,2 %, p = 0,005). Auch die übrigen Aufgaben werden, wenngleich nicht signifikant, von weiblichen Befragten für schwieriger gehalten. Einzig „einzuschätzen, inwieweit kulturelle Unterschiede das gegenseitige Verständnis erschweren“ (PGK11), wird von Ärzten in der Tendenz als schwieriger beurteilt (signifikanter Unterschied in der Kategorie „eher einfach“: Ärztinnen: 37,7 % vs. Ärzte: 24,8 %, p = 0,017). Zusammenhangsanalyse Die Korrelationskoeffizienten für den Zusammenhang zwischen der Vertrautheit mit dem GK-Konzept, der Beurteilung der Ausbildung und den Rahmenbedingungen mit den subjektiven Schwierigkeiten bei der Bewältigung der 8 schwierigsten Aufgaben im Rahmen der professionellen GK sind in Tab. dargestellt. Insgesamt deutet die Analyse auf eine schwache bis moderate Korrelationen hin (min. ρ = 0,11, max. ρ = 0,32). Befragte, die angeben, vertraut oder sehr vertraut mit dem Konzept zu sein, schätzen fast alle Aufgaben als leichter ein. Dies gilt auch für die Einschätzung der Ausbildung: Eine als besser beurteilte Ausbildung geht in beiden Gruppen durchgängig mit geringeren Schwierigkeiten bei der Aufgabenbewältigung einher. Pflegende, die über bessere zeitliche (R1) und räumliche (R2) Ressourcen verfügen und häufiger die Möglichkeit haben, zusätzliche Gespräche anzubieten (R4), schätzen die Aufgabenbewältigung fast immer als einfacher ein. Dies zeigt sich partiell auch bei den Ärzt:innen. Bei ihnen steht zudem die Möglichkeit, auf Dolmetschdienste oder digitale Übersetzungsoptionen zurückzugreifen (R5), durchgängig mit einer einfacheren Aufgabenbewältigung im Zusammenhang. Beide GP erreichen im Mittel zwischen 54,0 bis 73,8 Punkte in den 4 Aufgabenbereichen professioneller GK. Die niedrigsten Scores zeigen sich bei der professionellen digitalen GK, gefolgt von der Informations- und Wissensvermittlung mit durchschnittlich 61,9 Punkten. Etwas mehr Punkte werden im Bereich des Informations- und Wissensmanagements erzielt (65,0). Deutlich höher fällt die Punktzahl bei der patientenzentrierten Kommunikation aus (73,8), sie wird mit Abstand als am leichtesten eingeschätzt (Abb. ). Betrachtet man die beiden Berufsgruppen getrennt voneinander (Abb. ), zeigt sich ein ähnliches Muster: Sowohl die Pflegefachpersonen als auch die Ärzt:innen erreichen bei der professionellen digitalen GK die geringsten (54,5 bzw. 53,1), bei der patientenzentrierten Kommunikation die höchsten Scores (74,3 bzw. 72,7). Die Informations- und Wissensvermittlung (62,5 bzw. 60,5) und Informations- und Wissensmanagement (64,7 bzw. 65,8) nehmen die Plätze 2 und 3 in der Rangfolge der schwierigsten Aufgabenbereiche ein. Unter geschlechtsspezifischen Gesichtspunkten zeigen sich nur geringfügige Unterschiede. Einzig im Bereich der patientenzentrierten Kommunikation erreichen weibliche Pflegefachpersonen statistisch signifikant höhere Scores als männliche Befragte (MW: 75,2 vs. 70,0 Punkte, p < 0,01). Eine Betrachtung der 8 am häufigsten als „eher schwierig“ oder „sehr schwierig“ beurteilten Aufgaben (Abb. ) zeigt für die Gesamtstichprobe, dass die Befragten am schwierigsten einschätzen, „mit fehl- oder falschinformierten Patient:innen umzugehen“ (PGK21: 39,5 %), gefolgt davon, Patient:innen bei der Beurteilung der Vertrauenswürdigkeit digitaler Informationen zu unterstützen (PGK33: 35,0 %) und „einzuschätzen, inwieweit kulturelle Unterschiede das gegenseitige Verständnis erschweren“ (PGK11: 33,8 %). An 4. und 5. Stelle stehen die Aufgaben, „Patient:innen dabei zu unterstützen, die für sie relevanten digitalen Gesundheitsinformationen zu finden“ (PGK31: 32,1 %) sowie diese „zur Verbesserung ihres Gesundheitsproblems oder ihrer Gesundheit zu nutzen“ (PGK34: 26 %). „Einzuschätzen, inwieweit Patient:innen in der Lage sind, mit Informationen über Krankheits- und Gesundheitsthemen umzugehen“ (PGK10), wird von 24,7 % der Befragten als eher/sehr schwierig eingeschätzt und steht damit an 6. Stelle. Das Item, „Patient:innen dabei zu unterstützen, die gefundenen digitalen Informationen zu verstehen“ (PGK32: 24,5 %), steht an vorletzter Stelle in der Liste der als am schwersten eingeschätzten Aufgaben, noch vor der Aufgabe „bei der Informationsvermittlung mit unsicherer Evidenz umgehen“ (PGK18: 23,4 %). Bei der Untersuchung der geschlechtsspezifischen Unterschiede bei den 8 als am schwierigsten beurteilten Aufgaben (Abb. ) wird deutlich, dass die Differenzen innerhalb der Gruppe der Pflegenden sehr gering sind. Bei den Ärzt:innen zeigen sich größere Unterschiede. Das Item PGK21, „mit fehl- oder falschinformierten Patient:innen umzugehen“, wird von den befragten Ärztinnen statistisch signifikant als schwieriger eingeschätzt als von den Ärzten (Kategorie „eher schwierig“: 42,9 % vs. 31,8 %, p < 0,049). Dies gilt auch für Item PGK31, „Patient:innen dabei zu unterstützen, die für sie relevanten digitalen Gesundheitsinformationen zu finden“ (Kategorie „eher schwierig“: 37,2 % vs. 22,2 %, p = 0,005). Auch die übrigen Aufgaben werden, wenngleich nicht signifikant, von weiblichen Befragten für schwieriger gehalten. Einzig „einzuschätzen, inwieweit kulturelle Unterschiede das gegenseitige Verständnis erschweren“ (PGK11), wird von Ärzten in der Tendenz als schwieriger beurteilt (signifikanter Unterschied in der Kategorie „eher einfach“: Ärztinnen: 37,7 % vs. Ärzte: 24,8 %, p = 0,017). Die Korrelationskoeffizienten für den Zusammenhang zwischen der Vertrautheit mit dem GK-Konzept, der Beurteilung der Ausbildung und den Rahmenbedingungen mit den subjektiven Schwierigkeiten bei der Bewältigung der 8 schwierigsten Aufgaben im Rahmen der professionellen GK sind in Tab. dargestellt. Insgesamt deutet die Analyse auf eine schwache bis moderate Korrelationen hin (min. ρ = 0,11, max. ρ = 0,32). Befragte, die angeben, vertraut oder sehr vertraut mit dem Konzept zu sein, schätzen fast alle Aufgaben als leichter ein. Dies gilt auch für die Einschätzung der Ausbildung: Eine als besser beurteilte Ausbildung geht in beiden Gruppen durchgängig mit geringeren Schwierigkeiten bei der Aufgabenbewältigung einher. Pflegende, die über bessere zeitliche (R1) und räumliche (R2) Ressourcen verfügen und häufiger die Möglichkeit haben, zusätzliche Gespräche anzubieten (R4), schätzen die Aufgabenbewältigung fast immer als einfacher ein. Dies zeigt sich partiell auch bei den Ärzt:innen. Bei ihnen steht zudem die Möglichkeit, auf Dolmetschdienste oder digitale Übersetzungsoptionen zurückzugreifen (R5), durchgängig mit einer einfacheren Aufgabenbewältigung im Zusammenhang. Die Analyse zielte darauf, die professionelle GK zu untersuchen und zu fragen, wie die GP – konkret Pflegefachpersonen und Ärzt:innen – die Aufgaben einschätzen, die sich bei der Förderung von GK von Patient:innen in 4 zuvor konzeptualisierten Bereichen stellen. Dabei galt das Interesse den insgesamt am schwierigsten beurteilten Aufgaben und der Frage, welche berufs- und geschlechtsspezifischen Unterschiede sich dabei zeigen und ebenso, welche Bedeutung qualifikatorischen und organisatorischen Rahmenbedingungen bei der Aufgabenbewältigung zukommt. Gezeigt wurde, dass die Berufsgruppen in den 4 erfragten Aufgabenbereichen mit 54 bis 74 von 100 möglichen Punkten verhältnismäßig hohe Scores erreichen und ihre professionelle GK recht positiv einzuschätzen ist – positiver als nach der vorliegenden Literatur zu erwarten war (z. B. ). Bei der Einordnung ist zu beachten, dass es sich bei der vorliegenden Untersuchung um eine Online-Befragung handelte, in der vermutlich einzelne Teilgruppen, etwa weniger Interessierte oder weniger onlineaffine Personen, nicht teilgenommen haben, was die Ergebnisse positiv beeinflusst haben dürfte. Gleichzeitig deuten die Scores auf Optimierungspotenzial hin, da ein nicht unerheblicher Anteil an Punkten nicht erreicht wurde. Dies spiegelt sich auch in den untersuchten Schwierigkeiten wider: Auffällig ist, dass die Aufgaben, die beiden Berufsgruppen subjektiv die größten Schwierigkeiten bereiten, samt und sonders aus 2 Bereichen stammen, der „Informations- und Wissensvermittlung“ und der „professionellen digitalen GK“. Die in den beiden anderen Bereichen „Informations- und Wissensmanagement“ und „patientenzentrierte Kommunikation“ enthaltenen Aufgaben werden dementsprechend als leichter eingeschätzt. Dies könnte darauf zurückzuführen sein, dass die sich hier stellenden Aufgaben überwiegend als Routineaufgaben angesehen werden, die ohnehin im Alltag anfallen und bewältigt werden müssen. Zudem haben beide Aufgabenbereiche – insbesondere die Kommunikation – in den letzten Jahren verstärkte Aufmerksamkeit erfahren, um die seit vielen Jahren geäußerte Kritik aufzugreifen und Verbesserungen einzuleiten . Dagegen stellen die „Informations- und Wissensvermittlung“ und „professionelle digitale GK“ – verstanden als Förderung von Patient:innen im Umgang mit digitalen Informationen – weniger beachtete bzw. neuere Aufgabenbereiche dar, die noch stärker in die Ausbildungen der GP einbezogen werden sollten. Hinzu kommt, dass sie nicht unmittelbar mit der Bewältigung von Krankheit und Gesundheits- und Funktionsbeeinträchtigungen in Verbindung stehen und daher vermutlich schwerer in Einklang mit dem Selbst- und Aufgabenverständnis und der professionellen Identität zu bringen sind. Gleichwohl stellen sie drängende Aufgaben im Alltag der GP dar, wie u.a. qualitative Studien zeigen . Betrachtet man die 8 schwierigsten Aufgaben unter inhaltlichen Gesichtspunkten, wird deutlich, dass die zum Bereich „Informations- und Wissensvermittlung“ gehörenden Items sich entweder auf das Thema Herausforderungen bei der Informationsvermittlung beziehen oder sich mit der Ermittlung von Informationsvoraussetzungen befassen. Beides sind wichtige und anspruchsvolle Aufgaben im Rahmen systematischer Informationsvermittlungsprozesse, die in der Aus‑, Fort- und Weiterbildung der GP bislang eine bestenfalls periphere Rolle spielen und im Praxisalltag daher eher intuitiv und auf der Basis von persönlichem Erfahrungswissen wahrgenommen werden müssen. Dass hier Veränderungen erforderlich sind, wird durch die von beiden befragten Berufsgruppen als am schwierigsten beurteilte Aufgabe des Fragebogens unterstrichen: Fast 40 % betrachten es als (sehr) schwierig, mit fehl- oder falschinformierten Patient:innen umzugehen – eine Herausforderung, die in den letzten Jahren mit der um sich greifenden „Infodemie“ an Bedeutung gewonnen hat. Hier ist eine komplexe Aufgabe zu bewältigen, die nicht allein mit schlichten, faktenorientierten Richtigstellungen, etwa bei Falschinformationen, gelöst werden kann, sondern meist auch eine Intervention in grundlegende Annahmen und Vorstellungen im Zusammenhang mit Krankheiten verlangt, ebenso die Einleitung von Verlern- und Umlernprozessen bei Patient:innen. Das wiederum setzt die Fähigkeiten dazu voraus, Lernprozesse stimulieren und begleiten zu können, und erfordert spezielle edukative Kompetenzen , doch auch mehr Zeit als in alltäglichen Konsultationen hierzulande vorgesehen ist . Die Dringlichkeit, hier Veränderungen herbeizuführen, wird dadurch unterstrichen, dass diese Aufgabe in allen 3 Ländern, in denen die professionelle GK bislang erhoben wurde, auf ähnlich große Schwierigkeiten stößt . Die drittschwierigste Aufgabe (PGK11: „einzuschätzen, inwieweit kulturelle Unterschiede das gegenseitige Verständnis erschweren“) weist auf eine anders gelagerte Herausforderung hin. Mittlerweile verfügt gut jede vierte Person (28,7 %) in Deutschland über einen Migrationshintergrund, von denen rund 16 Mio. auf eine eigene Migrationserfahrung schauen . Menschen mit Migrationshintergrund stellen eine sehr heterogene Gruppe dar. Sie unterscheiden sich u. a. hinsichtlich ihrer Gesundheits- und Krankheitserfahrungen und -vorstellungen. Das Gesundheitssystem stärker an diese Entwicklung anzupassen und es diversitäts- und kultursensibler zu gestalten, gehört zu den seit Längerem bestehenden Forderungen , deren Relevanz durch die Ergebnisse der vorliegenden Analyse erneut gestützt wird. Auch das verlangt Veränderungen auf der Kompetenzebene, ebenso allerdings – wie auch die Korrelationsanalyse andeutet – Veränderungen auf struktureller Ebene (etwa den Ausbau von Dolmetsch- und Sprachmittlungsdiensten oder der technischen Ausstattung, die eine Vereinfachung der sprachlichen Verständigung ermöglicht ). Die sprachliche Verständigung zu erleichtern, dürfte künftig durch die fortschreitende Digitalisierung und die Fortschritte im Bereich künstlicher Intelligenz (KI) strukturell möglich werden – auch im Gesundheitswesen. Das setzt jedoch voraus, dass die Anpassung an die digitale Transformation und auch an die damit einhergehenden neuen Aufgaben dort rascher voranschreitet als bislang. Denn gerade dem deutschen Gesundheitssystem wird hier nach wie vor eine zu geringe Flexibilität und ein großer Entwicklungsrückstand attestiert . Wie wichtig es ist, diesen Rückstand aufzuholen, deutet die vorliegende Untersuchung erneut an. Denn alle gestellten Fragen zum Bereich „professionelle digitale GK“ – dem am schwierigsten eingeschätzten Aufgabenbereich – sind in der Rangliste der schwierigsten Aufgaben zu finden. Besonders schwer ist es für die befragten Berufsgruppen, Patient:innen zu unterstützen, die Vertrauenswürdigkeit digitaler Informationen einzuschätzen oder für sie relevante Informationen überhaupt ausfindig zu machen. Dieses Ergebnis erstaunt angesichts der verwirrenden Informationsvielfalt nicht, könnte sich jedoch ebenfalls durch weitere Fortschritte im Bereich KI verändern – positiv wie auch negativ . Ebenso gibt es Bemühungen, zu gemeinsamen Qualitätsstandards im Bereich digitaler Information zu gelangen . Konkret absehbar sind diese Entwicklungen bislang nicht. So oder so bleibt daher als Aufgabe, die professionelle digitale GK zu stärken – nicht zuletzt, um die GK von Patient:innen besser fördern und die Krankheitsbewältigung stärker unterstützen und erleichtern zu können. Berufs- und geschlechtsspezifische Unterschiede im Antwortverhalten sind insgesamt gering. Auch in Studien zur GK in der Allgemeinbevölkerung lassen sich, wenn überhaupt, nur geringe geschlechtsspezifische Unterschiede identifizieren . Die durchgeführte Analyse deutet dies ebenfalls für die professionelle GK an. Ein genauerer Blick auf die Einzelitems zeigt dennoch einige Differenzen, insbesondere in der Berufsgruppe der Ärzt:innen. Bei ihnen zeichnen sich die meisten Abweichungen im Antwortverhalten ab. Am größten und statistisch signifikant sind sie bei dem Item PGK31: „Patienten dabei zu unterstützen, die für sie relevanten digitalen Informationen zu finden“. Doch auch PGK21: „mit fehl- oder falschinformierten Patient:innen umzugehen“, fällt Ärztinnen deutlich schwerer als ihren männlichen Kollegen. Dies deutet an, dass die Geschlechterdifferenzen bei den Ärzt:innen durchaus beachtet werden sollten. Zugleich fällt auf, dass Ärztinnen tendenziell häufiger als ihre männlichen Kollegen Schwierigkeiten bei der Unterstützung von Patient:innen im Umgang mit digitalen Informationen angeben. Dies passt zu anderen Studienergebnissen, nach denen weibliche Befragte ihre digitalen Fähigkeiten generell als geringer einschätzen als männliche Befragte . Die Autor:innen führen dies auf eine höhere Technikaffinität bei Männern und bestehende Stereotype und Rollenmuster zurück, die sich möglicherweise auch im Antwortverhalten bei den Items zur professionellen digitalen GK widerspiegeln. Diese abzubauen und ein gleichwertiges digitales Empowerment für beide Geschlechter zu ermöglichen, scheint auch der professionellen digitalen GK zuträglich zu sein. Insgesamt zeigt die Analyse, dass es aufschlussreich ist, die subjektiven Schwierigkeiten beider Berufsgruppen bei der Förderung der GK ihrer Patient:innen genauer in den Blick zu nehmen und ihnen bei der Interventionsentwicklung Aufmerksamkeit zu schenken. Dies gilt auch für die festgestellten Zusammenhänge. Wenngleich sie eher schwach ausgeprägt sind und der weiteren Überprüfung bedürfen, deuten sie darauf, dass die professionelle GK mit den gegebenen Ausbildungs- und Rahmenbedingungen wie auch dem Ausmaß der Konzeptkenntnis in Beziehung steht – ein Ergebnis, das sich ähnlich in den anderen deutschsprachigen Ländern zeigt . Dies mag erwartbar erscheinen, unterstreicht aber die Wichtigkeit, die Kenntnis des GK-Konzepts bei den GP zu verbessern und die Qualifikations- und Rahmenbedingungen unter der Frage zu prüfen, ob sie geeignet sind, die GP in die Lage zu versetzen, mit neuen, im Zuge des gesellschaftlichen Wandels an Relevanz gewinnenden Aufgaben umzugehen. Stärken und Limitationen Mit der Analyse der professionellen GK und der hier vorgenommenen Teilanalyse erfolgte ein erster Schritt zur Untersuchung der bei der professionellen Förderung von GK bestehenden Schwierigkeiten und Optimierungspotenziale. Gleichzeitig wurde Weiterentwicklungsbedarf deutlich: So ist limitierend anzuführen, dass es sich bei der Befragung um eine Online-Erhebung handelte, die vermutlich mit Selektionseffekten einhergegangen ist und eine Repräsentativität der Stichprobe ausschließt. Zudem wurde die professionelle GK durch Selbsteinschätzungen erfasst, die nicht zwangsläufig die tatsächlichen Fähigkeiten widerspiegeln. Dies könnte auch Einfluss auf die untersuchten Zusammenhänge gehabt haben. Außerdem handelt es sich um eine Querschnittsbefragung, die keine Rückschlüsse auf kausale Zusammenhänge erlaubt. Mit der Analyse der professionellen GK und der hier vorgenommenen Teilanalyse erfolgte ein erster Schritt zur Untersuchung der bei der professionellen Förderung von GK bestehenden Schwierigkeiten und Optimierungspotenziale. Gleichzeitig wurde Weiterentwicklungsbedarf deutlich: So ist limitierend anzuführen, dass es sich bei der Befragung um eine Online-Erhebung handelte, die vermutlich mit Selektionseffekten einhergegangen ist und eine Repräsentativität der Stichprobe ausschließt. Zudem wurde die professionelle GK durch Selbsteinschätzungen erfasst, die nicht zwangsläufig die tatsächlichen Fähigkeiten widerspiegeln. Dies könnte auch Einfluss auf die untersuchten Zusammenhänge gehabt haben. Außerdem handelt es sich um eine Querschnittsbefragung, die keine Rückschlüsse auf kausale Zusammenhänge erlaubt. Eine hohe professionelle GK wird zunehmend als wichtig für die Förderung der GK der Bevölkerung und auch die Umsetzung von gesundheitskompetenten Organisationen diskutiert. Zwar fällt die professionelle GK recht positiv aus, doch machen die dargestellten Ergebnisse auch deutlich, dass Maßnahmen zur Verbesserung erforderlich sind. Auch geben sie Hinweise, wo dabei jeweils anzusetzen ist. Sie belegen zugleich, dass dem relationalen Verständnis von GK folgend ein doppelgleisiges Vorgehen bei der Entwicklung von Verbesserungsmaßnahmen sinnvoll ist, das sowohl auf die Stärkung professioneller Fähigkeiten wie auch auf strukturelle Veränderungen zielt. Tab. Z1 Bewertung der Ausbildung, Vertrautheit mit dem Gesundheitskompetenzkonzept und organisatorische Rahmenbedingungen insgesamt und nach Berufsgruppen |
Perceptions and tolerance of uncertainty: relationship to trust in COVID-19 health information and vaccine hesitancy | 09a1dc5b-250f-4c38-a9b8-dc53ffd0991a | 8990605 | Health Communication[mh] | Design and procedure This cross-sectional study utilized data from a larger online experiment designed to compare the effects of alternative strategies for communicating scientific uncertainty about the nature, transmission, prevention, and treatment of COVID-19 ([citation masked for review]). The strategy used in the control condition consisted of basic information about the nature and prevention of COVID-19, which was developed by a US state public health department and contained no explicit language on scientific uncertainty. In the alternative experimental conditions, this basic information was supplemented by language designed to communicate scientific uncertainty, with and without additional language aimed at “normalizing” uncertainty as an expected state of affairs, promoting hope, and promoting prosocial values. Because the objective of the current study was to examine the influence of pre-existing uncertainty perceptions about COVID-19 and trait-level tolerance of uncertainty, we tested our hypotheses within the control condition alone, to exclude any potential effects of language designed to convey scientific uncertainty or to alter people’s responses to it. Participants The study population consisted of a US national sample recruited by Qualtrics® panels from May 7 to June 11, 2020. During this time, the number of total coronavirus infections in the United States increased from > 1.2 million to > 1.6 million, total deaths increased from > 77,200 to > 98,000 (Han et al., ), and no effective treatments nor vaccines against COVID-19 were available. Panel members were eligible to participate if they were ≥ 18 years living in the US and belonged to Qualtrics® opt-in web survey panel. Study recruitment employed quotas aimed at balancing the study sample based on age, gender, race, geographic region, education, and income. Participants who reported a previous or current COVID-19 diagnosis were excluded. 303 participants were in the control condition that made up the subsample of interest in the current study. Participant demographic information is reported in Table . Measures Perceived uncertainty about COVID-19 Perceived uncertainty about COVID-19 was assessed using a 6-item scale (α = 0.71) developed for this study. This measure assessed participants’ perceptions of uncertainty arising from the sources discussed above, with two items each for perceived uncertainty due to probability (hereafter referred to as perceived indeterminacy), perceived ambiguity, and perceived complexity. The full measure can be found in Appendix . Likert scale response options ranged from 1 (strongly disagree) to 7 (strongly agree). Additionally, we computed subscales defining the different types of perceived uncertainty captured above (perceived indeterminacy, r = 0.62, ambiguity, r = 0.42, and complexity , r = 0.47). Uncertainty tolerance Uncertainty tolerance was measured using three scales assessing trait-level tolerance of uncertainty arising respectively from indeterminacy, ambiguity, and complexity. Pearson risk attitude (PRA) The PRA scale (Pearson et al., ) uses six items (α = 0.72) that measure tolerance of uncertainty arising from indeterminacy/probability (hereafter referred to as tolerance of risk). Higher scores on this scale indicate higher risk tolerance. Example items include: “I enjoy taking risks” and “I try to avoid situations that have uncertain outcomes” (reverse-coded). Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Ambiguity aversion in medicine (AA-Med) The AA-Med scale (Han et al., ) is comprised of 5 items (α = 0.70) measuring tolerance of uncertainty arising from ambiguity (hereafter referred to as tolerance of ambiguity) in medicine. Higher scores on the scale indicate lower tolerance of ambiguity. Example items include: “I would not have confidence in a medical test or treatment if experts had conflicting opinions about it” and “If experts had conflicting opinions about a medical test or treatment, I would still be willing to try it” (reverse-coded). Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Tolerance for ambiguity (TFA) The TFA scale (Geller et al., ) is a 7-item measure (α = 0.76) that arguably measures tolerance of uncertainty arising specifically from complexity (Han et al., )—e.g., “I don't like to work on a problem unless there is a possibility of getting a clear-cut and unambiguous answer” and “If I am uncertain about the responsibilities involved in a particular task, I get very anxious.” Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Higher scores on this scale can thus be interpreted as indicating lower tolerance of uncertainty arising from complexity (hereafter referred to as tolerance of complexity). Outcome variables Trust in COVID-19 Information Was measured with a single item measured after participants were exposed to the COVID-19 message. Participants responded to the item, “How much do you trust the information you just read?” on a scale from 1 (not at all) to 7 (completely). Vaccine hesitancy Was operationalized with two items examining different components of vaccine confidence/caution: preferences for further vaccine testing and intentions for vaccination. Preferences for vaccine delay As one element of vaccine hesitancy, we measured participants’ beliefs that vaccine release to the public should be delayed to allow further testing. The text for this item read, “COVID-19 researchers believe that it may take 1–2 years to develop a COVID-19 vaccine, but it could take several years to go through the normal testing for safety and effectiveness. Would you prefer that a full longer testing period be completed before a COVID-19 vaccine is made available to the public, or that the vaccine be released as soon as possible?” Participants responded on a scale from 1 (definitely release the vaccine as soon as possible) to 7 (definitely wait to release the vaccine until full testing has been completed). Vaccination intentions Vaccination intentions were measured with a single item: “If a vaccine becomes available for COVID-19, how likely would you be to get vaccinated against COVID-19?” Participants responded on a scale from 1 (definitely would not get a vaccination) to 7 (definitely would get a vaccination). Covariates: demographics and participant characteristics A variety of sociodemographic and other relevant participant characteristics were measured and used as covariates in the current analyses, to adjust for their potential confounding effects on the primary associations of interest. We specifically adjusted for race given prior evidence of higher vaccine hesitancy and distrust of healthcare institutions among Black adults in the US, arising at least in part to negative healthcare experiences and the historical abuse of Black individuals by the medical system (Webb Hooper et al., ). Covariates included political affiliation (Democrat, Republican, or Independent), race, age group (e.g., < 30, 30–39…70 +), gender, US region, education level (< 4-year college degree, ≥ 4-year college degree), and whether the participant was an essential worker during the COVID-19 pandemic (yes, no, unsure). Covariates were entered into regression models using dummy variables—Table indicates the number of categories that were included for each covariate. In ANOVA models examining differences in the outcomes of interest as a function of each covariate separately, age group ( F (5, 297) = 2.49, p = 0.03) and education ( F (1, 301) = 6.81, p = 0.01) were significantly associated with trust; political affiliation ( F (2, 300) = 4.50, p = 0.01), race ( F (5, 297) = 2.38, p = 0.04), and age ( F (5, 297) = 2.59, p = 0.03) were significantly associated with vaccine intentions; and age ( F (5, 297) = 2.31, p = 0.04) was significantly associated with vaccine testing beliefs.
This cross-sectional study utilized data from a larger online experiment designed to compare the effects of alternative strategies for communicating scientific uncertainty about the nature, transmission, prevention, and treatment of COVID-19 ([citation masked for review]). The strategy used in the control condition consisted of basic information about the nature and prevention of COVID-19, which was developed by a US state public health department and contained no explicit language on scientific uncertainty. In the alternative experimental conditions, this basic information was supplemented by language designed to communicate scientific uncertainty, with and without additional language aimed at “normalizing” uncertainty as an expected state of affairs, promoting hope, and promoting prosocial values. Because the objective of the current study was to examine the influence of pre-existing uncertainty perceptions about COVID-19 and trait-level tolerance of uncertainty, we tested our hypotheses within the control condition alone, to exclude any potential effects of language designed to convey scientific uncertainty or to alter people’s responses to it.
The study population consisted of a US national sample recruited by Qualtrics® panels from May 7 to June 11, 2020. During this time, the number of total coronavirus infections in the United States increased from > 1.2 million to > 1.6 million, total deaths increased from > 77,200 to > 98,000 (Han et al., ), and no effective treatments nor vaccines against COVID-19 were available. Panel members were eligible to participate if they were ≥ 18 years living in the US and belonged to Qualtrics® opt-in web survey panel. Study recruitment employed quotas aimed at balancing the study sample based on age, gender, race, geographic region, education, and income. Participants who reported a previous or current COVID-19 diagnosis were excluded. 303 participants were in the control condition that made up the subsample of interest in the current study. Participant demographic information is reported in Table .
Perceived uncertainty about COVID-19 Perceived uncertainty about COVID-19 was assessed using a 6-item scale (α = 0.71) developed for this study. This measure assessed participants’ perceptions of uncertainty arising from the sources discussed above, with two items each for perceived uncertainty due to probability (hereafter referred to as perceived indeterminacy), perceived ambiguity, and perceived complexity. The full measure can be found in Appendix . Likert scale response options ranged from 1 (strongly disagree) to 7 (strongly agree). Additionally, we computed subscales defining the different types of perceived uncertainty captured above (perceived indeterminacy, r = 0.62, ambiguity, r = 0.42, and complexity , r = 0.47). Uncertainty tolerance Uncertainty tolerance was measured using three scales assessing trait-level tolerance of uncertainty arising respectively from indeterminacy, ambiguity, and complexity. Pearson risk attitude (PRA) The PRA scale (Pearson et al., ) uses six items (α = 0.72) that measure tolerance of uncertainty arising from indeterminacy/probability (hereafter referred to as tolerance of risk). Higher scores on this scale indicate higher risk tolerance. Example items include: “I enjoy taking risks” and “I try to avoid situations that have uncertain outcomes” (reverse-coded). Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Ambiguity aversion in medicine (AA-Med) The AA-Med scale (Han et al., ) is comprised of 5 items (α = 0.70) measuring tolerance of uncertainty arising from ambiguity (hereafter referred to as tolerance of ambiguity) in medicine. Higher scores on the scale indicate lower tolerance of ambiguity. Example items include: “I would not have confidence in a medical test or treatment if experts had conflicting opinions about it” and “If experts had conflicting opinions about a medical test or treatment, I would still be willing to try it” (reverse-coded). Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Tolerance for ambiguity (TFA) The TFA scale (Geller et al., ) is a 7-item measure (α = 0.76) that arguably measures tolerance of uncertainty arising specifically from complexity (Han et al., )—e.g., “I don't like to work on a problem unless there is a possibility of getting a clear-cut and unambiguous answer” and “If I am uncertain about the responsibilities involved in a particular task, I get very anxious.” Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Higher scores on this scale can thus be interpreted as indicating lower tolerance of uncertainty arising from complexity (hereafter referred to as tolerance of complexity). Outcome variables Trust in COVID-19 Information Was measured with a single item measured after participants were exposed to the COVID-19 message. Participants responded to the item, “How much do you trust the information you just read?” on a scale from 1 (not at all) to 7 (completely). Vaccine hesitancy Was operationalized with two items examining different components of vaccine confidence/caution: preferences for further vaccine testing and intentions for vaccination. Preferences for vaccine delay As one element of vaccine hesitancy, we measured participants’ beliefs that vaccine release to the public should be delayed to allow further testing. The text for this item read, “COVID-19 researchers believe that it may take 1–2 years to develop a COVID-19 vaccine, but it could take several years to go through the normal testing for safety and effectiveness. Would you prefer that a full longer testing period be completed before a COVID-19 vaccine is made available to the public, or that the vaccine be released as soon as possible?” Participants responded on a scale from 1 (definitely release the vaccine as soon as possible) to 7 (definitely wait to release the vaccine until full testing has been completed). Vaccination intentions Vaccination intentions were measured with a single item: “If a vaccine becomes available for COVID-19, how likely would you be to get vaccinated against COVID-19?” Participants responded on a scale from 1 (definitely would not get a vaccination) to 7 (definitely would get a vaccination). Covariates: demographics and participant characteristics A variety of sociodemographic and other relevant participant characteristics were measured and used as covariates in the current analyses, to adjust for their potential confounding effects on the primary associations of interest. We specifically adjusted for race given prior evidence of higher vaccine hesitancy and distrust of healthcare institutions among Black adults in the US, arising at least in part to negative healthcare experiences and the historical abuse of Black individuals by the medical system (Webb Hooper et al., ). Covariates included political affiliation (Democrat, Republican, or Independent), race, age group (e.g., < 30, 30–39…70 +), gender, US region, education level (< 4-year college degree, ≥ 4-year college degree), and whether the participant was an essential worker during the COVID-19 pandemic (yes, no, unsure). Covariates were entered into regression models using dummy variables—Table indicates the number of categories that were included for each covariate. In ANOVA models examining differences in the outcomes of interest as a function of each covariate separately, age group ( F (5, 297) = 2.49, p = 0.03) and education ( F (1, 301) = 6.81, p = 0.01) were significantly associated with trust; political affiliation ( F (2, 300) = 4.50, p = 0.01), race ( F (5, 297) = 2.38, p = 0.04), and age ( F (5, 297) = 2.59, p = 0.03) were significantly associated with vaccine intentions; and age ( F (5, 297) = 2.31, p = 0.04) was significantly associated with vaccine testing beliefs.
Perceived uncertainty about COVID-19 was assessed using a 6-item scale (α = 0.71) developed for this study. This measure assessed participants’ perceptions of uncertainty arising from the sources discussed above, with two items each for perceived uncertainty due to probability (hereafter referred to as perceived indeterminacy), perceived ambiguity, and perceived complexity. The full measure can be found in Appendix . Likert scale response options ranged from 1 (strongly disagree) to 7 (strongly agree). Additionally, we computed subscales defining the different types of perceived uncertainty captured above (perceived indeterminacy, r = 0.62, ambiguity, r = 0.42, and complexity , r = 0.47).
Uncertainty tolerance was measured using three scales assessing trait-level tolerance of uncertainty arising respectively from indeterminacy, ambiguity, and complexity. Pearson risk attitude (PRA) The PRA scale (Pearson et al., ) uses six items (α = 0.72) that measure tolerance of uncertainty arising from indeterminacy/probability (hereafter referred to as tolerance of risk). Higher scores on this scale indicate higher risk tolerance. Example items include: “I enjoy taking risks” and “I try to avoid situations that have uncertain outcomes” (reverse-coded). Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Ambiguity aversion in medicine (AA-Med) The AA-Med scale (Han et al., ) is comprised of 5 items (α = 0.70) measuring tolerance of uncertainty arising from ambiguity (hereafter referred to as tolerance of ambiguity) in medicine. Higher scores on the scale indicate lower tolerance of ambiguity. Example items include: “I would not have confidence in a medical test or treatment if experts had conflicting opinions about it” and “If experts had conflicting opinions about a medical test or treatment, I would still be willing to try it” (reverse-coded). Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Tolerance for ambiguity (TFA) The TFA scale (Geller et al., ) is a 7-item measure (α = 0.76) that arguably measures tolerance of uncertainty arising specifically from complexity (Han et al., )—e.g., “I don't like to work on a problem unless there is a possibility of getting a clear-cut and unambiguous answer” and “If I am uncertain about the responsibilities involved in a particular task, I get very anxious.” Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Higher scores on this scale can thus be interpreted as indicating lower tolerance of uncertainty arising from complexity (hereafter referred to as tolerance of complexity).
The PRA scale (Pearson et al., ) uses six items (α = 0.72) that measure tolerance of uncertainty arising from indeterminacy/probability (hereafter referred to as tolerance of risk). Higher scores on this scale indicate higher risk tolerance. Example items include: “I enjoy taking risks” and “I try to avoid situations that have uncertain outcomes” (reverse-coded). Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree).
The AA-Med scale (Han et al., ) is comprised of 5 items (α = 0.70) measuring tolerance of uncertainty arising from ambiguity (hereafter referred to as tolerance of ambiguity) in medicine. Higher scores on the scale indicate lower tolerance of ambiguity. Example items include: “I would not have confidence in a medical test or treatment if experts had conflicting opinions about it” and “If experts had conflicting opinions about a medical test or treatment, I would still be willing to try it” (reverse-coded). Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree).
The TFA scale (Geller et al., ) is a 7-item measure (α = 0.76) that arguably measures tolerance of uncertainty arising specifically from complexity (Han et al., )—e.g., “I don't like to work on a problem unless there is a possibility of getting a clear-cut and unambiguous answer” and “If I am uncertain about the responsibilities involved in a particular task, I get very anxious.” Likert scale response options ranged from 1 (strongly disagree) to 6 (strongly agree). Higher scores on this scale can thus be interpreted as indicating lower tolerance of uncertainty arising from complexity (hereafter referred to as tolerance of complexity).
Trust in COVID-19 Information Was measured with a single item measured after participants were exposed to the COVID-19 message. Participants responded to the item, “How much do you trust the information you just read?” on a scale from 1 (not at all) to 7 (completely). Vaccine hesitancy Was operationalized with two items examining different components of vaccine confidence/caution: preferences for further vaccine testing and intentions for vaccination. Preferences for vaccine delay As one element of vaccine hesitancy, we measured participants’ beliefs that vaccine release to the public should be delayed to allow further testing. The text for this item read, “COVID-19 researchers believe that it may take 1–2 years to develop a COVID-19 vaccine, but it could take several years to go through the normal testing for safety and effectiveness. Would you prefer that a full longer testing period be completed before a COVID-19 vaccine is made available to the public, or that the vaccine be released as soon as possible?” Participants responded on a scale from 1 (definitely release the vaccine as soon as possible) to 7 (definitely wait to release the vaccine until full testing has been completed). Vaccination intentions Vaccination intentions were measured with a single item: “If a vaccine becomes available for COVID-19, how likely would you be to get vaccinated against COVID-19?” Participants responded on a scale from 1 (definitely would not get a vaccination) to 7 (definitely would get a vaccination).
Was measured with a single item measured after participants were exposed to the COVID-19 message. Participants responded to the item, “How much do you trust the information you just read?” on a scale from 1 (not at all) to 7 (completely).
Was operationalized with two items examining different components of vaccine confidence/caution: preferences for further vaccine testing and intentions for vaccination. Preferences for vaccine delay As one element of vaccine hesitancy, we measured participants’ beliefs that vaccine release to the public should be delayed to allow further testing. The text for this item read, “COVID-19 researchers believe that it may take 1–2 years to develop a COVID-19 vaccine, but it could take several years to go through the normal testing for safety and effectiveness. Would you prefer that a full longer testing period be completed before a COVID-19 vaccine is made available to the public, or that the vaccine be released as soon as possible?” Participants responded on a scale from 1 (definitely release the vaccine as soon as possible) to 7 (definitely wait to release the vaccine until full testing has been completed).
Vaccination intentions were measured with a single item: “If a vaccine becomes available for COVID-19, how likely would you be to get vaccinated against COVID-19?” Participants responded on a scale from 1 (definitely would not get a vaccination) to 7 (definitely would get a vaccination).
A variety of sociodemographic and other relevant participant characteristics were measured and used as covariates in the current analyses, to adjust for their potential confounding effects on the primary associations of interest. We specifically adjusted for race given prior evidence of higher vaccine hesitancy and distrust of healthcare institutions among Black adults in the US, arising at least in part to negative healthcare experiences and the historical abuse of Black individuals by the medical system (Webb Hooper et al., ). Covariates included political affiliation (Democrat, Republican, or Independent), race, age group (e.g., < 30, 30–39…70 +), gender, US region, education level (< 4-year college degree, ≥ 4-year college degree), and whether the participant was an essential worker during the COVID-19 pandemic (yes, no, unsure). Covariates were entered into regression models using dummy variables—Table indicates the number of categories that were included for each covariate. In ANOVA models examining differences in the outcomes of interest as a function of each covariate separately, age group ( F (5, 297) = 2.49, p = 0.03) and education ( F (1, 301) = 6.81, p = 0.01) were significantly associated with trust; political affiliation ( F (2, 300) = 4.50, p = 0.01), race ( F (5, 297) = 2.38, p = 0.04), and age ( F (5, 297) = 2.59, p = 0.03) were significantly associated with vaccine intentions; and age ( F (5, 297) = 2.31, p = 0.04) was significantly associated with vaccine testing beliefs.
We began by obtaining descriptive statistics and bivariate relationships for the primary variables of interest. Analyses to address the study hypotheses were conducted using multiple regression models examining the main effects and interactions of interest. Specific details are provided within the Results section. Analysis syntax and output are available at https://osf.io/m5nye/?view_only=9ee8d045678c4164a305c1e347d7c6c1 . Given that the sample size collected was determined based upon power considerations for the parent study, we conducted a post hoc power analysis using G*Power. For a multiple regression model with 23 predictors (e.g., the model testing general uncertainty perceptions and tolerance of risk, ambiguity, and complexity with all the included covariates dummy coded), the analytic sample size of n = 303 equates to power ranging from 0.69 to 0.97 to detect effect size f 2 ranging from 0.02 to 0.05 respectively (equivalent to effect sizes observed in the current study) for a 1 degree of freedom test at an alpha level of 0.05.
Correlations between uncertainty constructs and outcomes of interest Bivariate correlations between uncertainty perceptions, uncertainty tolerance measures, and outcomes of interest are presented in Table . Associations among perceived uncertainty about COVID-19, uncertainty tolerance, trust in COVID-19 information, and vaccine hesitancy To test our first hypothesis—that perceptions of COVID-19 uncertainty and uncertainty tolerance would each explain unique variance in trust in public health information and vaccine hesitancy—each outcome of interest was regressed on COVID-19 uncertainty perceptions (first, using the full uncertainty perception scale, ignoring type of uncertainty, for purposes of parsimony) and each type of uncertainty tolerance (tolerance of risk, ambiguity, and complexity), controlling for the covariates described above. Results from these models are presented in Table . Next, we ran an additional multiple regression model for each outcome, where perceptions of uncertainty were broken down into the three uncertainty subtypes—perceived indeterminacy, ambiguity, and complexity, to examine whether type of uncertainty perception was differentially associated with the dependent variables of interest. Results from these models are presented in Table . All results are summarized below. Perceived uncertainty about COVID-19 Perceptions of uncertainty were not significantly associated with trust in COVID-19 information. This remained true when the type of uncertainty perception (indeterminacy, ambiguity, and complexity) was considered. Perceptions of uncertainty were not significantly associated with vaccine intentions in models using overall uncertainty perceptions as the independent variable. These results were similar when uncertainty perception subtypes were considered: regardless of subtype, uncertainty perceptions were not associated with vaccine intentions. Finally, uncertainty perceptions were not significantly associated with vaccine testing preferences. Again, in the model separating specific uncertainty perceptions, perceived uncertainty was not associated with vaccine testing beliefs regardless of subtype. Trait-Level uncertainty tolerance Dependent variable: trust in information Neither trait-level tolerance of risk, ambiguity, nor complexity were associated with trust in information. Dependent variable: vaccine intentions Tolerance of both risk and ambiguity were significantly associated with vaccine intentions, demonstrating associations in opposite directions, such that higher tolerance of risk was associated with lower vaccine intentions ( p = 0.04), and lower tolerance of ambiguity ( higher ambiguity aversion) was associated with lower vaccine intentions ( p = 0.02). Tolerance of complexity was not significantly associated with vaccine intentions. In the regression models where uncertainty perception subtypes were considered separately, the association between risk tolerance and vaccine intentions became nonsignificant ( p = 0.08), but tolerance of ambiguity remained a significant predictor of vaccine intentions ( p = 0.02). Dependent variable: preferences for vaccine delay Neither tolerance of risk nor tolerance of complexity were not significantly associated with preferences for vaccine delay. However, tolerance of ambiguity was associated with these preferences: lower tolerance of ambiguity was associated with believing that vaccines should go through a relatively longer vs. shorter testing period ( p < 0.001). This association remained significant in the model separating type of uncertainty perception. Sensitivity analyses: main effects As noted earlier, in addition to the control condition that served as the analytical sample for the current paper, the parent study included two additional conditions that did not receive a theoretical manipulation of uncertainty: these conditions read the same basic information about the nature and prevention of the COVID-19 pandemic as those in the control condition (with no language explicitly discussing uncertainty), plus additional content that promoted either prosociality or hope. Given that these conditions did not receive an explicit manipulation of uncertainty, we conducted a sensitivity analysis in this larger subsample ( N = 901; n = 303 in control condition, n = 301 hope promoting condition, n = 297 in prosocial condition), while statistically adjusting for experimental condition, to evaluate the robustness of our findings. We began by examining whether the independent variables of interest differed across these three study conditions. Even though uncertainty was not explicitly manipulated in the three study conditions of interest, we found that perceptions of uncertainty about COVID-19 were lower in the no uncertainty + prosocial condition compared to the other two conditions: F (2, 898) = 2.93, p = 0.054, M diff hope promoting vs. prosocial = 0.18, p = 0.04, M diff control vs. prosocial = 0.20, p = 0.03. Individual differences in tolerance of uncertainty did not differ by condition (all p ’s < 0.52). The results of the sensitivity analyses in the larger subgroup of participants largely confirmed the above results from analyses of the non-uncertainty + control group. All significant relationships in the main study sample remained significant in the larger sample. The analyses in the larger sample also identified significant effects not observed in the smaller sample. Specifically, uncertainty perceptions and tolerance of complexity were both positively associated with trust in information, and tolerance for ambiguity was negatively associated with vaccine intentions in the larger sample for the analyses that did not separate type of uncertainty perception. In the analyses that separated type of uncertainty perception, perceived uncertainty due to ambiguity was negatively associated with trust, perceived uncertainty due to complexity was significantly positively associated with trust, and perceived uncertainty due to complexity was significantly positively associated with intentions (see Supplemental Tables 1 and 2). Interactions between perceptions and tolerance of uncertainty To test our hypothesis that the potential effects of uncertainty perceptions would be moderated by individuals’ trait-level tolerance of different types of uncertainty, we conducted three multiple regression models for each outcome that included a term for the two-way interaction between general uncertainty perceptions and each type of uncertainty tolerance. We included only the general measure of perceived uncertainty in the interactions for two reasons: (1) at the level of main effects, general uncertainty perceptions performed similarly compared to breaking this construct out by subtype—i.e., neither general uncertainty perceptions nor uncertainty perception subtypes were significantly associated with outcomes, and (2) probing interactions between all uncertainty perception subtypes and uncertainty tolerance subtypes would have involved a multiplicative increase in the number of statistical tests conducted, for which the study lacked power. Predictors were mean-centered. We observed significant interactions between uncertainty perceptions and tolerance, but only for trust in information. Tolerance of risk significantly moderated the effect of uncertainty perceptions on trust in information ( b = 0.196, 95% CI [0.068, 0.032], p = 0.003, partial η 2 = 0.03), such that the negative relationship between perceived uncertainty and trust in information about COVID-19 was stronger for individuals with lower tolerance of risk (see Fig. a). Specifically, in simple slopes analysis, we found that the relationship between uncertainty perceptions and trust was not significantly different from zero for individuals with mean levels of risk tolerance ( b = 0.04, p = 0.53); the simple slope for uncertainty perceptions was negative, but not significantly different from zero for individuals with risk tolerance one standard deviation below the mean ( b = − 0.15, p = 0.10); finally, the simple slope for uncertainty perceptions was negative and significantly different from zero for individuals with risk tolerance two standard deviations below the mean ( b = − 0.33, p = 0.02). The proportion of variance in trust explained by all the uncertainty-related predictors, including the interaction between perceived uncertainty and tolerance of risk, in this model was 0.04. Tolerance of ambiguity also moderated the relationship between uncertainty perceptions and trust in information, b = − 0.142, 95% CI [− 0.252, − 0.031], p = 0.012, partial η 2 = 0.02, such that greater perceived uncertainty was associated with higher trust for individuals with higher tolerance of ambiguity (less ambiguity aversion) (see Fig. b). Specifically, simple slopes analysis demonstrated that the relationship between uncertainty perceptions and trust was not significantly different from zero for individuals with mean levels of ambiguity aversion ( b = 0.04, p = 0.57), and the simple slopes for uncertainty perceptions were negative, but not significantly different from zero for individuals with ambiguity aversion one ( b = − 0.10, p = 0.25) and two ( b = − 0.23, p = 0.06) standard deviations above the mean; finally, individuals with ambiguity aversion scores one ( b = 0.17, p = 0.05) and two ( b = 0.31, p = 0.02) standard deviations below the mean had significant, positive simple slopes for uncertainty perceptions and trust. The proportion of variance explained by all the uncertainty-related predictors, including the interaction between perceived uncertainty and tolerance of risk, in this model was 0.03. Sensitivity analyses: interactions In sensitivity analyses conducted with the larger sample, the significant interactions between uncertainty perceptions and risk tolerance ( b = 0.16, 95% CI [0.94, 0.088], p < 0.001) and uncertainty perceptions and tolerance of ambiguity ( b = − 0.07, 95% CI [− 0.138, − 0.001], p = 0.046) were also present. Sensitivity analyses in the larger sample also demonstrated significant interactions between uncertainty perceptions and risk tolerance on vaccine intentions, such that uncertainty perceptions were more negatively associated with vaccine intentions among individuals with lower risk tolerance, b = 0.137, 95% CI [0.031, 0.244], p = 0.012. Additionally, tolerance of complexity moderated the effect of uncertainty perceptions on vaccine testing beliefs, such that perceived uncertainty was more positively associated with preferences for vaccine delay among individuals with lower tolerance of complexity ( b = 0.123, 95% CI [0.006, 0.239], p = 0.039).
Bivariate correlations between uncertainty perceptions, uncertainty tolerance measures, and outcomes of interest are presented in Table .
To test our first hypothesis—that perceptions of COVID-19 uncertainty and uncertainty tolerance would each explain unique variance in trust in public health information and vaccine hesitancy—each outcome of interest was regressed on COVID-19 uncertainty perceptions (first, using the full uncertainty perception scale, ignoring type of uncertainty, for purposes of parsimony) and each type of uncertainty tolerance (tolerance of risk, ambiguity, and complexity), controlling for the covariates described above. Results from these models are presented in Table . Next, we ran an additional multiple regression model for each outcome, where perceptions of uncertainty were broken down into the three uncertainty subtypes—perceived indeterminacy, ambiguity, and complexity, to examine whether type of uncertainty perception was differentially associated with the dependent variables of interest. Results from these models are presented in Table . All results are summarized below. Perceived uncertainty about COVID-19 Perceptions of uncertainty were not significantly associated with trust in COVID-19 information. This remained true when the type of uncertainty perception (indeterminacy, ambiguity, and complexity) was considered. Perceptions of uncertainty were not significantly associated with vaccine intentions in models using overall uncertainty perceptions as the independent variable. These results were similar when uncertainty perception subtypes were considered: regardless of subtype, uncertainty perceptions were not associated with vaccine intentions. Finally, uncertainty perceptions were not significantly associated with vaccine testing preferences. Again, in the model separating specific uncertainty perceptions, perceived uncertainty was not associated with vaccine testing beliefs regardless of subtype. Trait-Level uncertainty tolerance Dependent variable: trust in information Neither trait-level tolerance of risk, ambiguity, nor complexity were associated with trust in information. Dependent variable: vaccine intentions Tolerance of both risk and ambiguity were significantly associated with vaccine intentions, demonstrating associations in opposite directions, such that higher tolerance of risk was associated with lower vaccine intentions ( p = 0.04), and lower tolerance of ambiguity ( higher ambiguity aversion) was associated with lower vaccine intentions ( p = 0.02). Tolerance of complexity was not significantly associated with vaccine intentions. In the regression models where uncertainty perception subtypes were considered separately, the association between risk tolerance and vaccine intentions became nonsignificant ( p = 0.08), but tolerance of ambiguity remained a significant predictor of vaccine intentions ( p = 0.02). Dependent variable: preferences for vaccine delay Neither tolerance of risk nor tolerance of complexity were not significantly associated with preferences for vaccine delay. However, tolerance of ambiguity was associated with these preferences: lower tolerance of ambiguity was associated with believing that vaccines should go through a relatively longer vs. shorter testing period ( p < 0.001). This association remained significant in the model separating type of uncertainty perception. Sensitivity analyses: main effects As noted earlier, in addition to the control condition that served as the analytical sample for the current paper, the parent study included two additional conditions that did not receive a theoretical manipulation of uncertainty: these conditions read the same basic information about the nature and prevention of the COVID-19 pandemic as those in the control condition (with no language explicitly discussing uncertainty), plus additional content that promoted either prosociality or hope. Given that these conditions did not receive an explicit manipulation of uncertainty, we conducted a sensitivity analysis in this larger subsample ( N = 901; n = 303 in control condition, n = 301 hope promoting condition, n = 297 in prosocial condition), while statistically adjusting for experimental condition, to evaluate the robustness of our findings. We began by examining whether the independent variables of interest differed across these three study conditions. Even though uncertainty was not explicitly manipulated in the three study conditions of interest, we found that perceptions of uncertainty about COVID-19 were lower in the no uncertainty + prosocial condition compared to the other two conditions: F (2, 898) = 2.93, p = 0.054, M diff hope promoting vs. prosocial = 0.18, p = 0.04, M diff control vs. prosocial = 0.20, p = 0.03. Individual differences in tolerance of uncertainty did not differ by condition (all p ’s < 0.52). The results of the sensitivity analyses in the larger subgroup of participants largely confirmed the above results from analyses of the non-uncertainty + control group. All significant relationships in the main study sample remained significant in the larger sample. The analyses in the larger sample also identified significant effects not observed in the smaller sample. Specifically, uncertainty perceptions and tolerance of complexity were both positively associated with trust in information, and tolerance for ambiguity was negatively associated with vaccine intentions in the larger sample for the analyses that did not separate type of uncertainty perception. In the analyses that separated type of uncertainty perception, perceived uncertainty due to ambiguity was negatively associated with trust, perceived uncertainty due to complexity was significantly positively associated with trust, and perceived uncertainty due to complexity was significantly positively associated with intentions (see Supplemental Tables 1 and 2).
Perceptions of uncertainty were not significantly associated with trust in COVID-19 information. This remained true when the type of uncertainty perception (indeterminacy, ambiguity, and complexity) was considered. Perceptions of uncertainty were not significantly associated with vaccine intentions in models using overall uncertainty perceptions as the independent variable. These results were similar when uncertainty perception subtypes were considered: regardless of subtype, uncertainty perceptions were not associated with vaccine intentions. Finally, uncertainty perceptions were not significantly associated with vaccine testing preferences. Again, in the model separating specific uncertainty perceptions, perceived uncertainty was not associated with vaccine testing beliefs regardless of subtype.
Dependent variable: trust in information Neither trait-level tolerance of risk, ambiguity, nor complexity were associated with trust in information. Dependent variable: vaccine intentions Tolerance of both risk and ambiguity were significantly associated with vaccine intentions, demonstrating associations in opposite directions, such that higher tolerance of risk was associated with lower vaccine intentions ( p = 0.04), and lower tolerance of ambiguity ( higher ambiguity aversion) was associated with lower vaccine intentions ( p = 0.02). Tolerance of complexity was not significantly associated with vaccine intentions. In the regression models where uncertainty perception subtypes were considered separately, the association between risk tolerance and vaccine intentions became nonsignificant ( p = 0.08), but tolerance of ambiguity remained a significant predictor of vaccine intentions ( p = 0.02). Dependent variable: preferences for vaccine delay Neither tolerance of risk nor tolerance of complexity were not significantly associated with preferences for vaccine delay. However, tolerance of ambiguity was associated with these preferences: lower tolerance of ambiguity was associated with believing that vaccines should go through a relatively longer vs. shorter testing period ( p < 0.001). This association remained significant in the model separating type of uncertainty perception.
Neither trait-level tolerance of risk, ambiguity, nor complexity were associated with trust in information.
Tolerance of both risk and ambiguity were significantly associated with vaccine intentions, demonstrating associations in opposite directions, such that higher tolerance of risk was associated with lower vaccine intentions ( p = 0.04), and lower tolerance of ambiguity ( higher ambiguity aversion) was associated with lower vaccine intentions ( p = 0.02). Tolerance of complexity was not significantly associated with vaccine intentions. In the regression models where uncertainty perception subtypes were considered separately, the association between risk tolerance and vaccine intentions became nonsignificant ( p = 0.08), but tolerance of ambiguity remained a significant predictor of vaccine intentions ( p = 0.02).
Neither tolerance of risk nor tolerance of complexity were not significantly associated with preferences for vaccine delay. However, tolerance of ambiguity was associated with these preferences: lower tolerance of ambiguity was associated with believing that vaccines should go through a relatively longer vs. shorter testing period ( p < 0.001). This association remained significant in the model separating type of uncertainty perception.
As noted earlier, in addition to the control condition that served as the analytical sample for the current paper, the parent study included two additional conditions that did not receive a theoretical manipulation of uncertainty: these conditions read the same basic information about the nature and prevention of the COVID-19 pandemic as those in the control condition (with no language explicitly discussing uncertainty), plus additional content that promoted either prosociality or hope. Given that these conditions did not receive an explicit manipulation of uncertainty, we conducted a sensitivity analysis in this larger subsample ( N = 901; n = 303 in control condition, n = 301 hope promoting condition, n = 297 in prosocial condition), while statistically adjusting for experimental condition, to evaluate the robustness of our findings. We began by examining whether the independent variables of interest differed across these three study conditions. Even though uncertainty was not explicitly manipulated in the three study conditions of interest, we found that perceptions of uncertainty about COVID-19 were lower in the no uncertainty + prosocial condition compared to the other two conditions: F (2, 898) = 2.93, p = 0.054, M diff hope promoting vs. prosocial = 0.18, p = 0.04, M diff control vs. prosocial = 0.20, p = 0.03. Individual differences in tolerance of uncertainty did not differ by condition (all p ’s < 0.52). The results of the sensitivity analyses in the larger subgroup of participants largely confirmed the above results from analyses of the non-uncertainty + control group. All significant relationships in the main study sample remained significant in the larger sample. The analyses in the larger sample also identified significant effects not observed in the smaller sample. Specifically, uncertainty perceptions and tolerance of complexity were both positively associated with trust in information, and tolerance for ambiguity was negatively associated with vaccine intentions in the larger sample for the analyses that did not separate type of uncertainty perception. In the analyses that separated type of uncertainty perception, perceived uncertainty due to ambiguity was negatively associated with trust, perceived uncertainty due to complexity was significantly positively associated with trust, and perceived uncertainty due to complexity was significantly positively associated with intentions (see Supplemental Tables 1 and 2).
To test our hypothesis that the potential effects of uncertainty perceptions would be moderated by individuals’ trait-level tolerance of different types of uncertainty, we conducted three multiple regression models for each outcome that included a term for the two-way interaction between general uncertainty perceptions and each type of uncertainty tolerance. We included only the general measure of perceived uncertainty in the interactions for two reasons: (1) at the level of main effects, general uncertainty perceptions performed similarly compared to breaking this construct out by subtype—i.e., neither general uncertainty perceptions nor uncertainty perception subtypes were significantly associated with outcomes, and (2) probing interactions between all uncertainty perception subtypes and uncertainty tolerance subtypes would have involved a multiplicative increase in the number of statistical tests conducted, for which the study lacked power. Predictors were mean-centered. We observed significant interactions between uncertainty perceptions and tolerance, but only for trust in information. Tolerance of risk significantly moderated the effect of uncertainty perceptions on trust in information ( b = 0.196, 95% CI [0.068, 0.032], p = 0.003, partial η 2 = 0.03), such that the negative relationship between perceived uncertainty and trust in information about COVID-19 was stronger for individuals with lower tolerance of risk (see Fig. a). Specifically, in simple slopes analysis, we found that the relationship between uncertainty perceptions and trust was not significantly different from zero for individuals with mean levels of risk tolerance ( b = 0.04, p = 0.53); the simple slope for uncertainty perceptions was negative, but not significantly different from zero for individuals with risk tolerance one standard deviation below the mean ( b = − 0.15, p = 0.10); finally, the simple slope for uncertainty perceptions was negative and significantly different from zero for individuals with risk tolerance two standard deviations below the mean ( b = − 0.33, p = 0.02). The proportion of variance in trust explained by all the uncertainty-related predictors, including the interaction between perceived uncertainty and tolerance of risk, in this model was 0.04. Tolerance of ambiguity also moderated the relationship between uncertainty perceptions and trust in information, b = − 0.142, 95% CI [− 0.252, − 0.031], p = 0.012, partial η 2 = 0.02, such that greater perceived uncertainty was associated with higher trust for individuals with higher tolerance of ambiguity (less ambiguity aversion) (see Fig. b). Specifically, simple slopes analysis demonstrated that the relationship between uncertainty perceptions and trust was not significantly different from zero for individuals with mean levels of ambiguity aversion ( b = 0.04, p = 0.57), and the simple slopes for uncertainty perceptions were negative, but not significantly different from zero for individuals with ambiguity aversion one ( b = − 0.10, p = 0.25) and two ( b = − 0.23, p = 0.06) standard deviations above the mean; finally, individuals with ambiguity aversion scores one ( b = 0.17, p = 0.05) and two ( b = 0.31, p = 0.02) standard deviations below the mean had significant, positive simple slopes for uncertainty perceptions and trust. The proportion of variance explained by all the uncertainty-related predictors, including the interaction between perceived uncertainty and tolerance of risk, in this model was 0.03. Sensitivity analyses: interactions In sensitivity analyses conducted with the larger sample, the significant interactions between uncertainty perceptions and risk tolerance ( b = 0.16, 95% CI [0.94, 0.088], p < 0.001) and uncertainty perceptions and tolerance of ambiguity ( b = − 0.07, 95% CI [− 0.138, − 0.001], p = 0.046) were also present. Sensitivity analyses in the larger sample also demonstrated significant interactions between uncertainty perceptions and risk tolerance on vaccine intentions, such that uncertainty perceptions were more negatively associated with vaccine intentions among individuals with lower risk tolerance, b = 0.137, 95% CI [0.031, 0.244], p = 0.012. Additionally, tolerance of complexity moderated the effect of uncertainty perceptions on vaccine testing beliefs, such that perceived uncertainty was more positively associated with preferences for vaccine delay among individuals with lower tolerance of complexity ( b = 0.123, 95% CI [0.006, 0.239], p = 0.039).
In sensitivity analyses conducted with the larger sample, the significant interactions between uncertainty perceptions and risk tolerance ( b = 0.16, 95% CI [0.94, 0.088], p < 0.001) and uncertainty perceptions and tolerance of ambiguity ( b = − 0.07, 95% CI [− 0.138, − 0.001], p = 0.046) were also present. Sensitivity analyses in the larger sample also demonstrated significant interactions between uncertainty perceptions and risk tolerance on vaccine intentions, such that uncertainty perceptions were more negatively associated with vaccine intentions among individuals with lower risk tolerance, b = 0.137, 95% CI [0.031, 0.244], p = 0.012. Additionally, tolerance of complexity moderated the effect of uncertainty perceptions on vaccine testing beliefs, such that perceived uncertainty was more positively associated with preferences for vaccine delay among individuals with lower tolerance of complexity ( b = 0.123, 95% CI [0.006, 0.239], p = 0.039).
Scientific uncertainty about COVID-19 is prevalent, but the extent to which public perceptions of scientific uncertainty may explain variability in important pandemic control behaviors and their antecedents has not been previously investigated. In this study, we sought to examine the relationship between perceptions of scientific uncertainty about COVID-19, trait-level individual differences in uncertainty tolerance (and interactions between these two factors), and trust in COVID-19 information and COVID-19 vaccine hesitancy. We found that, on average, uncertainty perceptions about COVID-19 were not independently associated with trust in information or vaccine hesitancy. This was true when considering perceived uncertainty broadly, as well as when considering perceptions of specific types of uncertainty (i.e., indeterminacy, ambiguity, or complexity). However, consistent with hypotheses, we found that individual trait-level differences in tolerance of uncertainty were associated with vaccine hesitancy—though these associations differed depending on the type of uncertainty tolerance. Specifically, higher tolerance of risk and lower tolerance of ambiguity were both associated with lower vaccine intentions. Lower tolerance of ambiguity was also associated with stronger preferences for delaying vaccination. These findings are consistent with previous work demonstrating negative relationships between uncertainty tolerance and health cognition-related outcomes (Han et al., ; Strout et al., ), and supports the importance of considering the type of uncertainty tolerance, as different forms of uncertainty tolerance can have different effects (Han et al., ). The fact that we observed associations in the opposite directions for tolerance of risk and tolerance of ambiguity on vaccine intentions was unexpected. It is possible that individuals with high risk tolerance may be predisposed to focus on the risk posed by COVID-19 itself (as opposed to risk related to vaccines); these individuals may thus have been less interested in vaccination because of their willingness to accept the risk posed by COVID-19. On the other hand, individuals with lower tolerance of ambiguity had lower vaccine intentions, suggesting that among these individuals, aversion to ambiguity may have been directed toward ambiguities about the vaccine, rather than aspects of COVID-19 itself. Notably, we also did not observe significant associations between either uncertainty perceptions or tolerance of uncertainty and trust in information (i.e., when not considering interactive effects between these factors). The reasons for these null findings are not clear; they may reflect either methodological shortcomings of our study (e.g., measurement error, limited power to detect associations) or the influence of unmeasured variables that may moderate or mediate the relationship between uncertainty perceptions, tolerance, and outcomes. Examples include personality traits (e.g., dispositional optimism and hope), perceptions of the effectiveness of alternative risk-reducing behaviors, and past experiences with illness and health care. Notably, however, our findings are consistent with recent work from van der Bles and colleagues (2020), which found minor or null relationships between uncertainty and trust in science. Together, these findings may offer some reassurance to public health officials and policymakers that communicating scientific uncertainty may not diminish public trust—at least at a broad, population level. Although we did not observe significant associations between either uncertainty perceptions or tolerance of uncertainty and trust in COVID-19 information, we did find that the association between uncertainty perceptions and trust was moderated by uncertainty tolerance. Consistent with hypotheses, we found that perceived uncertainty about COVID-19 had a stronger negative association with trust in information among participants having a lower tolerance of risk and ambiguity. These findings relate to previous work showing that individual differences in uncertainty tolerance may modify relationships between uncertainty perceptions and relevant outcomes in health contexts (Anderson et al., ). In this case, our findings suggest that individual differences in tolerance of uncertainty may be an important determinant of the effects of scientific uncertainty on public trust in the science, and may explain the inconsistent or null findings of past work. Notably, we did not observe similar moderating effects for the vaccine hesitancy outcomes. Although the cross-sectional nature of our study restricts causal inferences, it is conceivable that although perceptions of scientific uncertainty about the COVID-19 pandemic may not lead to lower trust in public health information or vaccine hesitancy for all individuals, perceived uncertainty may reduce trust (i.e., an element of hesitancy) particularly for individuals who are especially averse to uncertainty. If these findings are confirmed, they may help future inform public health communication or other interventions to increase COVID-19 vaccination uptake. We acknowledge several important limitations of the current study. Above all, the cross-sectional design of the study limits causal claims about the observed associations. We therefore cannot rule out alternative causal explanations; for example, it is conceivable that vaccine-hesitant individuals may endorse higher tolerance of risk and lower tolerance of ambiguity as a means of rationalizing their opposition to vaccination (manifesting motivated reasoning). We believe this explanation is less likely, given that uncertainty tolerance is thought to be a stable trait-level characteristic; however, further experimental and longitudinal studies are needed to establish the causal directions of the associations between these variables. Our post hoc power analysis indicated that the subsample of participants that was used in the primary analyses for the current paper was also likely insufficient for testing main/interaction effects with small effect sizes—thus, some of the null effects we observed may have resulted from a lack of statistical power to observe significant associations. Additionally, since we conducted multiple statistical tests (the independent and interactive effects of multiple independent variables on three separate dependent variables), we cannot rule out Type I errors; our findings thus need to be replicated in larger studies. Furthermore, perceived uncertainty was queried with regard to the COVID-19 pandemic more broadly, rather than about COVID-19 vaccination itself, and trust in information was queried regarding the message participants read for the study specifically (e.g., rather than trust in public health experts or scientists more broadly). Although we believe that the current measures were reasonable proxies for the constructs they represent, it is possible that our pattern of results would have differed had these items been alternatively worded. Similarly, responses to the question on preferences for vaccine delay may have been biased by the response anchor, “definitely release the vaccines as soon as possible,” which may have implied to participants that no testing would be completed. Finally, the study was conducted in June 2020—before vaccines or treatments had been released to the public. The subsequent evolution of social norms and mandates for various COVID-19 preventive behaviors, as well as other systemic factors influencing vaccination behavior, may reduce to some degree the generalizability of our findings to present circumstances. These limitations are offset by a number of strengths. The overall study sample was sociodemographically diverse, enhancing the external validity of our findings. Additionally, although our primary analytic sample was relatively small, the sensitivity analysis conducted in a larger subset of our sample confirmed all of the associations observed in the smaller sample, supporting the robustness of our findings. To summarize, we explored whether and how both perceptions of uncertainty about COVID-19 and individual, trait-level differences in uncertainty tolerance might affect trust in public health information and vaccine hesitancy—two important outcomes in efforts to control the COVID pandemic. We found that overall perceptions of uncertainty about COVID-19 were not related to trust or vaccine hesitancy, but that uncertainty is linked to lower trust among individuals who are less tolerant of uncertainty. Additionally, individual differences in tolerance of uncertainty were associated with vaccine hesitancy in nuanced ways. These results suggest that uncertainty tolerance might play a critical role in moderating the public’s responses to scientific uncertainty about COVID-19, and endorse the value of further research to better understand its mechanisms. Our findings also suggest the need to better account for trait-level differences in uncertainty tolerance among individuals in the design of public health interventions and communication strategies aimed at promoting vaccination uptake and other risk-reducing measures for COVID-19 and other public health threats.
Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 22 KB)
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Effects of Self-focused Augmented Reality on Health Perceptions During the COVID-19 Pandemic: A Web-Based Between-Subject Experiment | 3593cb83-2e5a-43fa-afea-d21350aaf441 | 8244728 | Health Communication[mh] | Overview With self-focused augmented reality (AR) usage increasing in recent years , the utilization of this technology has a potential in addressing health communication and behavior interventions challenges. Whereas AR technology layers digital content onto the real world , self-focused AR visually augments the self, layering digital content onto the self. One technology enabling self-focused AR is video filters, which superimpose computer-generated content onto a user using their web or smartphone camera (eg, Snapchat Lenses and AR effects on Instagram ). For example, Snapchat’s Time Machine AR lens morphs a user’s reflection to display what the user might look like at different ages. Smart mirrors, another self-focused AR technology, combine digital screens (eg, an LCD [liquid-crystal display] monitor) with semitransparent glass. A smart mirror looks and functions similarly to a traditional mirror but with digital content displayed in the foreground . Real-world applications of smart mirrors are on display in vehicles , dressing rooms , and home gyms . For example, the MIRROR home gym displays fitness instruction layered on top of the user’s reflection. Both AR video filters and smart mirrors provide new opportunities for displaying health behavior communication to the public. In response to the recent increase in self-focused AR usage , we investigated the potential impact of self-focused AR within the health domain. For behavior change researchers, the effect of layering health threats along with mitigative behaviors and their results on top of a user may be of particular interest. For example, would using AR to layer a set of very healthy teeth due to good oral hygiene on dental patients impact their behavior? Could using AR in rearview mirrors to overlay scratches and bruises on top of drivers and riders encourage seat belt usage? We now have self-focused AR technologies in the hands of millions, presenting the opportunity to visually show individuals the impact of their decisions before they make them. Research in psychology suggests that heightening self-focused attention (manipulated by using a mirror or video camera) has implications for perception, affective experiences (emotions, feelings, and moods), and behavior . Objective self-awareness occurs when an individual places attention on themself, viewing themself as a social object. Objective self-awareness theory posits that self-focused attention heightens the awareness of the gap between one’s perceived “real self” and “ideal self,” resulting in negative affect. For example, if one desires good health and believes that exercise is vital to maintain one’s health and yet does not exercise, heightening objective self-awareness will likely result in negative emotions. The increased negative affect resulting from the awareness of discrepancies leads to either (1) the avoidance of self-focused attention and the discrepancy or (2) actions to reduce the discrepancy . This theory suggests that self-focused AR might impact behavior. We investigated which perceptions could be involved when individuals experience self-focused AR within a health context. Research prototypes have explored self-focus and self-focused AR technologies for health behavior change. However, few have investigated how the design of interventions that aim to increase self-focused attention might impact health perceptions and emotions. Similarly, prior studies did not consider the potential of combining self-focused AR with vicarious reinforcement, that is, reinforcement from observing others’ behavior and the results of those actions. This paper draws upon insights from objective self-awareness theory and social cognitive theory to inform hypotheses about the relationships between predictors of health behavior change and self-focused AR. We present findings from an online experiment on the impact of combining self-focused AR with vicarious reinforcement, visualizing the cause and effect of risk-mitigating behavior layered onto one’s reflection. Our study took place during the COVID-19 pandemic, focusing on hand hygiene behavior as an effective measure against pathogen transmission . We discuss the implications of the results in light of the public health emergency, addressing the following research question: how does reinforcement in self-focused AR impact health perceptions during a pandemic? Background Various health behavior change models highlight the roles of predictors of intentions such as risk perceptions (perceived threat severity and threat susceptibility) and outcome expectancy. Drawing on objective self-awareness theory and social cognitive theory, we postulated that health behavior change–themed self-focused AR could impact these predictors of intention. Research suggests that self-focused attention can result in action consistency with behavioral standards . Objective self-awareness theory posits that self-focused attention will result in negative affect through the increased awareness of contradicting beliefs about one’s self or discrepancies between belief and behavior . If negative affect is experienced, and one does not avoid the self-focused attention, they will attempt to reduce the discrepancy to reduce the negative affect, such as by changing their behavior. To further illustrate this, recall the individual from the example earlier who values their health and believes that exercise is vital for their health but does not exercise. According to objective self-awareness theory, an increase in self-focused attention would result in an attempt to reduce the discrepancy, which may result in exercise behavior. We propose that when increasing self-focused attention in the context of a health threat, especially during a pandemic, that the negative affect experienced will include fear and will increase to levels higher than if self-focused attention was not activated. Research suggests that fear may play a large role in health behavior, especially during public health emergencies such as the current pandemic. Harper et al found COVID-19 fear scores to be a positive predictor of behavior change. Fear may also impact behavior as it relates to risk perception. Risk perception, an individual’s perceived susceptibility to or severity of a threat, is included in many health behavior change theories . Li found perceived threat (measured by averaging threat susceptibility and severity) to be a positive predictor of fear. Affective factors are believed to play a role in the formation of risk perception . While risk perceptions can increase fear, fear has also been found to induce higher risk perceptions . Self-focused AR content layering onto the body increases the sense of spatial presence (ie, the object “being there”), potentially heightening fear if the object is threatening. Due to the combination of self-focus and spatial presence, we suggest that health threat–related self-focused AR may impact levels of fear, perceived threat severity, and perceived threat susceptibility. It is important to consider the potential negative impact of heightening fear and risk perceptions. While Harper et al found increased fear to be associated with higher behavioral adherence, they also found fear to be correlated with decreased physical and environmental quality of life and warned about mental health implications. Fear can also have an adverse effect on behavior. The Extended Parallel Process Model (EPPM) outlines the importance of a balance of fear and efficacy for health communication campaigns to be effective. While fear can be a motivator for behavior, where the fear/efficacy balance is disrupted, individuals may use cognitive defense mechanisms instead of behavior as a means of fear control. In this case, not only would the behavior change method be ineffective, but it could result in the adverse effect of prompting the development of these defense mechanisms. Based on the EPPM, Li tested a model for protective behaviors during a public health emergency with a study during the Ebola outbreak of 2014. Fear controls measured included negative reactance to messages, message minimizing, and defensive avoidance. Li found perceived threat to have a significant effect on fear and fear controls but did not find self-efficacy to be a successful moderator of that relationship. This suggests that although fear may be an effective strategy to encourage health behavior adherence, certain levels of fear may lead individuals to minimize health behavior messaging to control their fear instead of engaging in behavior change. We take these findings and the EPPM into consideration, as our study directly layers a health threat onto participants, which could result in excessive levels of fear triggering adverse fear control mechanisms. We expected self-focused AR, in the form of AR video filters, to heighten both fear and fear control mechanisms when displaying a health threat. Our study investigated the impact of vicarious reinforcement outcome expectancy when combined with self-focused AR. Research suggests that outcome expectancy mediates the impact of self-focused attention on behavior. For someone who has been made aware of a discrepancy between “actual state” and “desired state,” if they don’t believe a suggested behavior change will result in the “desire state,” they are more likely to change the “desired state” . When the “desired state” is health related, this can have adverse implications. Outcome expectancy can be impacted by experiencing vicarious reinforcement. Vicarious reinforcement occurs when a reinforcing effect for an individual takes place by observing others’ behavior and the results of their actions . Bandura et al found that children who were exposed to media displaying aggressive behavior that was rewarded showed more imitative aggressive behavior than those who saw aggressive behavior that was punished. Bandura’s social cognitive theory details how behaviors can be formed by observing a model engage in a behavior. Bandura suggests that due to limited contact with physical and social environments, people rely largely on vicarious experiences to form their idea of reality. In our study, vicarious reinforcement consists of visual representations of pathogens (ie, germs), which are made visible on avatar hands. As a hand hygiene animation plays, covering all the steps of proper handwashing, these pathogens disappear from the avatar’s hands. We predicted that this experience would heighten one’s perception of outcome expectancy. Given that health behavior models, such as The Health Action Process Approach attribute outcome expectancies to the formation of intention, we found this valuable to investigate in our study. We proposed that the vicarious experience described above would directly affect outcome expectancy. Related Work While studies combining self-focused attention and vicarious reinforcement have yet to see much direct utilization in human-computer interaction research, a few studies on health smart mirrors , self-representation , and spatial presence suggest an impact of self-focused attention on perceptions and behavior. While applications of smart mirrors for health care are limited, exploratory research prototypes have shown their potential to detect emotional states, monitor physiological parameters, and encourage behavior change. The Wize Mirror encouraged users to improve their lifestyle to mitigate cardiometabolic risk assessed by tracking physical face signs )eg, skin color, subcutaneous fat, facial expressions). Medical Mirror utilized computer vision and advanced signal processing within a smart mirror design to encourage people to keep track of their vital signs regularly. Fit Mirror increased user’s motivation, happiness, and fitness for the day by integrating exercising and challenging others into their morning routine. Although the rise of smart mirrors has resulted in studies exploring the use of these devices in health care, there is a lack of research investigating how self-focus specifically plays a role in influencing health behavior change. The studies mentioned above lack a control condition in which all design features are present except the mirror to study the direct impact of seeing one’s self-reflection. A recent study by Jung et al used a projector and a mirror to show participants their bodies with x-ray visualization of smoking lungs. A separate condition displayed the same content but on a mannequin. Both conditions were compared to a control, which displayed the information on a screen in 2D. The researchers found that spatial augmented reality increases spatial presence, the perceptual illusion that the real world and the mediated world are “equally present.” In addition, they found that higher levels of spatial presence were associated with a negative emotional change toward cigarettes and cigarette cessation campaign engagement intention. These findings support the idea that displaying the consequences of health behaviors on top of the user’s own body can impact behavior change constructs regarding emotions and intention. Our study aimed to contribute to this line of research by exploring additional behavior change constructs. One notable finding from Jung et al was that the mannequin condition also resulted in higher levels of spatial presence. However, an analysis comparing emotions and intentions reported for those in the mannequin condition compared to the control was not reported. Gaining a better understanding of how objects that can serve as external self-representations, such as avatars and mannequins, could help develop more feasible design interventions when mapping AR elements directly on the body would be complex. Yee and Bailenson found that self-representations can help form our behaviors, even when this representation is digital, such as in the case of avatars. Yee and Bailenson call this the Proteus effect and provided support from two experiments. In study one, participants were provided with an avatar that was previously rated as high, medium, or low on an attractiveness scale. They were asked to interact with another character (in a virtual reality environment) after looking at themselves in a mirror. Those in the high attractiveness condition disclosed more information and moved closer to the other character. The second experiment, testing avatar height in an ultimatum game, found that those in the tall condition were more likely to offer an unfair split. Those in the short condition were more likely to accept an unfair split. These findings suggest that augmentations to self-representation, as an avatar, may impact one’s behavior. Fox and Bailenson studied whether vicarious reinforcement with a user’s avatar had an effect on physical exercise. Seeing one’s avatar benefit from exercise behavior and experience consequences from not engaging in the behavior encouraged the observer to engage in the behavior. These results suggest that vicarious reinforcement using avatars may be effective. Based on the studies mentioned above, we expected that the display of health threats on an avatar representation of the self in an AR environment will impact levels of threat severity, susceptibility, fear, and message minimization. Our study expands on existing research by investigating how health behavior self-focused AR may impact specific predictors of behavioral intentions and what negative implications may exist in regard to fear control responses. This Study In our research, we examined the impact of self-focused AR and vicarious reinforcement on perception and emotion as it relates to hand washing health beliefs and behavioral intentions. Below, we present our hypotheses: H1: The combination of self-focused AR and vicarious reinforcement will result in higher levels of perceived positive outcome expectancy, perceived threat severity and susceptibility, fear, and message minimization when compared to a control. H2: Using avatar representations in self-focused AR with vicarious reinforcement will result in higher levels of threat severity, susceptibility, fear, and message minimization compared to a control.
With self-focused augmented reality (AR) usage increasing in recent years , the utilization of this technology has a potential in addressing health communication and behavior interventions challenges. Whereas AR technology layers digital content onto the real world , self-focused AR visually augments the self, layering digital content onto the self. One technology enabling self-focused AR is video filters, which superimpose computer-generated content onto a user using their web or smartphone camera (eg, Snapchat Lenses and AR effects on Instagram ). For example, Snapchat’s Time Machine AR lens morphs a user’s reflection to display what the user might look like at different ages. Smart mirrors, another self-focused AR technology, combine digital screens (eg, an LCD [liquid-crystal display] monitor) with semitransparent glass. A smart mirror looks and functions similarly to a traditional mirror but with digital content displayed in the foreground . Real-world applications of smart mirrors are on display in vehicles , dressing rooms , and home gyms . For example, the MIRROR home gym displays fitness instruction layered on top of the user’s reflection. Both AR video filters and smart mirrors provide new opportunities for displaying health behavior communication to the public. In response to the recent increase in self-focused AR usage , we investigated the potential impact of self-focused AR within the health domain. For behavior change researchers, the effect of layering health threats along with mitigative behaviors and their results on top of a user may be of particular interest. For example, would using AR to layer a set of very healthy teeth due to good oral hygiene on dental patients impact their behavior? Could using AR in rearview mirrors to overlay scratches and bruises on top of drivers and riders encourage seat belt usage? We now have self-focused AR technologies in the hands of millions, presenting the opportunity to visually show individuals the impact of their decisions before they make them. Research in psychology suggests that heightening self-focused attention (manipulated by using a mirror or video camera) has implications for perception, affective experiences (emotions, feelings, and moods), and behavior . Objective self-awareness occurs when an individual places attention on themself, viewing themself as a social object. Objective self-awareness theory posits that self-focused attention heightens the awareness of the gap between one’s perceived “real self” and “ideal self,” resulting in negative affect. For example, if one desires good health and believes that exercise is vital to maintain one’s health and yet does not exercise, heightening objective self-awareness will likely result in negative emotions. The increased negative affect resulting from the awareness of discrepancies leads to either (1) the avoidance of self-focused attention and the discrepancy or (2) actions to reduce the discrepancy . This theory suggests that self-focused AR might impact behavior. We investigated which perceptions could be involved when individuals experience self-focused AR within a health context. Research prototypes have explored self-focus and self-focused AR technologies for health behavior change. However, few have investigated how the design of interventions that aim to increase self-focused attention might impact health perceptions and emotions. Similarly, prior studies did not consider the potential of combining self-focused AR with vicarious reinforcement, that is, reinforcement from observing others’ behavior and the results of those actions. This paper draws upon insights from objective self-awareness theory and social cognitive theory to inform hypotheses about the relationships between predictors of health behavior change and self-focused AR. We present findings from an online experiment on the impact of combining self-focused AR with vicarious reinforcement, visualizing the cause and effect of risk-mitigating behavior layered onto one’s reflection. Our study took place during the COVID-19 pandemic, focusing on hand hygiene behavior as an effective measure against pathogen transmission . We discuss the implications of the results in light of the public health emergency, addressing the following research question: how does reinforcement in self-focused AR impact health perceptions during a pandemic?
Various health behavior change models highlight the roles of predictors of intentions such as risk perceptions (perceived threat severity and threat susceptibility) and outcome expectancy. Drawing on objective self-awareness theory and social cognitive theory, we postulated that health behavior change–themed self-focused AR could impact these predictors of intention. Research suggests that self-focused attention can result in action consistency with behavioral standards . Objective self-awareness theory posits that self-focused attention will result in negative affect through the increased awareness of contradicting beliefs about one’s self or discrepancies between belief and behavior . If negative affect is experienced, and one does not avoid the self-focused attention, they will attempt to reduce the discrepancy to reduce the negative affect, such as by changing their behavior. To further illustrate this, recall the individual from the example earlier who values their health and believes that exercise is vital for their health but does not exercise. According to objective self-awareness theory, an increase in self-focused attention would result in an attempt to reduce the discrepancy, which may result in exercise behavior. We propose that when increasing self-focused attention in the context of a health threat, especially during a pandemic, that the negative affect experienced will include fear and will increase to levels higher than if self-focused attention was not activated. Research suggests that fear may play a large role in health behavior, especially during public health emergencies such as the current pandemic. Harper et al found COVID-19 fear scores to be a positive predictor of behavior change. Fear may also impact behavior as it relates to risk perception. Risk perception, an individual’s perceived susceptibility to or severity of a threat, is included in many health behavior change theories . Li found perceived threat (measured by averaging threat susceptibility and severity) to be a positive predictor of fear. Affective factors are believed to play a role in the formation of risk perception . While risk perceptions can increase fear, fear has also been found to induce higher risk perceptions . Self-focused AR content layering onto the body increases the sense of spatial presence (ie, the object “being there”), potentially heightening fear if the object is threatening. Due to the combination of self-focus and spatial presence, we suggest that health threat–related self-focused AR may impact levels of fear, perceived threat severity, and perceived threat susceptibility. It is important to consider the potential negative impact of heightening fear and risk perceptions. While Harper et al found increased fear to be associated with higher behavioral adherence, they also found fear to be correlated with decreased physical and environmental quality of life and warned about mental health implications. Fear can also have an adverse effect on behavior. The Extended Parallel Process Model (EPPM) outlines the importance of a balance of fear and efficacy for health communication campaigns to be effective. While fear can be a motivator for behavior, where the fear/efficacy balance is disrupted, individuals may use cognitive defense mechanisms instead of behavior as a means of fear control. In this case, not only would the behavior change method be ineffective, but it could result in the adverse effect of prompting the development of these defense mechanisms. Based on the EPPM, Li tested a model for protective behaviors during a public health emergency with a study during the Ebola outbreak of 2014. Fear controls measured included negative reactance to messages, message minimizing, and defensive avoidance. Li found perceived threat to have a significant effect on fear and fear controls but did not find self-efficacy to be a successful moderator of that relationship. This suggests that although fear may be an effective strategy to encourage health behavior adherence, certain levels of fear may lead individuals to minimize health behavior messaging to control their fear instead of engaging in behavior change. We take these findings and the EPPM into consideration, as our study directly layers a health threat onto participants, which could result in excessive levels of fear triggering adverse fear control mechanisms. We expected self-focused AR, in the form of AR video filters, to heighten both fear and fear control mechanisms when displaying a health threat. Our study investigated the impact of vicarious reinforcement outcome expectancy when combined with self-focused AR. Research suggests that outcome expectancy mediates the impact of self-focused attention on behavior. For someone who has been made aware of a discrepancy between “actual state” and “desired state,” if they don’t believe a suggested behavior change will result in the “desire state,” they are more likely to change the “desired state” . When the “desired state” is health related, this can have adverse implications. Outcome expectancy can be impacted by experiencing vicarious reinforcement. Vicarious reinforcement occurs when a reinforcing effect for an individual takes place by observing others’ behavior and the results of their actions . Bandura et al found that children who were exposed to media displaying aggressive behavior that was rewarded showed more imitative aggressive behavior than those who saw aggressive behavior that was punished. Bandura’s social cognitive theory details how behaviors can be formed by observing a model engage in a behavior. Bandura suggests that due to limited contact with physical and social environments, people rely largely on vicarious experiences to form their idea of reality. In our study, vicarious reinforcement consists of visual representations of pathogens (ie, germs), which are made visible on avatar hands. As a hand hygiene animation plays, covering all the steps of proper handwashing, these pathogens disappear from the avatar’s hands. We predicted that this experience would heighten one’s perception of outcome expectancy. Given that health behavior models, such as The Health Action Process Approach attribute outcome expectancies to the formation of intention, we found this valuable to investigate in our study. We proposed that the vicarious experience described above would directly affect outcome expectancy.
While studies combining self-focused attention and vicarious reinforcement have yet to see much direct utilization in human-computer interaction research, a few studies on health smart mirrors , self-representation , and spatial presence suggest an impact of self-focused attention on perceptions and behavior. While applications of smart mirrors for health care are limited, exploratory research prototypes have shown their potential to detect emotional states, monitor physiological parameters, and encourage behavior change. The Wize Mirror encouraged users to improve their lifestyle to mitigate cardiometabolic risk assessed by tracking physical face signs )eg, skin color, subcutaneous fat, facial expressions). Medical Mirror utilized computer vision and advanced signal processing within a smart mirror design to encourage people to keep track of their vital signs regularly. Fit Mirror increased user’s motivation, happiness, and fitness for the day by integrating exercising and challenging others into their morning routine. Although the rise of smart mirrors has resulted in studies exploring the use of these devices in health care, there is a lack of research investigating how self-focus specifically plays a role in influencing health behavior change. The studies mentioned above lack a control condition in which all design features are present except the mirror to study the direct impact of seeing one’s self-reflection. A recent study by Jung et al used a projector and a mirror to show participants their bodies with x-ray visualization of smoking lungs. A separate condition displayed the same content but on a mannequin. Both conditions were compared to a control, which displayed the information on a screen in 2D. The researchers found that spatial augmented reality increases spatial presence, the perceptual illusion that the real world and the mediated world are “equally present.” In addition, they found that higher levels of spatial presence were associated with a negative emotional change toward cigarettes and cigarette cessation campaign engagement intention. These findings support the idea that displaying the consequences of health behaviors on top of the user’s own body can impact behavior change constructs regarding emotions and intention. Our study aimed to contribute to this line of research by exploring additional behavior change constructs. One notable finding from Jung et al was that the mannequin condition also resulted in higher levels of spatial presence. However, an analysis comparing emotions and intentions reported for those in the mannequin condition compared to the control was not reported. Gaining a better understanding of how objects that can serve as external self-representations, such as avatars and mannequins, could help develop more feasible design interventions when mapping AR elements directly on the body would be complex. Yee and Bailenson found that self-representations can help form our behaviors, even when this representation is digital, such as in the case of avatars. Yee and Bailenson call this the Proteus effect and provided support from two experiments. In study one, participants were provided with an avatar that was previously rated as high, medium, or low on an attractiveness scale. They were asked to interact with another character (in a virtual reality environment) after looking at themselves in a mirror. Those in the high attractiveness condition disclosed more information and moved closer to the other character. The second experiment, testing avatar height in an ultimatum game, found that those in the tall condition were more likely to offer an unfair split. Those in the short condition were more likely to accept an unfair split. These findings suggest that augmentations to self-representation, as an avatar, may impact one’s behavior. Fox and Bailenson studied whether vicarious reinforcement with a user’s avatar had an effect on physical exercise. Seeing one’s avatar benefit from exercise behavior and experience consequences from not engaging in the behavior encouraged the observer to engage in the behavior. These results suggest that vicarious reinforcement using avatars may be effective. Based on the studies mentioned above, we expected that the display of health threats on an avatar representation of the self in an AR environment will impact levels of threat severity, susceptibility, fear, and message minimization. Our study expands on existing research by investigating how health behavior self-focused AR may impact specific predictors of behavioral intentions and what negative implications may exist in regard to fear control responses.
In our research, we examined the impact of self-focused AR and vicarious reinforcement on perception and emotion as it relates to hand washing health beliefs and behavioral intentions. Below, we present our hypotheses: H1: The combination of self-focused AR and vicarious reinforcement will result in higher levels of perceived positive outcome expectancy, perceived threat severity and susceptibility, fear, and message minimization when compared to a control. H2: Using avatar representations in self-focused AR with vicarious reinforcement will result in higher levels of threat severity, susceptibility, fear, and message minimization compared to a control.
To study the effects of self-focused AR on behavioral intention and perception, we conducted an online experiment. Participants interacted with a web application that displayed health information regarding the coronavirus and a hand hygiene animation . Five conditions differed in their inclusion of self-focused attention and vicarious reinforcement. Experimental Conditions We conducted a between-subjects experiment where users interacted with a web application and then responded to an online questionnaire. The study followed a posttest-only control group design to avoid a testing threat to internal validity. The design of the web application differed depending on the intervention condition each participant was randomly allocated to. All five conditions displayed the same information about COVID-19, including how it is spread and preventative measures as described by the US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) . This information was followed by a hand hygiene animation, the display of which differed based on the participant’s assigned condition. Control Condition: No Self-focused AR or Vicarious Reinforcement In the control condition , we displayed an animation of a 12-step handwashing technique following standards outlined by the WHO , accompanied by captions to describe each movement (“Rub hands palm to palm”). Reinforcement In the reinforcement condition , the handwashing animation described in the control condition was accompanied by an additional animation showing germs disappearing from a pair of illustrated hands as the handwashing animation progressed. These animations were synced so that the appropriate areas of the hands displayed were affected based on the specific stage of the handwashing animation a viewer was watching. For example, the thumb cleaning animation segment was paired with germs disappearing from the thumbs. Self-focused AR The self-focus condition utilized the participant’s web camera to display their self-reflection, serving as the stimulus for self-focused attention. The handwashing animation was layered on top of the viewer’s reflection. This reflection was shown in real time and was created using the participant’s web camera. Self-focus AR × Reinforcement The self-focus AR × reinforcement condition visualized germs directly on the participant’s hands. Instructions at the beginning of the animation directed participants on where to place their hands. The handwashing animation was displayed in between their hands. As the animation progresses, the user saw the germs disappear from the reflection of their own hands. Avatar In the avatar condition , participants viewed an animation showing germs disappearing from a pair of illustrated hands layered on top of the user’s reflection. These are referred to as avatar hands, as they are meant to represent the user’s hands. The perspective displayed was that which is seen if the individual were to hold up their hands and look at them. Participants Participants were recruited via Prolific (a crowdsourcing platform ) and were compensated $2.50 for their time. Participation was limited to those residing in the United States, who spoke fluent English, and were ≥18 years of age. Pilot testing revealed technical challenges that could interfere with the study, mainly involving web camera use verification. To address this, prior to being recruited for the study, Prolific members were invited to a prescreener that verified (1) their access to an acceptable browser for the study (Safari, Chrome, and Firefox) and (2) that camera permissions worked with their technical setup. Prolific IDs for those who passed the screener were collected, and access to the main study was restricted to those IDs. Procedure The experiment took place between August 6-21, 2020. In all five conditions, after receiving consent, we described the experiment as a study on health information presentation and provided instructions to review the information given carefully. In the three self-focused-AR–based conditions, we displayed information on how to set up the web camera for the study. All conditions provided information about COVID-19. Details focused on how the virus is spread and preventative measures as described by the CDC . Participants next viewed an animation detailing the steps of proper hand hygiene as described by the WHO . This was followed by a questionnaire to collect demographic information and measure health knowledge and perception, which concluded the study. The questionnaire included questions to check whether the participant is paying attention. Three multiple-choice questions asked the user about the information displayed in the study (eg, What is a recommended preventative measure to reduce the spread of the coronavirus?). To validate that self-focused AR interventions were delivered correctly using the camera, participants in these conditions were informed, prior to the study, that screenshots would be collected randomly throughout the animation. The screenshots were reviewed to ensure that participant’s reflections were displayed to them and that those in the self-focus AR × reinforcement condition had their hands within the view as instructed. Only those who followed the instructions, verified by screenshots, were included in the final data set. Variables and Measures We collected measures of self-reported health beliefs, behavioral intention, and self-reported perceptions of COVID-19 , along with demographic data. These measures were adapted from Schwarzer and Li . Items in this study were all measured on a 7-point Likert-type scale ranging from 1 (strongly disagree) to 7 (strongly agree). Although adapted from previous research, cross loading was a concern due to the rewording of items and the difference in factors present compared to the adapted questionnaires. For example, we added items to measure opinions about perceived threat severity and susceptibility of family and friends. To examine the validity and reliability of our measures, we conducted exploratory and confirmatory factor analysis using a split-sample approach, with one half to develop a model and the other half to validate. Factors loaded as expected . Data on demographics and the COVID-19 risk of a severe illness of loved ones were collected. Participants were asked to report their age, gender, and the state in which they currently reside. In addition, they were asked to report if they have a family member or friend who is at high risk of severe illness if they are infected with COVID-19. It was noted that one is considered high risk if they are ≥65 years and/or have underlying medical conditions. Statistical Analysis Analysis of our data using histograms and the Shapiro-Wilk test showed that the data were not normally distributed. Shapiro-Wilk P values ranged from 6.35e-08 (efficacy) to 1.927e-25 (intention). Therefore, hypothesis testing was conducted using the nonparametric Wilcoxon-Mann-Whitney test, comparing perceived threat severity, susceptibility, outcome expectancy, fear, and message minimization scores between intervention conditions. If condition pairs had the same distribution shape, medians were compared. If the shapes were different, the mean ranks were compared. Additionally, mediation models for dependent variables and design conditions with significant findings were tested. A bootstrapping method using PROCESS macro models 4 and 6 , 5000 bootstrap samples, and percentile bootstrap CIs were used. Significance was established at P <.05. Statistical analysis was performed using Python (Python Software Foundation) , and the pandas (Community) and SciPy (Enthought) libraries were used to conduct the Wilcoxon-Mann-Whitney test. SPSS software (IBM Corp) was resourced, in which the PROCESS macro was implemented to test mediation.
We conducted a between-subjects experiment where users interacted with a web application and then responded to an online questionnaire. The study followed a posttest-only control group design to avoid a testing threat to internal validity. The design of the web application differed depending on the intervention condition each participant was randomly allocated to. All five conditions displayed the same information about COVID-19, including how it is spread and preventative measures as described by the US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) . This information was followed by a hand hygiene animation, the display of which differed based on the participant’s assigned condition. Control Condition: No Self-focused AR or Vicarious Reinforcement In the control condition , we displayed an animation of a 12-step handwashing technique following standards outlined by the WHO , accompanied by captions to describe each movement (“Rub hands palm to palm”). Reinforcement In the reinforcement condition , the handwashing animation described in the control condition was accompanied by an additional animation showing germs disappearing from a pair of illustrated hands as the handwashing animation progressed. These animations were synced so that the appropriate areas of the hands displayed were affected based on the specific stage of the handwashing animation a viewer was watching. For example, the thumb cleaning animation segment was paired with germs disappearing from the thumbs. Self-focused AR The self-focus condition utilized the participant’s web camera to display their self-reflection, serving as the stimulus for self-focused attention. The handwashing animation was layered on top of the viewer’s reflection. This reflection was shown in real time and was created using the participant’s web camera. Self-focus AR × Reinforcement The self-focus AR × reinforcement condition visualized germs directly on the participant’s hands. Instructions at the beginning of the animation directed participants on where to place their hands. The handwashing animation was displayed in between their hands. As the animation progresses, the user saw the germs disappear from the reflection of their own hands. Avatar In the avatar condition , participants viewed an animation showing germs disappearing from a pair of illustrated hands layered on top of the user’s reflection. These are referred to as avatar hands, as they are meant to represent the user’s hands. The perspective displayed was that which is seen if the individual were to hold up their hands and look at them.
In the control condition , we displayed an animation of a 12-step handwashing technique following standards outlined by the WHO , accompanied by captions to describe each movement (“Rub hands palm to palm”).
In the reinforcement condition , the handwashing animation described in the control condition was accompanied by an additional animation showing germs disappearing from a pair of illustrated hands as the handwashing animation progressed. These animations were synced so that the appropriate areas of the hands displayed were affected based on the specific stage of the handwashing animation a viewer was watching. For example, the thumb cleaning animation segment was paired with germs disappearing from the thumbs.
The self-focus condition utilized the participant’s web camera to display their self-reflection, serving as the stimulus for self-focused attention. The handwashing animation was layered on top of the viewer’s reflection. This reflection was shown in real time and was created using the participant’s web camera.
The self-focus AR × reinforcement condition visualized germs directly on the participant’s hands. Instructions at the beginning of the animation directed participants on where to place their hands. The handwashing animation was displayed in between their hands. As the animation progresses, the user saw the germs disappear from the reflection of their own hands.
In the avatar condition , participants viewed an animation showing germs disappearing from a pair of illustrated hands layered on top of the user’s reflection. These are referred to as avatar hands, as they are meant to represent the user’s hands. The perspective displayed was that which is seen if the individual were to hold up their hands and look at them.
Participants were recruited via Prolific (a crowdsourcing platform ) and were compensated $2.50 for their time. Participation was limited to those residing in the United States, who spoke fluent English, and were ≥18 years of age. Pilot testing revealed technical challenges that could interfere with the study, mainly involving web camera use verification. To address this, prior to being recruited for the study, Prolific members were invited to a prescreener that verified (1) their access to an acceptable browser for the study (Safari, Chrome, and Firefox) and (2) that camera permissions worked with their technical setup. Prolific IDs for those who passed the screener were collected, and access to the main study was restricted to those IDs.
The experiment took place between August 6-21, 2020. In all five conditions, after receiving consent, we described the experiment as a study on health information presentation and provided instructions to review the information given carefully. In the three self-focused-AR–based conditions, we displayed information on how to set up the web camera for the study. All conditions provided information about COVID-19. Details focused on how the virus is spread and preventative measures as described by the CDC . Participants next viewed an animation detailing the steps of proper hand hygiene as described by the WHO . This was followed by a questionnaire to collect demographic information and measure health knowledge and perception, which concluded the study. The questionnaire included questions to check whether the participant is paying attention. Three multiple-choice questions asked the user about the information displayed in the study (eg, What is a recommended preventative measure to reduce the spread of the coronavirus?). To validate that self-focused AR interventions were delivered correctly using the camera, participants in these conditions were informed, prior to the study, that screenshots would be collected randomly throughout the animation. The screenshots were reviewed to ensure that participant’s reflections were displayed to them and that those in the self-focus AR × reinforcement condition had their hands within the view as instructed. Only those who followed the instructions, verified by screenshots, were included in the final data set.
We collected measures of self-reported health beliefs, behavioral intention, and self-reported perceptions of COVID-19 , along with demographic data. These measures were adapted from Schwarzer and Li . Items in this study were all measured on a 7-point Likert-type scale ranging from 1 (strongly disagree) to 7 (strongly agree). Although adapted from previous research, cross loading was a concern due to the rewording of items and the difference in factors present compared to the adapted questionnaires. For example, we added items to measure opinions about perceived threat severity and susceptibility of family and friends. To examine the validity and reliability of our measures, we conducted exploratory and confirmatory factor analysis using a split-sample approach, with one half to develop a model and the other half to validate. Factors loaded as expected . Data on demographics and the COVID-19 risk of a severe illness of loved ones were collected. Participants were asked to report their age, gender, and the state in which they currently reside. In addition, they were asked to report if they have a family member or friend who is at high risk of severe illness if they are infected with COVID-19. It was noted that one is considered high risk if they are ≥65 years and/or have underlying medical conditions.
Analysis of our data using histograms and the Shapiro-Wilk test showed that the data were not normally distributed. Shapiro-Wilk P values ranged from 6.35e-08 (efficacy) to 1.927e-25 (intention). Therefore, hypothesis testing was conducted using the nonparametric Wilcoxon-Mann-Whitney test, comparing perceived threat severity, susceptibility, outcome expectancy, fear, and message minimization scores between intervention conditions. If condition pairs had the same distribution shape, medians were compared. If the shapes were different, the mean ranks were compared. Additionally, mediation models for dependent variables and design conditions with significant findings were tested. A bootstrapping method using PROCESS macro models 4 and 6 , 5000 bootstrap samples, and percentile bootstrap CIs were used. Significance was established at P <.05. Statistical analysis was performed using Python (Python Software Foundation) , and the pandas (Community) and SciPy (Enthought) libraries were used to conduct the Wilcoxon-Mann-Whitney test. SPSS software (IBM Corp) was resourced, in which the PROCESS macro was implemented to test mediation.
Overview A total of 502 individuals participated in the study. Of this, 335 participants met the attention and screenshot verification checks (see Procedure section) and were included in the analysis. Of the 335 participants, 77 were randomly assigned to the control condition, 61 to the self-focused AR condition, 70 to the reinforcement condition, 63 to the self-focus AR × reinforcement condition, and 64 to the avatar condition. Although our study focused on predictors of behavioral intentions, we began with results pertaining to intention to provide context for further discussion. A significant difference between design conditions and the control was not found (self-focused AR: P =.42; reinforcement: P =.43; self-focus AR × reinforcement: P =.41; avatar: P =.43). Effects of Self-focused AR With Vicarious Reinforcement in Regards to Perceived Fear, Threat Severity, Threat Susceptibility, Outcome Expectancy, and Message Minimization (Hypothesis 1) Hypothesis 1 proposed that a combination of vicarious reinforcement and self-focused AR would result in higher levels of fear, perceived threat severity, susceptibility, outcome expectancy, and message minimization compared to the control group. Compared with participants in the control ( P =.43), message minimization scores of those in the self-focus AR × reinforcement condition were not significantly different. This was also the case for outcome expectancy ( P =.41) and fear ( P =.23) . However, perceived threat severity and susceptibility had significant findings. For perceived threat severity, median scores for the self-focus AR × reinforcement and control groups were 6.25 and 6.00, respectively ; the two groups’ distributions differed significantly (Mann-Whitney U =1983, P =.03). Regarding perceived threat susceptibility, median scores for the self-focus AR × reinforcement and control groups were 6.00 and 5.33 ; the two groups’ distributions differed significantly (Mann-Whitney U =1897.0, P =.01). Our results partially supported H1 regarding perceived threat severity and susceptibility; however, we did not find support for outcome expectancy, fear, and message minimization. Additional analysis revealed that the use of self-focused AR and vicarious reinforcement individually did not impact measured predictors of intention, except for in the case of fear. When compared with participants in the control condition, those in the reinforcement condition (without self-focused AR) did not have a significant difference in medians for outcome expectancy ( P =.48), threat severity ( P =.39), susceptibility ( P =.40), fear ( P =.10), and message minimization ( P =.47). Conversely, when compared to the control, those in the self-focused AR condition (without vicarious reinforcement) did not have a significant difference in medians for outcome expectancy ( P =.26), threat severity ( P =.21), susceptibility ( P =.45), and message minimization ( P =.39). Median scores for the self-focused AR and control groups were 5.0 and 5.3 ( U =1950.5, P =.04). A mediation model was used to test whether self-focus AR × reinforcement affects behavioral intention through perceived threat susceptibility and perceived threat severity . We found a significant indirect effect of self-focus AR × reinforcement on intention with perceived threat severity as the only mediator (b=.06, 95% CI 0.02-0.12, SE 0.02), but not with perceived threat susceptibility as the only mediator. In addition, a significant indirect effect of self-focus AR × reinforcement on intention was found when both perceived threat susceptibility and threat severity were included as serial mediators (b=.06, 95% CI 0.02-0.12, SE 0.03). These results indicate that although the self-focus AR × reinforcement condition does not directly affect intention in this study, its effect on threat susceptibility and threat severity results in an indirect effect on intention. A separate mediation model was used to investigate whether self-focus AR × reinforcement affects perceived threat severity through perceived threat susceptibility. A significant indirect effect of self-focus AR × reinforcement on perceived threat severity through perceived threat susceptibility was found (b=.26, 95% CI 0.08-0.45, SE 0.09). Although fear was not significantly different from the control, we investigated whether fear or message minimization impacted intentions for those in the self-focus AR × reinforcement condition. First, a mediation model was used to test whether self-focus AR × reinforcement affects perceived fear through perceived threat susceptibility and perceived threat severity . A significant indirect effect of self-focus AR × reinforcement on fear was found with perceived threat severity as the only mediator (b=.16, 95% CI 0.05-0.29, SE 0.06) and with perceived threat susceptibility as the only mediator (b=.13, 95% CI 0.04-0.22, SE 0.05). In addition, a significant indirect effect of self-focus AR × reinforcement on fear was found when both perceived threat susceptibility and threat severity were included as serial mediators (b=.15, 95% CI 0.05-0.28, SE 0.06). Next, a mediation model was used to test whether the self-focus AR × reinforcement condition affects behavioral intention through fear. A significant indirect effect of the self-focus AR × reinforcement condition on intention was found with fear as the mediator (b=.07, 95% CI 0.01-0.16, SE 0.04). An additional model tested whether the self-focus AR × reinforcement condition affects message minimization through fear, threat severity, or threat severity. A significant negative indirect effect of the self-focus AR × reinforcement condition on message minimization was found with severity as the mediator (b=–.07, 95% CI –0.16 to –0.008, SE 0.04. A negative serial mediation effect with susceptibility and severity was also found (b=–.07, 95% CI –0.16 to –0.008, SE 0.04). Effects of Using an Avatar on Outcome Expectancy, Fear, Threat Severity, Threat Susceptibility, and Message Minimization (Hypothesis 2) Hypothesis 2 proposed that combining vicarious reinforcement and self-focused AR while using an avatar would result in higher levels of positive outcome expectancies, fear, perceived threat severity, perceived threat susceptibility, and message minimization when compared to a control. Compared to the control, those in the avatar condition did not have significantly different levels of outcome expectancy ( P =.42), severity ( P =.49), susceptibility ( P =.15), fear ( P =.23), or message minimization ( P =.17). User Feedback At the end of the questionnaire, we asked participants to enter optional free-form text about the study. Themes among the responses included impact on knowledge, risk perception formation, and challenges with the self-focus AR × reinforcement condition. Several participants in the reinforcement condition provided generalized statements that the study was educational and helpful. The following comments from the self-focused AR group provided more details. I thought the handwashing animation together with the illustration of where dirt is cleaned from the hands was very informative. P1 Some participants learned a new technique or strategy: I learned some new handwashing techniques! (Particularly, locking your hands together by curling your fingers into each other to get the backs of the fingers). P2 I hadn’t thought about separately lathering and washing my thumbs. P3 Comments on personal concerns about risk indicate that future work measuring these variables may consider the time spent around others vs alone and personal risk: I answered questions knowing that my husband’s and my job allow us to work from home, which decreases our risk significantly, and that most of my family lives in a rural area, also less susceptible to infection. P4 I know that I take it way more seriously due to the cancer treatment drug I take than most of my friends and peers because if I get it, I am not strong enough to fight it off. I think that factors in way more than friends and family risk, at least for me personally. P5 Lastly, a few participants in the self-focus AR × reinforcement group expressed confusion about the design. P7, for example, expressed difficulty in positioning themselves on the screen. It was an interesting survey, but the instruction given for the hand part was kind of hard to complete because the outline of the hands and the picture did not match. However, I tried my best to make it work. P7 Found the movements in the video hard to follow along with, but I tried my best! P8 The handwashing directions confused me. At first, I didn’t understand that I wasn’t supposed to mimic the exact instructions. P9 The responses of P8 and P9 suggest that participants may have practiced along with the video animation. Practicing was not a requirement of participation but appeared to be a trend among those in this condition.
A total of 502 individuals participated in the study. Of this, 335 participants met the attention and screenshot verification checks (see Procedure section) and were included in the analysis. Of the 335 participants, 77 were randomly assigned to the control condition, 61 to the self-focused AR condition, 70 to the reinforcement condition, 63 to the self-focus AR × reinforcement condition, and 64 to the avatar condition. Although our study focused on predictors of behavioral intentions, we began with results pertaining to intention to provide context for further discussion. A significant difference between design conditions and the control was not found (self-focused AR: P =.42; reinforcement: P =.43; self-focus AR × reinforcement: P =.41; avatar: P =.43).
Hypothesis 1 proposed that a combination of vicarious reinforcement and self-focused AR would result in higher levels of fear, perceived threat severity, susceptibility, outcome expectancy, and message minimization compared to the control group. Compared with participants in the control ( P =.43), message minimization scores of those in the self-focus AR × reinforcement condition were not significantly different. This was also the case for outcome expectancy ( P =.41) and fear ( P =.23) . However, perceived threat severity and susceptibility had significant findings. For perceived threat severity, median scores for the self-focus AR × reinforcement and control groups were 6.25 and 6.00, respectively ; the two groups’ distributions differed significantly (Mann-Whitney U =1983, P =.03). Regarding perceived threat susceptibility, median scores for the self-focus AR × reinforcement and control groups were 6.00 and 5.33 ; the two groups’ distributions differed significantly (Mann-Whitney U =1897.0, P =.01). Our results partially supported H1 regarding perceived threat severity and susceptibility; however, we did not find support for outcome expectancy, fear, and message minimization. Additional analysis revealed that the use of self-focused AR and vicarious reinforcement individually did not impact measured predictors of intention, except for in the case of fear. When compared with participants in the control condition, those in the reinforcement condition (without self-focused AR) did not have a significant difference in medians for outcome expectancy ( P =.48), threat severity ( P =.39), susceptibility ( P =.40), fear ( P =.10), and message minimization ( P =.47). Conversely, when compared to the control, those in the self-focused AR condition (without vicarious reinforcement) did not have a significant difference in medians for outcome expectancy ( P =.26), threat severity ( P =.21), susceptibility ( P =.45), and message minimization ( P =.39). Median scores for the self-focused AR and control groups were 5.0 and 5.3 ( U =1950.5, P =.04). A mediation model was used to test whether self-focus AR × reinforcement affects behavioral intention through perceived threat susceptibility and perceived threat severity . We found a significant indirect effect of self-focus AR × reinforcement on intention with perceived threat severity as the only mediator (b=.06, 95% CI 0.02-0.12, SE 0.02), but not with perceived threat susceptibility as the only mediator. In addition, a significant indirect effect of self-focus AR × reinforcement on intention was found when both perceived threat susceptibility and threat severity were included as serial mediators (b=.06, 95% CI 0.02-0.12, SE 0.03). These results indicate that although the self-focus AR × reinforcement condition does not directly affect intention in this study, its effect on threat susceptibility and threat severity results in an indirect effect on intention. A separate mediation model was used to investigate whether self-focus AR × reinforcement affects perceived threat severity through perceived threat susceptibility. A significant indirect effect of self-focus AR × reinforcement on perceived threat severity through perceived threat susceptibility was found (b=.26, 95% CI 0.08-0.45, SE 0.09). Although fear was not significantly different from the control, we investigated whether fear or message minimization impacted intentions for those in the self-focus AR × reinforcement condition. First, a mediation model was used to test whether self-focus AR × reinforcement affects perceived fear through perceived threat susceptibility and perceived threat severity . A significant indirect effect of self-focus AR × reinforcement on fear was found with perceived threat severity as the only mediator (b=.16, 95% CI 0.05-0.29, SE 0.06) and with perceived threat susceptibility as the only mediator (b=.13, 95% CI 0.04-0.22, SE 0.05). In addition, a significant indirect effect of self-focus AR × reinforcement on fear was found when both perceived threat susceptibility and threat severity were included as serial mediators (b=.15, 95% CI 0.05-0.28, SE 0.06). Next, a mediation model was used to test whether the self-focus AR × reinforcement condition affects behavioral intention through fear. A significant indirect effect of the self-focus AR × reinforcement condition on intention was found with fear as the mediator (b=.07, 95% CI 0.01-0.16, SE 0.04). An additional model tested whether the self-focus AR × reinforcement condition affects message minimization through fear, threat severity, or threat severity. A significant negative indirect effect of the self-focus AR × reinforcement condition on message minimization was found with severity as the mediator (b=–.07, 95% CI –0.16 to –0.008, SE 0.04. A negative serial mediation effect with susceptibility and severity was also found (b=–.07, 95% CI –0.16 to –0.008, SE 0.04).
Hypothesis 2 proposed that combining vicarious reinforcement and self-focused AR while using an avatar would result in higher levels of positive outcome expectancies, fear, perceived threat severity, perceived threat susceptibility, and message minimization when compared to a control. Compared to the control, those in the avatar condition did not have significantly different levels of outcome expectancy ( P =.42), severity ( P =.49), susceptibility ( P =.15), fear ( P =.23), or message minimization ( P =.17).
At the end of the questionnaire, we asked participants to enter optional free-form text about the study. Themes among the responses included impact on knowledge, risk perception formation, and challenges with the self-focus AR × reinforcement condition. Several participants in the reinforcement condition provided generalized statements that the study was educational and helpful. The following comments from the self-focused AR group provided more details. I thought the handwashing animation together with the illustration of where dirt is cleaned from the hands was very informative. P1 Some participants learned a new technique or strategy: I learned some new handwashing techniques! (Particularly, locking your hands together by curling your fingers into each other to get the backs of the fingers). P2 I hadn’t thought about separately lathering and washing my thumbs. P3 Comments on personal concerns about risk indicate that future work measuring these variables may consider the time spent around others vs alone and personal risk: I answered questions knowing that my husband’s and my job allow us to work from home, which decreases our risk significantly, and that most of my family lives in a rural area, also less susceptible to infection. P4 I know that I take it way more seriously due to the cancer treatment drug I take than most of my friends and peers because if I get it, I am not strong enough to fight it off. I think that factors in way more than friends and family risk, at least for me personally. P5 Lastly, a few participants in the self-focus AR × reinforcement group expressed confusion about the design. P7, for example, expressed difficulty in positioning themselves on the screen. It was an interesting survey, but the instruction given for the hand part was kind of hard to complete because the outline of the hands and the picture did not match. However, I tried my best to make it work. P7 Found the movements in the video hard to follow along with, but I tried my best! P8 The handwashing directions confused me. At first, I didn’t understand that I wasn’t supposed to mimic the exact instructions. P9 The responses of P8 and P9 suggest that participants may have practiced along with the video animation. Practicing was not a requirement of participation but appeared to be a trend among those in this condition.
This study explored the impact of self-focus and vicarious reinforcement design interventions on psychological predictors of behavior change during the COVID-19 pandemic. Our results showed that combining self-focused AR with vicarious reinforcement increases perceived threat severity and threat susceptibility and could potentially impact behavioral intentions. Behavior Intention and Outcome Expectancy Our results did not show any direct effects on behavioral intention . Given the severity of COVID-19, we expected these findings to result from a ceiling effect, as similar studies on behavior messaging strategies have found during COVID-19 . A surprising finding was the lack of impact of the design interventions on outcome expectancy; however, this also appears to result from a ceiling effect , possibly resulting from the surge of news messaging surrounding hand hygiene’s role in mitigating risk during COVID-19. News segments showed the impact of hand hygiene using blacklights to simulate how hand washing reduces viruses’ presence, which may have helped combat any disbelief. Due to the ceiling effect, our findings require replication studies post–COVID-19 or studies that include intention and outcome expectancy measures less susceptible to the ceiling effect to investigate the impact of self-focused AR and vicarious reinforcement. Perceived Threat Severity, Threat Susceptibility, Fear, and Intention Our findings also revealed interesting relationships between the self-focus AR × reinforcement condition, perceived threat severity, threat susceptibility, fear, and intention. Mediation models showed the self-focus AR × reinforcement condition to positively affect intention and fear through increased perceived threat susceptibility and threat severity. We found self-focus AR × reinforcement to increase perceived threat severity through increased perceived susceptibility. Lastly, we found self-focus AR × reinforcement to indirectly affect intention with fear as the mediator. These results suggest that design strategies that layer a health threat directly on an individual’s reflection may increase one’s perceived threat susceptibility, threat, severity, fear, and indirectly behavioral intention. While such strategies might help meet behavior change design objectives, it is essential to note the potential consequences of designs that increase fear, especially in the context of a public health emergency. Based on the EPPM, Li tested a model for protective behaviors during a public health emergency with a study during the Ebola outbreak of 2014. The study found perceived threat to have a significant effect on fear and fear controls. Our study partially supports these findings, indicating an impact of threat severity on fear but not a significant positive effect of fear on message minimization (a fear control mechanism). The danger/fear control responses and the impact of self-focused AR likely varies for each health behavior context, as levels of fear will be different for each health threat. While our study did not show adverse effects, researchers and designers should still use caution if utilizing similar design techniques to effect behavior change. More research is needed on the adverse effects of fear concerning triggering fear control mechanisms through design interventions. In addition, increased fear could have mental health implications. Harper et al found fear of COVID-19 to be a positive predictor of behavior change and fear to be correlated with decreased physical and environmental quality of life. Given our findings, designers must investigate the extent to which a design strategy that involves self-focused AR with a health threat increases fear. Independent Use of Self-focused Attention and Vicarious Reinforcement Self-focused AR and vicarious reinforcement embedded as features independently (versus combined) did not show a significant result on any of the tested predictors of behavior change except fear. We provide a few possible explanations for these results. First, regarding mirror self-focus, threat severity, and susceptibility, there may have been too large of a time gap between when participants reviewed the health information provided and when they looked at their self-reflection. In the conditions combining the features, there was content on the screen, reminding participants of the threat at hand. Future work may account for this difference by providing text-based information over one’s reflection. Second, regarding vicarious reinforcement and outcome expectancy, indicates the presence of a ceiling effect. This study may need to be replicated for a different health threat or mitigating behavior that wouldn’t have as many positive, strong outcome expectancy beliefs. Limitations and Future Work It is important to note that this research took place during a long-term global public health emergency with restrictions on lifestyles that can take a while to get adjusted to. Health perceptions related to current circumstances are subject to change throughout the lifecycle of a pandemic. Our findings warrant replication studies that consider changes in severity, government mandates, social perceptions, and the availability and range of tools for risk mitigation (vaccines/medication, personal protective equipment, etc). The data used in this study are self-reported and susceptible to response biases, specifically social desirability bias. Due to the severity of the pandemic, government mandates, news coverage, and social discussions may have increased the pressure to respond in ways that align with social norms. Future work should aim to use methods to decrease the impact of this limitation. It is important to note that focusing on individual constructs may create an ineffective design system if the construct only works in combination with other constructs . This should be taken into consideration as self-focused AR is explored in the future, possibly adding measures for other behavior change constructs such as normative beliefs and social facilitation. While our study indicates that combining self-focused AR with vicarious reinforcement may affect health behavior change by influencing threat severity and susceptibility, we lack a data-driven explanation of why. Future work may benefit from the inclusion of quantitative measures for self-focused attention to compare with severity and susceptibility scores. Future work may lend itself to developing experimental methods to explore the extent to which self-monitoring, reflective thinking, self-evaluation, and emotion management naturally occur (or do not occur) when using self-focused AR. Conducting these experiments will provide deeper insights into how self-focused AR impacts the psychological mechanism related to behavior change and possibly inspire experiments on how the combination of self-focused attention and other design features could enhance this effect. According to user feedback, future work should also consider accounting for time spent around others vs alone and personal health when measuring perceived threat severity. Responses also indicate that when measuring perceptions about new messaging, participants should be instructed to respond based on their preferred news source to limit confusion related to the different opinions they hold for individual new sources. Two comments from the user feedback indicated that participants might have been actually practicing handwashing movements while viewing the animation. During the screenshot verification process, it was noted whether a participant was observed practicing along with the handwashing video. Practicing was not a requirement of participation and was not mentioned in any instructions provided to them. A total of 32 out of 63 participants were observed practicing along with the video in the self-focus AR × reinforcement condition. The self-focused AR and avatar conditions had 7 and 6 individuals observed practicing, respectively. These results cannot be used to make any claims due to the study’s technical setup. Those in the self-focus AR × reinforcement group were instructed to have their hands in view of the camera. Those in other conditions may have practiced off-camera. However, as practicing may affect behavior, this is another potential area for future research. Conclusion As self-focused AR technologies grow in popularity, it is important to understand how such experiences could impact perceptions, emotions, and behavioral intentions. Previous research has explored self-focused AR to varying degrees revealing a potential impact on health behavior. Our study expands upon this work by combining self-focused AR and vicarious reinforcement. Doing so helped to reveal insights about the impact of each feature on perceptions and emotions as they relate to behavior change. We found that displaying germs disappearing directly from the user’s self-reflection during a handwashing animation will result in higher scores for perceived threat severity and susceptibility when compared to the control or conditions that implemented self-reflection and a display of germs disappearing separately. Increased perceived severity and susceptibility were found to increase behavioral intention. These findings indicate that combining self-focused AR with vicarious reinforcement may be an effective strategy for health communication designers. However, we also voice concern about the possible adverse effects of heightening levels of fear as a design strategy. While our study did not show concerning results, prior research indicates that heightening fear as a health communication strategy can lead to defensive reactions (versus changing behavior) and can lower quality of life . We recommend that this be taken into consideration by designers whenever augmenting self-focused attention with a health threat, especially during a public health emergency, as fear may already be at concerningly high levels. Future research should further investigate the role of fear, perceived threat severity, and threat susceptibility when using self-focused AR in health contexts and design strategies for maintaining the well-being of the user while inspiring behavior change.
Our results did not show any direct effects on behavioral intention . Given the severity of COVID-19, we expected these findings to result from a ceiling effect, as similar studies on behavior messaging strategies have found during COVID-19 . A surprising finding was the lack of impact of the design interventions on outcome expectancy; however, this also appears to result from a ceiling effect , possibly resulting from the surge of news messaging surrounding hand hygiene’s role in mitigating risk during COVID-19. News segments showed the impact of hand hygiene using blacklights to simulate how hand washing reduces viruses’ presence, which may have helped combat any disbelief. Due to the ceiling effect, our findings require replication studies post–COVID-19 or studies that include intention and outcome expectancy measures less susceptible to the ceiling effect to investigate the impact of self-focused AR and vicarious reinforcement.
Our findings also revealed interesting relationships between the self-focus AR × reinforcement condition, perceived threat severity, threat susceptibility, fear, and intention. Mediation models showed the self-focus AR × reinforcement condition to positively affect intention and fear through increased perceived threat susceptibility and threat severity. We found self-focus AR × reinforcement to increase perceived threat severity through increased perceived susceptibility. Lastly, we found self-focus AR × reinforcement to indirectly affect intention with fear as the mediator. These results suggest that design strategies that layer a health threat directly on an individual’s reflection may increase one’s perceived threat susceptibility, threat, severity, fear, and indirectly behavioral intention. While such strategies might help meet behavior change design objectives, it is essential to note the potential consequences of designs that increase fear, especially in the context of a public health emergency. Based on the EPPM, Li tested a model for protective behaviors during a public health emergency with a study during the Ebola outbreak of 2014. The study found perceived threat to have a significant effect on fear and fear controls. Our study partially supports these findings, indicating an impact of threat severity on fear but not a significant positive effect of fear on message minimization (a fear control mechanism). The danger/fear control responses and the impact of self-focused AR likely varies for each health behavior context, as levels of fear will be different for each health threat. While our study did not show adverse effects, researchers and designers should still use caution if utilizing similar design techniques to effect behavior change. More research is needed on the adverse effects of fear concerning triggering fear control mechanisms through design interventions. In addition, increased fear could have mental health implications. Harper et al found fear of COVID-19 to be a positive predictor of behavior change and fear to be correlated with decreased physical and environmental quality of life. Given our findings, designers must investigate the extent to which a design strategy that involves self-focused AR with a health threat increases fear.
Self-focused AR and vicarious reinforcement embedded as features independently (versus combined) did not show a significant result on any of the tested predictors of behavior change except fear. We provide a few possible explanations for these results. First, regarding mirror self-focus, threat severity, and susceptibility, there may have been too large of a time gap between when participants reviewed the health information provided and when they looked at their self-reflection. In the conditions combining the features, there was content on the screen, reminding participants of the threat at hand. Future work may account for this difference by providing text-based information over one’s reflection. Second, regarding vicarious reinforcement and outcome expectancy, indicates the presence of a ceiling effect. This study may need to be replicated for a different health threat or mitigating behavior that wouldn’t have as many positive, strong outcome expectancy beliefs.
It is important to note that this research took place during a long-term global public health emergency with restrictions on lifestyles that can take a while to get adjusted to. Health perceptions related to current circumstances are subject to change throughout the lifecycle of a pandemic. Our findings warrant replication studies that consider changes in severity, government mandates, social perceptions, and the availability and range of tools for risk mitigation (vaccines/medication, personal protective equipment, etc). The data used in this study are self-reported and susceptible to response biases, specifically social desirability bias. Due to the severity of the pandemic, government mandates, news coverage, and social discussions may have increased the pressure to respond in ways that align with social norms. Future work should aim to use methods to decrease the impact of this limitation. It is important to note that focusing on individual constructs may create an ineffective design system if the construct only works in combination with other constructs . This should be taken into consideration as self-focused AR is explored in the future, possibly adding measures for other behavior change constructs such as normative beliefs and social facilitation. While our study indicates that combining self-focused AR with vicarious reinforcement may affect health behavior change by influencing threat severity and susceptibility, we lack a data-driven explanation of why. Future work may benefit from the inclusion of quantitative measures for self-focused attention to compare with severity and susceptibility scores. Future work may lend itself to developing experimental methods to explore the extent to which self-monitoring, reflective thinking, self-evaluation, and emotion management naturally occur (or do not occur) when using self-focused AR. Conducting these experiments will provide deeper insights into how self-focused AR impacts the psychological mechanism related to behavior change and possibly inspire experiments on how the combination of self-focused attention and other design features could enhance this effect. According to user feedback, future work should also consider accounting for time spent around others vs alone and personal health when measuring perceived threat severity. Responses also indicate that when measuring perceptions about new messaging, participants should be instructed to respond based on their preferred news source to limit confusion related to the different opinions they hold for individual new sources. Two comments from the user feedback indicated that participants might have been actually practicing handwashing movements while viewing the animation. During the screenshot verification process, it was noted whether a participant was observed practicing along with the handwashing video. Practicing was not a requirement of participation and was not mentioned in any instructions provided to them. A total of 32 out of 63 participants were observed practicing along with the video in the self-focus AR × reinforcement condition. The self-focused AR and avatar conditions had 7 and 6 individuals observed practicing, respectively. These results cannot be used to make any claims due to the study’s technical setup. Those in the self-focus AR × reinforcement group were instructed to have their hands in view of the camera. Those in other conditions may have practiced off-camera. However, as practicing may affect behavior, this is another potential area for future research.
As self-focused AR technologies grow in popularity, it is important to understand how such experiences could impact perceptions, emotions, and behavioral intentions. Previous research has explored self-focused AR to varying degrees revealing a potential impact on health behavior. Our study expands upon this work by combining self-focused AR and vicarious reinforcement. Doing so helped to reveal insights about the impact of each feature on perceptions and emotions as they relate to behavior change. We found that displaying germs disappearing directly from the user’s self-reflection during a handwashing animation will result in higher scores for perceived threat severity and susceptibility when compared to the control or conditions that implemented self-reflection and a display of germs disappearing separately. Increased perceived severity and susceptibility were found to increase behavioral intention. These findings indicate that combining self-focused AR with vicarious reinforcement may be an effective strategy for health communication designers. However, we also voice concern about the possible adverse effects of heightening levels of fear as a design strategy. While our study did not show concerning results, prior research indicates that heightening fear as a health communication strategy can lead to defensive reactions (versus changing behavior) and can lower quality of life . We recommend that this be taken into consideration by designers whenever augmenting self-focused attention with a health threat, especially during a public health emergency, as fear may already be at concerningly high levels. Future research should further investigate the role of fear, perceived threat severity, and threat susceptibility when using self-focused AR in health contexts and design strategies for maintaining the well-being of the user while inspiring behavior change.
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3D bioprinting of complex tissues in vitro: state-of-the-art and future perspectives | 976ba5a4-3856-4a46-8bcd-7265c629b900 | 8850226 | Pharmacology[mh] | In vitro tissue models have greatly advanced our understanding of the pharmacological and toxicological processes of a wide range of treatments and chemicals (Davila et al. ). Such models are efficient, low-cost, and non-cruel recapitulations of native tissues, and their development has sped the discovery of various medications (Madorran et al. ), as well as the development of environmental pollution prevention and labor protection approaches. In vitro tissue models have evolved from simple two-dimensional (2D) monocultures into more advanced three-dimensional (3D) structures, such as organoids, dynamic culture systems, micro-tissues, organ-on-chip devices, and other combinations (Braun et al. ; Duval et al. ). Accurate recapitulation of native physiology, such as cell composition, biophysical and biochemical signaling, as well as microarchitecture, could result in greater substantive response when drawing correlations between in vitro and in vivo conditions (Lelièvre et al. ). 3D bioprinting has emerged as an intriguing approach for the production of complex in vitro models, by which means cells and/or their supporting scaffold are precisely deposited, localized, or joined in user-defined geometries and dimensions. With an ever-expanding range of available biomaterials (Yu et al. , ) and biocompatible processes (Ashammakhi et al. ), 3D bioprinting has aided in the tailored control over microarchitecture, extracellular matrix (ECM) construction, and cell deposition for the establishment of in vitro models, particularly the recapitulation of complex tissues (Ma et al. , ), and has resulted in significant accomplishments in moving the field forward in recent years. Here, we present a state-of-the-art review on the in vitro complex tissue model constructions based on 3D bioprinting. We begin with an overview of 3D printing techniques, biomaterials and their use in in vitro tissue construction, and then move on to discussing pioneering work in cancer, heart, liver, and muscle in vitro models for biological studies, drug screening, and toxicity investigations. In the concluding section, we also explore the applications, challenges, and future perspectives of 3D bioprinting technologies and tissue modeling.
3D bioprinting technology 3D bioprinting refers to a type of additive manufacturing, specifically a layer-by-layer fabrication technique that was originally born out of a need for rapid prototyping and has since enjoyed advancement into a fast, customizable fabrication method across many fields. 3D bioprinting technology allows for flexibility in both material choice and design paradigm—in the context of tissue engineering, the ability to incorporate biomaterials and cells inherently allows for 3D bioprinting. As 3D bioprinting becomes more ubiquitous, more research into bioprinting techniques has emerged, allowing for the fabrication of a wide range of biocompatible constructs, and cell-encapsulated tissues, and organ models. Inkjet-based A typical inkjet-based bio-printer is shown in Fig. A (Patel ). It dispenses droplets of low-viscosity bio-ink from a ‘printhead’ containing arrays of small nozzle apertures to form patterns and then stabilizes the structure by photo-crosslinking or thermal gelation (Yu et al. , ). Typical inkjet printer designs include: a bioink storage chamber(s), actuators to both guide bioink(s) to the nozzle and form the droplets, and stage/control systems for three-axial movement. There are three main categories of inkjet bioprinting methods: continuous-inkjet, droplet-on-demand, and electro-hydrodynamic jet bioprinting, all of which differ in their method of bioink droplet deployment control. The continuous-inkjet method extrudes streams of bioink droplets, which lack precise droplet control. The droplet-on-demand bioprinter improves droplet control by creating individual droplets at required times by pressurizing the bioink storage chamber, using thermal, piezoelectric, or electrostatic-based actuators. The electro-hydrodynamic jet bioprinter generates droplets by pulling the bio-ink through the nozzle instead of a pushing method with droplet-on-demand bioprinters (Derakhshanfar et al. ; Gudapati et al. ). In general, inkjet-based 3D bioprinting has the benefit of precise material deposition with a reasonable printing speed. The utilization of bioink droplets results in minimal material waste and low cost, with a high cell viability (except in thermal inkjet bioprinting). The bioink chamber and nozzle design also allow efficient material replacement and multi-material printing. However, a drawback to inkjet printing is that small nozzle apertures place a lower limit on cell densities in the bioinks—higher cell densities can cause nozzle clogging. Additionally, the low viscosity required for printing may result in limited mechanical strength in the final printed structure. Furthermore, the droplet form of the material causes a limited printing resolution (Gudapati et al. ; Mobaraki et al. ). Extrusion-based Extrusion-based bioprinting is another widely used bioprinting technology (Fig. B). Similar to inkjet printing, extrusion-based printing also extrudes material from a nozzle printhead, but instead of dispensing individual droplets, a typical extrusion printer extrudes a continuous flow of viscous bioink filament. The bioink viscosity used in extrusion bioprinting ranges from 30 to 60 kPa s (Derakhshanfar et al. ; Mobaraki et al. ). Higher-viscosity bioinks allow for the use of correspondingly higher viscous biomaterials and higher-density cell encapsulation, both of which are beneficial for tissue and organ fabrication. Extrusion-based bioprinter resolutions are limited by the size of the nozzle aperture and material characteristics, and also experience slower printing speeds due to the scanning nature of its printing movement. For both the extrusion-based and inkjet-based bioprinting, supporting structures are needed if creating overhanging 3D structures, which may introduce longer printing times as well as material/cell wastage. The overall fabrication time will vary based on the complexity of the 3D structure (Yu et al. , ). Laser polymerization-based Laser-based bioprinting methods feature the use of a precisely controlled, focused laser beam to effect patterned photopolymerization in a prepolymer solution. Among them, two-photon polymerization (TPP) bioprinting uses femtosecond laser pulses to achieve submicron level precise printing (Fig. C) (Claeyssens et al. ; Zhang and Chen ; Xing et al. ). The high precision afforded by TPP printing allows for the production of micro- and nano-scale tissue scaffolds and vasculatures. However, to ensure precise synchronization between the motion and laser pulses, the TPP printing speed is often limited, and thus requires long fabrication times for complex and/or large structures (Yu et al. , ). After printing, the un-polymerized residual solution will need to be removed to reveal the printed structure, which causes material waste; however, this material may function as a soft, supportive structure during the actual printing process, thus eliminates the need for designated supporting structures (Yu et al. , ). TPP printing allows for a variety of material viscosities, which is beneficial for the varied conditions of tissue and organ printing, but higher laser powers can cause thermal damage to cells, resulting in reduced cell viability (Derakhshanfar et al. ; Hopp ). Digital light processing (DLP)-based DLP-based bioprinting is an emerging photopolymerization-based bioprinting technique that addresses the primary limitations of previous bioprinting methods—speed and spatial resolution for tissue and organ fabrication. Compared to other methods that require point-by-point or line-by-line scanning to create an individual layer, DLP-based bioprinting drastically reduces the printing time by enabling the projection of an entire 2D design plane at once. A DLP printer’s core piece of hardware is a digital micro-mirror array device (DMD), a programmable micro-opto-electromechanical chip with an array of micro-mirrors—this can be used to spatially pattern an incoming light source, such as a 365 nm ultraviolet or 405 nm visible light, to photo-polymerize a vat of prepolymer solution (Fig. D). The XY resolution of the printed structure is defined by the projection from an individual micro-mirror on the DMD chip, which may be as low as 3–5 µm (and can vary based on intermediate optics and prepolymer characteristics) (Lu et al. ), with a typical layer-by-layer Z-resolution limited by the material refill process between subsequent layers (Yu et al. , ). The Chen group later developed a dynamic optical projection stereolithography (DOPsL) to continuously move the Z -axis during printing, resulting in smooth side walls for printed structures (Zhang et al. ), and applied this for pre-vascularized tissue printing (Zhu et al. ). Further additions to printing efficiency have been researched as well, to go beyond layer-by-layer into volumetric additive manufacturing, where the entire volume is printed at once. One approach is the holographic volumetric 3D fabrication system, which uses three orthogonal light beams with phase-only patterns to print the entire 3D structure in a single step (Shusteff et al. ). Another example is the computed axial lithography (CAL) technology, which utilized a DLP projector to illuminate their prepolymer vat from a single side, axially rotating the vat while modulating the projected light’s phase pattern (Kelly et al. ). Such volumetric additive manufacturing fabrication techniques are not limited in printing time by the Z -axis, but spatial resolution can suffer due to limitations in holographic precision. Recent efforts by Saha et al. to address these spatial resolution limitations utilize femtosecond projection two-photon lithography (FP-TPL), which combines the high precision afforded by TPP with the high-throughput nature of DLP-based printing, allowing simultaneous spatial and temporal focusing. Overall, DLP-based bioprinting enables rapid micro-scale bio-fabrication by plane-wise or even volume-wise printing with the use of a DMD. The high-throughput nature of the process is greatly beneficial for the fabrication of tissues and organs, taking into consideration the time-sensitive nature of live cells and tissues. Additionally, the micro-scale precision afforded by DLP-based printing is similarly beneficial for producing the fine features of native physiology, such as multi-tissue compartments and microvasculature. The relatively low power of the light sources and exposure times typically used also ensure cell viability (Ruskowitz and Deforest ). One drawback to DLP-based bioprinting is its pre-filling of a vet of bioink which if not used and recycled, would go to biological waste. Biomaterials in 3D printing Biomaterials form the basis of the matrix or substrate of the final printed structure, and are critical in the in vitro modeling of complex tissues—they provide crucial physical and chemical signals, and can have significant impacts on cell activities, such as adhesion, metabolism, proliferation, differentiation, and migration. For example, the stiffness of the extracellular matrix as a biophysical cue has been found to be deterministic in the viability, differentiation, and migration of a variety of cell types (Gasiorowski et al. ). Material porosity crucially aids in the transfer of substances, cell proliferation, and migration (Li et al. ). The texture of the substrate surface, such as bio-macromolecule alignment and micro/nanostructure, influences adhesion, migration, and maturation, having a substantial impact on tissue organization, remodeling, and development (Fleszar et al. ; Liu et al. ). Meanwhile, biochemical stimuli are conveyed via the material's backbone or network. The presence of cell adhesion motifs, such as RGD and GFOGER, for example, is crucial for the modeling with adherent cells. Bioactive small molecules, such as metal ions (Yang et al. ) and protein imitating peptides (Liu et al. ), can be integrated into the material to trigger specific biochemical signaling pathways for specific purposes. Therefore, in addition to taking into account the printing technique, the biomaterials used for in vitro tissue modeling via 3D printing must be carefully chosen based on the tissue of interest, as well as the study's purpose and scientific inquiry. Here, we review the most commonly used biomaterials for 3D bioprinting, with a focus on their characteristics and applications. Gelatin From a chemistry perspective, gelatin is a polypeptide, generated from the hydrolysis of collagen. Gelatin has become one of the most widely used materials in tissue engineering, due to the abundance of cell-adherent RGD motifs in its backbone, excellent biocompatibility, good biodegradability, and low immunogenicity. Its moderate translucency, viscoelasticity and strength also make it appropriate for various bio-fabrication and bioprinting methods. GelMA, a gelatin-based biomaterial in which the primary amines in the lysine backbone of gelatin are replaced with methacrylate groups to facilitate photo-initiated free-radical polymerization, is one of the most common (van Hoorick et al. ). For example, GelMA-based micro-constructs with encapsulated conjunctival stem cells were created using DLP-based bioprinting (Zhong et al. , ), where the GelMA provided a nurturing 3D environment that maintained stem cell phenotype and differentiation potency while maintaining the vitality. Click chemistry methods have been emerging as a way to modify gelatin because of its high efficiency, high selectivity, and minimal side reactions at mild reaction conditions. Gelatin, for example, was modified with norbornene and thiol to allow for photo-reactive thiol-ene crosslinking (Yu et al. , ). The highly selective reaction enabled high-order programmable bio-functionalization and tailored regulation of the mechanical properties of the hydrogel matrix. Click-crosslinked gelatin can also be obtained through the Diels–Alder reaction (García-Astrain et al. ) and the carbodiimide reaction (Cammarata et al. ). Hyaluronic acid Hyaluronic acid (HA) is a glycosaminoglycan composed of d -glucuronic acid and N -acetyl- d -glucosamine units. The abundance of reactive groups in its backbone allowed various crosslinking and chemical modification strategies to accommodate it for different printing methods. For example, hyaluronic acid glycidyl methacrylate (HAGM) has been synthesized for photo-assisted 3D printing (Liu et al. ). Furthermore, adamantane and beta-cyclodextrin were linked to HA to enable a non-covalent guest–host assembly, which introduces shear-thinning behavior into the hydrogel and aids the extrusion-based printing by adding a temporary mechanical support in the printed structure (Ouyang et al. ). In native physiology, HA is a key component of the ECM in cartilage, the eyeball, the brain, and a variety of other tissues, as well as a participant in tissue microenvironment, cell signaling, and tumor progression. Thus, in certain cases, HA is required for in vitro modeling of complicated tissue. To capture the HA-rich ECM in cartilage tissue, for example, methacrylated HA was introduced to the bioink as a matrix for in vitro chondrogenesis, resulting in enhanced tissue organization (Mouser et al. ). In another example, HAGM was used to print the scaffold to support the quiescence state of the limbal stem/progenitor cells (LSC), while the cells remained active when encapsulated in GelMA. The distinct states of the cells in different biomaterials enabled the fabrication of dual-state cells in a single construct, addressing a better mimicry of the native LSC niche (Zhong et al. , ). For further applications, HA can be modified for click chemistry crosslinking via a variety of mechanisms. De-cellularized extracellular matrix (dECM) De-cellularized extracellular matrix (dECM) of the tissue of interest can be manufactured and applied for 3D printing for enhanced recapitulation of native physiological microenvironments. dECM is not only made up of biopolymers like collagen, fibrin, and glycosaminoglycans as a supporting framework, but also retains the host tissue’s native biochemical signaling molecules. While dECM’s thermal gelling nature makes it suitable for extrusion-based bioprinting, its poor mechanical strength makes it difficult to use as a bio-scaffold on its own—as a result, substantial efforts have been made to composite dECM with other materials. For DLP printing of an in vitro liver lobule tissue, liver dECM was produced and combined 1:1 with GelMA. With the complex biochemical cues given, a stable physiological-mimicking environment for HepG2 3D culture was achieved (Ma et al. , ). Alginate Alginate is a brown algae-derived copolymer comprising beta- d -mannuronate and alpha- l -guluronate. Alginate is a popular choice for complex in vitro tissue modeling due to its simultaneous nature of high biocompatibility and bio-inertness. As a result, alginate makes a good choice for investigations that require minimal degradation as well as precise control of scaffold/substrate stiffness. Alginate possesses the appropriate rheological properties and ion cross-linking capabilities for inkjet and extrusion printing, and can also be chemically modified using 2-aminoethyl methacrylate hydrochloride (AEMA) for photo-assisted printing methods for a broader applicability. Alginate can also build an interpenetrating polymer network (IPN) with other hydrogel materials like collagen and dextran to provide even more customized stiffness and multiplexed bioactivity. For example, alginate was incorporated with collagen to generate the IPN with a tunable storage modulus of 49–419 Pa, the variation of which triggered a reversible state of cancer-associated fibroblast between inflammatory state and myofibroblastic state (Cao et al. ). Synthetic polymers Synthetic polymers, in contrast to biopolymers formed from natural sources, are of tremendous interest due to the inherent consistency and feature tailorability that come with scalable industrial manufacturing. One example is polyethylene glycol (PEG), a synthetic polymer extensively used in tissue engineering applications due to its biocompatibility, non-immunogenic degradation and high capacity for modification. Many functional groups, such as protein-mimicking peptides, growth factors, signaling molecules, and significantly, acrylate groups for photo-polymerization, can be added due to the abundance of reactive hydroxyl groups in the backbone. The mechanical properties of a PEG-based hydrogel, on the other hand, are determined by its molecular weight, the concentration and density of crosslinking synergistically, implying that the substrate's stiffness can be customized by demand. The Young’s modulus of PEG diacrylate (PEGDA) ranges from 668 Pa to 2 kPa as the molecular weight varies from 575 to 20,000 (Vannozzi et al. ). Polymers made from various monomers could be used for specific requirements in the substrate or scaffold for in vitro complicated tissue modeling. For example, poly (glycerol-co-sebacate), polyurethane, or polycaprolactone can be used to form an elastic substrate, collectively providing a varied range of elastic strength and degradation profile.
3D bioprinting refers to a type of additive manufacturing, specifically a layer-by-layer fabrication technique that was originally born out of a need for rapid prototyping and has since enjoyed advancement into a fast, customizable fabrication method across many fields. 3D bioprinting technology allows for flexibility in both material choice and design paradigm—in the context of tissue engineering, the ability to incorporate biomaterials and cells inherently allows for 3D bioprinting. As 3D bioprinting becomes more ubiquitous, more research into bioprinting techniques has emerged, allowing for the fabrication of a wide range of biocompatible constructs, and cell-encapsulated tissues, and organ models. Inkjet-based A typical inkjet-based bio-printer is shown in Fig. A (Patel ). It dispenses droplets of low-viscosity bio-ink from a ‘printhead’ containing arrays of small nozzle apertures to form patterns and then stabilizes the structure by photo-crosslinking or thermal gelation (Yu et al. , ). Typical inkjet printer designs include: a bioink storage chamber(s), actuators to both guide bioink(s) to the nozzle and form the droplets, and stage/control systems for three-axial movement. There are three main categories of inkjet bioprinting methods: continuous-inkjet, droplet-on-demand, and electro-hydrodynamic jet bioprinting, all of which differ in their method of bioink droplet deployment control. The continuous-inkjet method extrudes streams of bioink droplets, which lack precise droplet control. The droplet-on-demand bioprinter improves droplet control by creating individual droplets at required times by pressurizing the bioink storage chamber, using thermal, piezoelectric, or electrostatic-based actuators. The electro-hydrodynamic jet bioprinter generates droplets by pulling the bio-ink through the nozzle instead of a pushing method with droplet-on-demand bioprinters (Derakhshanfar et al. ; Gudapati et al. ). In general, inkjet-based 3D bioprinting has the benefit of precise material deposition with a reasonable printing speed. The utilization of bioink droplets results in minimal material waste and low cost, with a high cell viability (except in thermal inkjet bioprinting). The bioink chamber and nozzle design also allow efficient material replacement and multi-material printing. However, a drawback to inkjet printing is that small nozzle apertures place a lower limit on cell densities in the bioinks—higher cell densities can cause nozzle clogging. Additionally, the low viscosity required for printing may result in limited mechanical strength in the final printed structure. Furthermore, the droplet form of the material causes a limited printing resolution (Gudapati et al. ; Mobaraki et al. ). Extrusion-based Extrusion-based bioprinting is another widely used bioprinting technology (Fig. B). Similar to inkjet printing, extrusion-based printing also extrudes material from a nozzle printhead, but instead of dispensing individual droplets, a typical extrusion printer extrudes a continuous flow of viscous bioink filament. The bioink viscosity used in extrusion bioprinting ranges from 30 to 60 kPa s (Derakhshanfar et al. ; Mobaraki et al. ). Higher-viscosity bioinks allow for the use of correspondingly higher viscous biomaterials and higher-density cell encapsulation, both of which are beneficial for tissue and organ fabrication. Extrusion-based bioprinter resolutions are limited by the size of the nozzle aperture and material characteristics, and also experience slower printing speeds due to the scanning nature of its printing movement. For both the extrusion-based and inkjet-based bioprinting, supporting structures are needed if creating overhanging 3D structures, which may introduce longer printing times as well as material/cell wastage. The overall fabrication time will vary based on the complexity of the 3D structure (Yu et al. , ). Laser polymerization-based Laser-based bioprinting methods feature the use of a precisely controlled, focused laser beam to effect patterned photopolymerization in a prepolymer solution. Among them, two-photon polymerization (TPP) bioprinting uses femtosecond laser pulses to achieve submicron level precise printing (Fig. C) (Claeyssens et al. ; Zhang and Chen ; Xing et al. ). The high precision afforded by TPP printing allows for the production of micro- and nano-scale tissue scaffolds and vasculatures. However, to ensure precise synchronization between the motion and laser pulses, the TPP printing speed is often limited, and thus requires long fabrication times for complex and/or large structures (Yu et al. , ). After printing, the un-polymerized residual solution will need to be removed to reveal the printed structure, which causes material waste; however, this material may function as a soft, supportive structure during the actual printing process, thus eliminates the need for designated supporting structures (Yu et al. , ). TPP printing allows for a variety of material viscosities, which is beneficial for the varied conditions of tissue and organ printing, but higher laser powers can cause thermal damage to cells, resulting in reduced cell viability (Derakhshanfar et al. ; Hopp ). Digital light processing (DLP)-based DLP-based bioprinting is an emerging photopolymerization-based bioprinting technique that addresses the primary limitations of previous bioprinting methods—speed and spatial resolution for tissue and organ fabrication. Compared to other methods that require point-by-point or line-by-line scanning to create an individual layer, DLP-based bioprinting drastically reduces the printing time by enabling the projection of an entire 2D design plane at once. A DLP printer’s core piece of hardware is a digital micro-mirror array device (DMD), a programmable micro-opto-electromechanical chip with an array of micro-mirrors—this can be used to spatially pattern an incoming light source, such as a 365 nm ultraviolet or 405 nm visible light, to photo-polymerize a vat of prepolymer solution (Fig. D). The XY resolution of the printed structure is defined by the projection from an individual micro-mirror on the DMD chip, which may be as low as 3–5 µm (and can vary based on intermediate optics and prepolymer characteristics) (Lu et al. ), with a typical layer-by-layer Z-resolution limited by the material refill process between subsequent layers (Yu et al. , ). The Chen group later developed a dynamic optical projection stereolithography (DOPsL) to continuously move the Z -axis during printing, resulting in smooth side walls for printed structures (Zhang et al. ), and applied this for pre-vascularized tissue printing (Zhu et al. ). Further additions to printing efficiency have been researched as well, to go beyond layer-by-layer into volumetric additive manufacturing, where the entire volume is printed at once. One approach is the holographic volumetric 3D fabrication system, which uses three orthogonal light beams with phase-only patterns to print the entire 3D structure in a single step (Shusteff et al. ). Another example is the computed axial lithography (CAL) technology, which utilized a DLP projector to illuminate their prepolymer vat from a single side, axially rotating the vat while modulating the projected light’s phase pattern (Kelly et al. ). Such volumetric additive manufacturing fabrication techniques are not limited in printing time by the Z -axis, but spatial resolution can suffer due to limitations in holographic precision. Recent efforts by Saha et al. to address these spatial resolution limitations utilize femtosecond projection two-photon lithography (FP-TPL), which combines the high precision afforded by TPP with the high-throughput nature of DLP-based printing, allowing simultaneous spatial and temporal focusing. Overall, DLP-based bioprinting enables rapid micro-scale bio-fabrication by plane-wise or even volume-wise printing with the use of a DMD. The high-throughput nature of the process is greatly beneficial for the fabrication of tissues and organs, taking into consideration the time-sensitive nature of live cells and tissues. Additionally, the micro-scale precision afforded by DLP-based printing is similarly beneficial for producing the fine features of native physiology, such as multi-tissue compartments and microvasculature. The relatively low power of the light sources and exposure times typically used also ensure cell viability (Ruskowitz and Deforest ). One drawback to DLP-based bioprinting is its pre-filling of a vet of bioink which if not used and recycled, would go to biological waste.
A typical inkjet-based bio-printer is shown in Fig. A (Patel ). It dispenses droplets of low-viscosity bio-ink from a ‘printhead’ containing arrays of small nozzle apertures to form patterns and then stabilizes the structure by photo-crosslinking or thermal gelation (Yu et al. , ). Typical inkjet printer designs include: a bioink storage chamber(s), actuators to both guide bioink(s) to the nozzle and form the droplets, and stage/control systems for three-axial movement. There are three main categories of inkjet bioprinting methods: continuous-inkjet, droplet-on-demand, and electro-hydrodynamic jet bioprinting, all of which differ in their method of bioink droplet deployment control. The continuous-inkjet method extrudes streams of bioink droplets, which lack precise droplet control. The droplet-on-demand bioprinter improves droplet control by creating individual droplets at required times by pressurizing the bioink storage chamber, using thermal, piezoelectric, or electrostatic-based actuators. The electro-hydrodynamic jet bioprinter generates droplets by pulling the bio-ink through the nozzle instead of a pushing method with droplet-on-demand bioprinters (Derakhshanfar et al. ; Gudapati et al. ). In general, inkjet-based 3D bioprinting has the benefit of precise material deposition with a reasonable printing speed. The utilization of bioink droplets results in minimal material waste and low cost, with a high cell viability (except in thermal inkjet bioprinting). The bioink chamber and nozzle design also allow efficient material replacement and multi-material printing. However, a drawback to inkjet printing is that small nozzle apertures place a lower limit on cell densities in the bioinks—higher cell densities can cause nozzle clogging. Additionally, the low viscosity required for printing may result in limited mechanical strength in the final printed structure. Furthermore, the droplet form of the material causes a limited printing resolution (Gudapati et al. ; Mobaraki et al. ).
Extrusion-based bioprinting is another widely used bioprinting technology (Fig. B). Similar to inkjet printing, extrusion-based printing also extrudes material from a nozzle printhead, but instead of dispensing individual droplets, a typical extrusion printer extrudes a continuous flow of viscous bioink filament. The bioink viscosity used in extrusion bioprinting ranges from 30 to 60 kPa s (Derakhshanfar et al. ; Mobaraki et al. ). Higher-viscosity bioinks allow for the use of correspondingly higher viscous biomaterials and higher-density cell encapsulation, both of which are beneficial for tissue and organ fabrication. Extrusion-based bioprinter resolutions are limited by the size of the nozzle aperture and material characteristics, and also experience slower printing speeds due to the scanning nature of its printing movement. For both the extrusion-based and inkjet-based bioprinting, supporting structures are needed if creating overhanging 3D structures, which may introduce longer printing times as well as material/cell wastage. The overall fabrication time will vary based on the complexity of the 3D structure (Yu et al. , ).
Laser-based bioprinting methods feature the use of a precisely controlled, focused laser beam to effect patterned photopolymerization in a prepolymer solution. Among them, two-photon polymerization (TPP) bioprinting uses femtosecond laser pulses to achieve submicron level precise printing (Fig. C) (Claeyssens et al. ; Zhang and Chen ; Xing et al. ). The high precision afforded by TPP printing allows for the production of micro- and nano-scale tissue scaffolds and vasculatures. However, to ensure precise synchronization between the motion and laser pulses, the TPP printing speed is often limited, and thus requires long fabrication times for complex and/or large structures (Yu et al. , ). After printing, the un-polymerized residual solution will need to be removed to reveal the printed structure, which causes material waste; however, this material may function as a soft, supportive structure during the actual printing process, thus eliminates the need for designated supporting structures (Yu et al. , ). TPP printing allows for a variety of material viscosities, which is beneficial for the varied conditions of tissue and organ printing, but higher laser powers can cause thermal damage to cells, resulting in reduced cell viability (Derakhshanfar et al. ; Hopp ).
DLP-based bioprinting is an emerging photopolymerization-based bioprinting technique that addresses the primary limitations of previous bioprinting methods—speed and spatial resolution for tissue and organ fabrication. Compared to other methods that require point-by-point or line-by-line scanning to create an individual layer, DLP-based bioprinting drastically reduces the printing time by enabling the projection of an entire 2D design plane at once. A DLP printer’s core piece of hardware is a digital micro-mirror array device (DMD), a programmable micro-opto-electromechanical chip with an array of micro-mirrors—this can be used to spatially pattern an incoming light source, such as a 365 nm ultraviolet or 405 nm visible light, to photo-polymerize a vat of prepolymer solution (Fig. D). The XY resolution of the printed structure is defined by the projection from an individual micro-mirror on the DMD chip, which may be as low as 3–5 µm (and can vary based on intermediate optics and prepolymer characteristics) (Lu et al. ), with a typical layer-by-layer Z-resolution limited by the material refill process between subsequent layers (Yu et al. , ). The Chen group later developed a dynamic optical projection stereolithography (DOPsL) to continuously move the Z -axis during printing, resulting in smooth side walls for printed structures (Zhang et al. ), and applied this for pre-vascularized tissue printing (Zhu et al. ). Further additions to printing efficiency have been researched as well, to go beyond layer-by-layer into volumetric additive manufacturing, where the entire volume is printed at once. One approach is the holographic volumetric 3D fabrication system, which uses three orthogonal light beams with phase-only patterns to print the entire 3D structure in a single step (Shusteff et al. ). Another example is the computed axial lithography (CAL) technology, which utilized a DLP projector to illuminate their prepolymer vat from a single side, axially rotating the vat while modulating the projected light’s phase pattern (Kelly et al. ). Such volumetric additive manufacturing fabrication techniques are not limited in printing time by the Z -axis, but spatial resolution can suffer due to limitations in holographic precision. Recent efforts by Saha et al. to address these spatial resolution limitations utilize femtosecond projection two-photon lithography (FP-TPL), which combines the high precision afforded by TPP with the high-throughput nature of DLP-based printing, allowing simultaneous spatial and temporal focusing. Overall, DLP-based bioprinting enables rapid micro-scale bio-fabrication by plane-wise or even volume-wise printing with the use of a DMD. The high-throughput nature of the process is greatly beneficial for the fabrication of tissues and organs, taking into consideration the time-sensitive nature of live cells and tissues. Additionally, the micro-scale precision afforded by DLP-based printing is similarly beneficial for producing the fine features of native physiology, such as multi-tissue compartments and microvasculature. The relatively low power of the light sources and exposure times typically used also ensure cell viability (Ruskowitz and Deforest ). One drawback to DLP-based bioprinting is its pre-filling of a vet of bioink which if not used and recycled, would go to biological waste.
Biomaterials form the basis of the matrix or substrate of the final printed structure, and are critical in the in vitro modeling of complex tissues—they provide crucial physical and chemical signals, and can have significant impacts on cell activities, such as adhesion, metabolism, proliferation, differentiation, and migration. For example, the stiffness of the extracellular matrix as a biophysical cue has been found to be deterministic in the viability, differentiation, and migration of a variety of cell types (Gasiorowski et al. ). Material porosity crucially aids in the transfer of substances, cell proliferation, and migration (Li et al. ). The texture of the substrate surface, such as bio-macromolecule alignment and micro/nanostructure, influences adhesion, migration, and maturation, having a substantial impact on tissue organization, remodeling, and development (Fleszar et al. ; Liu et al. ). Meanwhile, biochemical stimuli are conveyed via the material's backbone or network. The presence of cell adhesion motifs, such as RGD and GFOGER, for example, is crucial for the modeling with adherent cells. Bioactive small molecules, such as metal ions (Yang et al. ) and protein imitating peptides (Liu et al. ), can be integrated into the material to trigger specific biochemical signaling pathways for specific purposes. Therefore, in addition to taking into account the printing technique, the biomaterials used for in vitro tissue modeling via 3D printing must be carefully chosen based on the tissue of interest, as well as the study's purpose and scientific inquiry. Here, we review the most commonly used biomaterials for 3D bioprinting, with a focus on their characteristics and applications. Gelatin From a chemistry perspective, gelatin is a polypeptide, generated from the hydrolysis of collagen. Gelatin has become one of the most widely used materials in tissue engineering, due to the abundance of cell-adherent RGD motifs in its backbone, excellent biocompatibility, good biodegradability, and low immunogenicity. Its moderate translucency, viscoelasticity and strength also make it appropriate for various bio-fabrication and bioprinting methods. GelMA, a gelatin-based biomaterial in which the primary amines in the lysine backbone of gelatin are replaced with methacrylate groups to facilitate photo-initiated free-radical polymerization, is one of the most common (van Hoorick et al. ). For example, GelMA-based micro-constructs with encapsulated conjunctival stem cells were created using DLP-based bioprinting (Zhong et al. , ), where the GelMA provided a nurturing 3D environment that maintained stem cell phenotype and differentiation potency while maintaining the vitality. Click chemistry methods have been emerging as a way to modify gelatin because of its high efficiency, high selectivity, and minimal side reactions at mild reaction conditions. Gelatin, for example, was modified with norbornene and thiol to allow for photo-reactive thiol-ene crosslinking (Yu et al. , ). The highly selective reaction enabled high-order programmable bio-functionalization and tailored regulation of the mechanical properties of the hydrogel matrix. Click-crosslinked gelatin can also be obtained through the Diels–Alder reaction (García-Astrain et al. ) and the carbodiimide reaction (Cammarata et al. ). Hyaluronic acid Hyaluronic acid (HA) is a glycosaminoglycan composed of d -glucuronic acid and N -acetyl- d -glucosamine units. The abundance of reactive groups in its backbone allowed various crosslinking and chemical modification strategies to accommodate it for different printing methods. For example, hyaluronic acid glycidyl methacrylate (HAGM) has been synthesized for photo-assisted 3D printing (Liu et al. ). Furthermore, adamantane and beta-cyclodextrin were linked to HA to enable a non-covalent guest–host assembly, which introduces shear-thinning behavior into the hydrogel and aids the extrusion-based printing by adding a temporary mechanical support in the printed structure (Ouyang et al. ). In native physiology, HA is a key component of the ECM in cartilage, the eyeball, the brain, and a variety of other tissues, as well as a participant in tissue microenvironment, cell signaling, and tumor progression. Thus, in certain cases, HA is required for in vitro modeling of complicated tissue. To capture the HA-rich ECM in cartilage tissue, for example, methacrylated HA was introduced to the bioink as a matrix for in vitro chondrogenesis, resulting in enhanced tissue organization (Mouser et al. ). In another example, HAGM was used to print the scaffold to support the quiescence state of the limbal stem/progenitor cells (LSC), while the cells remained active when encapsulated in GelMA. The distinct states of the cells in different biomaterials enabled the fabrication of dual-state cells in a single construct, addressing a better mimicry of the native LSC niche (Zhong et al. , ). For further applications, HA can be modified for click chemistry crosslinking via a variety of mechanisms. De-cellularized extracellular matrix (dECM) De-cellularized extracellular matrix (dECM) of the tissue of interest can be manufactured and applied for 3D printing for enhanced recapitulation of native physiological microenvironments. dECM is not only made up of biopolymers like collagen, fibrin, and glycosaminoglycans as a supporting framework, but also retains the host tissue’s native biochemical signaling molecules. While dECM’s thermal gelling nature makes it suitable for extrusion-based bioprinting, its poor mechanical strength makes it difficult to use as a bio-scaffold on its own—as a result, substantial efforts have been made to composite dECM with other materials. For DLP printing of an in vitro liver lobule tissue, liver dECM was produced and combined 1:1 with GelMA. With the complex biochemical cues given, a stable physiological-mimicking environment for HepG2 3D culture was achieved (Ma et al. , ). Alginate Alginate is a brown algae-derived copolymer comprising beta- d -mannuronate and alpha- l -guluronate. Alginate is a popular choice for complex in vitro tissue modeling due to its simultaneous nature of high biocompatibility and bio-inertness. As a result, alginate makes a good choice for investigations that require minimal degradation as well as precise control of scaffold/substrate stiffness. Alginate possesses the appropriate rheological properties and ion cross-linking capabilities for inkjet and extrusion printing, and can also be chemically modified using 2-aminoethyl methacrylate hydrochloride (AEMA) for photo-assisted printing methods for a broader applicability. Alginate can also build an interpenetrating polymer network (IPN) with other hydrogel materials like collagen and dextran to provide even more customized stiffness and multiplexed bioactivity. For example, alginate was incorporated with collagen to generate the IPN with a tunable storage modulus of 49–419 Pa, the variation of which triggered a reversible state of cancer-associated fibroblast between inflammatory state and myofibroblastic state (Cao et al. ). Synthetic polymers Synthetic polymers, in contrast to biopolymers formed from natural sources, are of tremendous interest due to the inherent consistency and feature tailorability that come with scalable industrial manufacturing. One example is polyethylene glycol (PEG), a synthetic polymer extensively used in tissue engineering applications due to its biocompatibility, non-immunogenic degradation and high capacity for modification. Many functional groups, such as protein-mimicking peptides, growth factors, signaling molecules, and significantly, acrylate groups for photo-polymerization, can be added due to the abundance of reactive hydroxyl groups in the backbone. The mechanical properties of a PEG-based hydrogel, on the other hand, are determined by its molecular weight, the concentration and density of crosslinking synergistically, implying that the substrate's stiffness can be customized by demand. The Young’s modulus of PEG diacrylate (PEGDA) ranges from 668 Pa to 2 kPa as the molecular weight varies from 575 to 20,000 (Vannozzi et al. ). Polymers made from various monomers could be used for specific requirements in the substrate or scaffold for in vitro complicated tissue modeling. For example, poly (glycerol-co-sebacate), polyurethane, or polycaprolactone can be used to form an elastic substrate, collectively providing a varied range of elastic strength and degradation profile.
From a chemistry perspective, gelatin is a polypeptide, generated from the hydrolysis of collagen. Gelatin has become one of the most widely used materials in tissue engineering, due to the abundance of cell-adherent RGD motifs in its backbone, excellent biocompatibility, good biodegradability, and low immunogenicity. Its moderate translucency, viscoelasticity and strength also make it appropriate for various bio-fabrication and bioprinting methods. GelMA, a gelatin-based biomaterial in which the primary amines in the lysine backbone of gelatin are replaced with methacrylate groups to facilitate photo-initiated free-radical polymerization, is one of the most common (van Hoorick et al. ). For example, GelMA-based micro-constructs with encapsulated conjunctival stem cells were created using DLP-based bioprinting (Zhong et al. , ), where the GelMA provided a nurturing 3D environment that maintained stem cell phenotype and differentiation potency while maintaining the vitality. Click chemistry methods have been emerging as a way to modify gelatin because of its high efficiency, high selectivity, and minimal side reactions at mild reaction conditions. Gelatin, for example, was modified with norbornene and thiol to allow for photo-reactive thiol-ene crosslinking (Yu et al. , ). The highly selective reaction enabled high-order programmable bio-functionalization and tailored regulation of the mechanical properties of the hydrogel matrix. Click-crosslinked gelatin can also be obtained through the Diels–Alder reaction (García-Astrain et al. ) and the carbodiimide reaction (Cammarata et al. ).
Hyaluronic acid (HA) is a glycosaminoglycan composed of d -glucuronic acid and N -acetyl- d -glucosamine units. The abundance of reactive groups in its backbone allowed various crosslinking and chemical modification strategies to accommodate it for different printing methods. For example, hyaluronic acid glycidyl methacrylate (HAGM) has been synthesized for photo-assisted 3D printing (Liu et al. ). Furthermore, adamantane and beta-cyclodextrin were linked to HA to enable a non-covalent guest–host assembly, which introduces shear-thinning behavior into the hydrogel and aids the extrusion-based printing by adding a temporary mechanical support in the printed structure (Ouyang et al. ). In native physiology, HA is a key component of the ECM in cartilage, the eyeball, the brain, and a variety of other tissues, as well as a participant in tissue microenvironment, cell signaling, and tumor progression. Thus, in certain cases, HA is required for in vitro modeling of complicated tissue. To capture the HA-rich ECM in cartilage tissue, for example, methacrylated HA was introduced to the bioink as a matrix for in vitro chondrogenesis, resulting in enhanced tissue organization (Mouser et al. ). In another example, HAGM was used to print the scaffold to support the quiescence state of the limbal stem/progenitor cells (LSC), while the cells remained active when encapsulated in GelMA. The distinct states of the cells in different biomaterials enabled the fabrication of dual-state cells in a single construct, addressing a better mimicry of the native LSC niche (Zhong et al. , ). For further applications, HA can be modified for click chemistry crosslinking via a variety of mechanisms.
De-cellularized extracellular matrix (dECM) of the tissue of interest can be manufactured and applied for 3D printing for enhanced recapitulation of native physiological microenvironments. dECM is not only made up of biopolymers like collagen, fibrin, and glycosaminoglycans as a supporting framework, but also retains the host tissue’s native biochemical signaling molecules. While dECM’s thermal gelling nature makes it suitable for extrusion-based bioprinting, its poor mechanical strength makes it difficult to use as a bio-scaffold on its own—as a result, substantial efforts have been made to composite dECM with other materials. For DLP printing of an in vitro liver lobule tissue, liver dECM was produced and combined 1:1 with GelMA. With the complex biochemical cues given, a stable physiological-mimicking environment for HepG2 3D culture was achieved (Ma et al. , ).
Alginate is a brown algae-derived copolymer comprising beta- d -mannuronate and alpha- l -guluronate. Alginate is a popular choice for complex in vitro tissue modeling due to its simultaneous nature of high biocompatibility and bio-inertness. As a result, alginate makes a good choice for investigations that require minimal degradation as well as precise control of scaffold/substrate stiffness. Alginate possesses the appropriate rheological properties and ion cross-linking capabilities for inkjet and extrusion printing, and can also be chemically modified using 2-aminoethyl methacrylate hydrochloride (AEMA) for photo-assisted printing methods for a broader applicability. Alginate can also build an interpenetrating polymer network (IPN) with other hydrogel materials like collagen and dextran to provide even more customized stiffness and multiplexed bioactivity. For example, alginate was incorporated with collagen to generate the IPN with a tunable storage modulus of 49–419 Pa, the variation of which triggered a reversible state of cancer-associated fibroblast between inflammatory state and myofibroblastic state (Cao et al. ).
Synthetic polymers, in contrast to biopolymers formed from natural sources, are of tremendous interest due to the inherent consistency and feature tailorability that come with scalable industrial manufacturing. One example is polyethylene glycol (PEG), a synthetic polymer extensively used in tissue engineering applications due to its biocompatibility, non-immunogenic degradation and high capacity for modification. Many functional groups, such as protein-mimicking peptides, growth factors, signaling molecules, and significantly, acrylate groups for photo-polymerization, can be added due to the abundance of reactive hydroxyl groups in the backbone. The mechanical properties of a PEG-based hydrogel, on the other hand, are determined by its molecular weight, the concentration and density of crosslinking synergistically, implying that the substrate's stiffness can be customized by demand. The Young’s modulus of PEG diacrylate (PEGDA) ranges from 668 Pa to 2 kPa as the molecular weight varies from 575 to 20,000 (Vannozzi et al. ). Polymers made from various monomers could be used for specific requirements in the substrate or scaffold for in vitro complicated tissue modeling. For example, poly (glycerol-co-sebacate), polyurethane, or polycaprolactone can be used to form an elastic substrate, collectively providing a varied range of elastic strength and degradation profile.
Along with the evolution of 3D bioprinting techniques and biomaterials, in vitro models of a variety of tissues, organs and diseases have been developed, evaluated, optimized and brought into use. Here, taking cancer, heart, liver and muscle as examples, we review the pioneering and representative work in the field. Cancer Cancer remains a significant public health issue worldwide due to its high occurrence, mortality, and economic impact. 3D bioprinting offers an opportunity to advance the in vitro modeling of various cancers types, such as brain cancer (Tang et al. , ), pancreatic cancer (Hakobyan et al. ), liver cancer (Ma et al. , ), lung cancer, colorectal cancer, ovarian cancer (Xu et al. ), breast cancer (Hribar et al. ; Zhu et al. ), and metastatic models (Meng et al. ; Zhu et al. ), owing to its ability to recapitulate the complex cellular and material heterogeneities. Brain cancer Tang et al. developed multicellular glioblastoma (GBM) models using DLP-based bioprinting (Tang et al. ). Patient-derived GBM stem cells (GSCs), macrophages, neural progenitor cells, and astrocytes were fabricated into a defined spatial organization to form a brain tumor within brain architecture, recapitulating immune interactions and functional dependencies in 3D microenvironment (Fig. A). Tumor cells demonstrated higher drug resistance and invasion capacity with the inclusion of macrophages. Extrusion-based bioprinting was also used to generate GBM models composed of GSCs, patient-derived GBM-associated stromal cells, and microglia in an alginate-based hydrogel (Hermida et al. ). Alginate hydrogels were modified with RGDs for better cell attachment. The 3D GBM models demonstrated enhanced resistance to cisplatin which failed in many clinical trials but showed promising efficacy in 2D cell cultures. 3D models have a potential application for more reliable efficacy evaluations. Pancreatic cancer Hakobyan et al. used laser-assisted bioprinting (LAB) for generation of pancreatic ductal adenocarcinoma (PDAC) spheroid arrays consisted of both acinar and ductal cells in GelMA (Hakobyan et al. ). These models were used for interrogation of different factors that contribute to the precursor PDAC lesions at the early stage PDAC onset and progression. Xu et al. developed PDAC models with biomimetic materials consisted of surface-engineered cellulose nanofibrils (CNFs) and photo-crosslinkable galactoglucomannan methacrylates (GGMMAs), and a UV-assisted extrusion-based printing techniques. The bioinks demonstrated promising biocompatibility and supported pancreatic cancer cell and dermal fibroblast proliferation. Utama et al. reported a drop-on-demand method to rapidly form PDAC models in 96-well format (Utama et al. ). Tunable biological and mechanical properties were enabled by the 4-arm PEG-based polymers that can form hydrogels within seconds. Lung cancer Mondal et al. utilized extrusion-based printing and a sodium alginate–gelatin hydrogel to develop non-small cell lung cancer (NSCLC) co-culture models with patient-derived xenograft cells and cancer-associated fibroblasts (Mondal et al. ). The methods demonstrated high printability and good cell viability. After two weeks of in vitro culture, the NSCLC spheroids could reach a diameter ranging from 50 to 1,100 µm, creating hypoxic cores within the spheroids for further research. Cellular crosstalk created by the co-culture system was confirmed by upregulation of specific genes, such as vimentin and α-SMA. Wang et al. reported a method that combined low-temperature molding and 3D bioprinting technique to fabricate a lung cancer model (Wang et al. ). A biomimetic 3D hydrogel grid scaffold was generated with gelatin, sodium alginate, and lung cancer cells A549/95-D. Cell proliferation plateaued after two weeks of culture and had a viability over 90%. Colorectal cancer Chen et al. reported a 3D printed colorectal cancer (CRC) model that closely mimicked the physiological functions and cellular crosstalk between the tumor cells and tumor-associated stromal cells (Chen et al. ). Co-culture of colorectal cancer cells, cancer-associated fibroblasts, and tumor-associated endothelial cells on bio-printed scaffolds reprogrammed normal stromal cells into tumor-associated phenotypes. Cellular processes and vascularization were observed, and could help elucidate oncogenesis factors and evaluate the efficiency of potential drugs. Tariq et al. utilized 3D magnetic bioprinted CRC models to investigate P-glycoprotein associated multidrug resistance (MDR) in cancer treatment (Tariq et al. ). The authors delivered siRNA, designed against MDR1 gene to silence the gene in Caco-2 cells and studied the role of MDR-1 gene in both 2D and 3D culture conditions. The 3D model was compared to 2D culture and demonstrated that the knockdown of MDR1 gene in colorectal carcinoma cells can significantly reduce the tumor cell migration in both 2D cell culture and 3D bioprinted models. Breast cancer Langer et al. reported generation of a multicellular scaffold-free tumor tissue representing subtypes of breast cancer and pancreatic cancer using 3D printing. The multiple cell types within the printed structure could self-organize into biomimetic morphologies and secreted their own ECMs to reform the tissues. Incorporation of patient-derived cells into the models offeres a translational tool for investigating the therapeutic responses, potential oncogenic endpoints, and crosstalk between different cell types relevant to individual patients. Vinson et al. investigated epithelial-adipose interactions in breast cancer using a 3D-printed breast cancer model. Patient-derived breast cancer cells MCF-7 and MDA-MB-231 and differentiated adipocytes were spatially patterned by laser direct-write bioprinting technique. Investigations of early onset of cancer cell invasion through cellular and tissue-level interactions in the adipose tissue were enabled by the 3D models. Duchamp et al. established a sacrificial bioprinting strategy to generate biomimetic mammary duct cancer models to study the oncogenesis and invasion processes of breast cancer. The models were first generated with GelMA into duct-like structures, and the channels were then populated with MCF-7 cells, which was reported to have relatively low invasiveness. Agarose was used as a sacrificial material for convenient extraction. The breast cancer model could be cultured over 24 days and outward invasion of cancer cells into the duct-like matrix was observed. This proof-of-concept model demonstrates the potential value of 3D printed models in studying the mechanism of oncogenesis of breast cancer. Heart According to a study by the American Heart Association , cardiovascular disease (CVD) affects nearly 50% of the US population and accounted for more than 360 billion dollars in consumer costs from 2016 to 2017, thus making CVD of significant medical, scientific, and economic importance. To help delve into and improve research surrounding CVD, significant effort has been invested into state-of-the-art solutions, from 3D-printed cardiac patches for damaged hearts to engineered heart tissues (EHTs) for evaluating drug efficacy and toxicity (Dvir et al. ). When evaluating a drug, researchers have commonly used traditional 2D cultures and/or animal models. Due to the lack of chemical and biophysical cues a 2D culture receives from its environment; specifically the cell–extracellular matrix, cell–cell, and tissue-level interactions; these cells are unable to properly recapitulate the response of a mature adult heart (Veldhuizen et al. ; Zuppinger ). On the other end of the spectrum, murine models are appealing as they can capture these environmental cues. However, inter-species differences in ion channels, biological pathways and pharmacokinetic properties negatively impact the predictive ability of these models for human hearts (Mathur et al. ). The need for a better predictive model of drug toxicity is paramount, as evidenced by the fact that 45% of post-approval drug withdrawal from the market is related to cardiovascular system issues (Ferri et al. ). To address these issues, researchers have developed various cardiac models and EHTs. Using a variety of methods, researchers have formed tissues with measurable functionality (beating frequency and force) against drugs or toxins (Mathur et al. ; Nam et al. ; Veldhuizen et al. ). One such EHT is the flexible cardiac thin film, developed by the Parker group (Grosberg et al. ). The model was created by seeding cardiac cells onto a thin sheet of fibronectin-stamped polydimethylsiloxane (PDMS), with the stamped fibronectin encouraging an anisotropic cardiac orientation, which is important for optimal force generation. The contractility of the tissue was then evaluated by measuring the “curl” of the thin film that occurred during tissue contraction. After developing this initial model, the group then expanded on the original study with multiple derivations and improvements of the original model (Ahn et al. ; Lind et al. ; McCain et al. ; Wang et al. ). In one compelling study, the group investigated Barth syndrome, a cardiomyopathic disease caused by mutated TAZ (the gene Tafazzin), in Cas9-edited induced pluripotent stem cell cardiomyocytes (iPSC-CMs). Using the in vitro model to analyze sarcomere assembly, contractile stress generation, and ROS (reactive oxygen species) production differences in the modified iPSC-CM compared to the wild type, the researchers were able to successfully show that both the reintroduction of wild-type TAZ and that suppression of ROS by the mitochondria reversed cardiomyopathic symptoms (Wang et al. ). However, many researchers have created much thicker tissues, as opposed to thin films, to better recapitulate a mature heart. One of the most common EHTs used in the field for thick tissues is a pillar design, where cardiac cells are attached to two anchor points, either pillars or wires (Hinson et al. ; Liu et al. ; Ma et al. ; Miller et al. ; Ronaldson-Bouchard et al. ; Tiburcy et al. ; Williams et al. ). These anchors are commonly formed from molded PDMS, which is then immersed in a suspension of cells and extracellular matrix (ECM), naturally forming a 3D tissue over multiple days of culture. Like the thin films, these models have also been specialized for disease types. For example, Hinson et al. investigated the effect of different titin protein mutations, an important protein for sarcomere functionality. Using the 3D pillar model, the researchers were able to examine the impact of different titin variants on tissue contractility (Hinson et al. ). In another study, Williams et al. developed an arrhythmic cardiac model by dosing culture models with methyl-beta cyclodextrin (MBCD). The MBCD induced arrhythmic behavior in the tissue, which subsequently lost calcium handling ability and exhibited fibrotic activity. This impacted the function of the tissue even after removal of MBCD from culture (Williams et al. ). However, these thicker EHTs rely on passive tension for the cardiac cells to self-organize into an anisotropic orientation. To directly address this, some researchers have incorporated 3D bioprinting into the model by directly printing encapsulated cardiac cells into small lines stretching between two pillars (Fig. B) (Liu et al. ; Miller et al. ). This is significant, as alignment has been shown to increase the maturity of the iPSC-CMs, which thereby increases the ability of the cells to recapitulate the adult heart during drug and toxin testing (Guo and Pu ; Hirt et al. ). Moreover, since the cells are directly printed, the tissues can be aligned in a small spatial footprint, enabling high-throughput testing on a 96-well plate, as opposed to the 24- or 48-well format used by other pillar models (Miller et al. ). Applications of 3D printing have also expanded into full heart and chamber models (Lee et al. ; Wang et al. ). However, the field still has a long way to go before regularly using these models for in vitro testing. In particular, the immaturity of iPSC-CMs, which are the preferred cardiomyocyte source, is a persistent issue (Hirt et al. ; Ronaldson-Bouchard et al. ). To fully repair or even recapitulate an adult heart, we need to have a cell source that is robust and mature. Nevertheless, 3D models have undoubtedly advanced the field of human cardiac research and will continue to do so as new techniques are developed. Liver The liver functions natively as the primary site for metabolism and detoxification in the body, thus making it a popular tissue for in vitro modeling of screening drugs, mechanistic studies, and liver regeneration. A wide range of biomaterials, such as gelatin, alginate, GelMA, dECM, hyaluronan, and collagen in different combinations, as well as different cell sources, such as primary hepatocytes, liver cancer cell lines, and stem cell-derived hepatocytes, have been utilized for creating biomimetic structures of liver (Ma et al. ). Kang et al. developed a vascularized liver lobule array model with hepatic cells and endothelial cells using preset extrusion bioprinting (Kang et al. ). Briefly, a preset cartridge with hepatic, endothelial, and lumen regions was fabricated and injected with bioink with hepatic cells, endothelial cells, and sacrificial materials, respectively. Spatially patterned models of different cell types demonstrated improved functional properties of liver, including higher albumin secretion and urea production, compared to mixed cell types with no spatial organization. Endothelial cells provided structural integrity of the model after culturing for a week. Grix et al. reported a perfusion-enabled liver model with twelve micro-channels open at both sides in a hexagonal structure (Grix et al. ). HepaRG cells and human stellate cells were patterned using stereolithography. Stable expression of tight junctions and metabolism markers was observed in the model. The channels within the bioprinted liver were shown to be perfusable. Efforts have been made on developing more biomimetic bioink for liver tissue engineering. Several groups reported the benefits of using liver dECM for generating liver models, in terms of improved printability, mechanical properties, as well as biological properties (Kim et al. ; Lee et al. ; Yu et al. developed photo-crosslinkable liver-specific dECM bioinks to generate complex patient-specific liver models. HiPSC-derived hepatocytes were patterned into hexagonal microscale architectures and demonstrated high cell viability and improved maturation in dECM bioinks compared to collagen bioink (Fig. E–G). Mao et al. also reported using DLP-based bioprinting and GelMA/dECM to generate a micro-liver tissue with improved hepatic function restoration. Human-induced hepatocytes demonstrated improved viability and functionality in bioink with dECM compared to bioink with only GelMA. Mazzocchi et al. improved the printability of collagen I by mixing it with thiolated hyaluronic acid at various ratios. Primary hepatocytes and stellate cells were printed using the composite bioink which remained viable for two weeks. Gori et al. developed a thermo-responsive hydrogel with alginate and sacrificial Pluronic materials for culturing hepatic cells in 3D constructs. The Pluronic materials improved the diffusion properties of the hydrogel and supported better cell viability. Goulart et al. compared using single cell dispersion, 2D HiPSC-hepatocytes, and HiPSC-hepatocyte spheroids for 3D printing with non-parenchymal cells. Single cell format had the worst performance, demonstrating reduced viability and hepatic functions after 18-day cultivation period. Loss of hepatic phenotype was also observed in single cell models. In contrast, spheroid-based models demonstrated improved functionality and stability. Engineered human liver models have a steadily increased use in the pharmaceutical industry due to their improved functionality, maturation, and steady metabolism compared to 2D-cultured cells (Underhill and Khetani ). The drug acetaminophen demonstrated significantly increased sensitivity in 3D-printed liver models than their 2D counterparts (Gori et al. ). The liver models can also serve as tools for viral study and infectious viruses. Bioprinted liver models using optimized bioink consisting of alginate, gelatin, and dECM were efficiently transduced with adeno-associated virus (AAV) and supported adenovirus replication (Hiller et al. ). Muscle Skeletal muscle is the most abundant tissue in the human body and is innervated by motor neurons through neuromuscular junctions (NMJs), crucial for both locomotion and the coordination of tasks through directional force generation via contraction and relaxation of myofibers (Grefte et al. ). Healthy skeletal muscle tissue possesses the ability to regenerate upon injury through the migration, proliferation, and differentiation of nearby satellite cells into mature functional myofibers (Greising et al. ), but can lose this regenerative capacity due to a number of factors, including but not limited to traumatic injury, aging, or diseases, such as amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), spinal muscular atrophy, and myasthenia gravis (MG), which can lead to a reduction in quality of life (Larkindale et al. , Cappello and Francolini ). To understand the underlying mechanisms of muscular disorders to come up with novel therapeutic intervention strategies, research efforts have been made toward developing physiologically relevant in vitro skeletal muscle models to overcome the limitations imposed by current animal models, such as species–species pathological differences and response. Microfluidic device serves as suitable candidates for developing in vitro NMJ platforms, where muscle and motor neuron cells can be cultured in separate chambers with their own media reservoirs, but have a bridge connecting them, allowing for axonal sprouting to recapitulate a functional NMJ. Santhanam et al. developed a microfluidic in vitro NMJ model for drug toxicity testing where human skeletal myoblasts were co-cultured with human motoneurons (MNs) in separate PDMS chambers with a microtunnel array connecting the two chambers as described, allowing for axonal outgrowth. The MNs were electrically stimulated via electrodes, with the resulting myotube contractions exhibiting dose-dependent responses to the toxins—Bungarotoxin, BOTOX ® , and curare (Santhanam et al. ). Osaki et al. utilized a similar setup to develop the first ALS-on-a-chip model by co-culturing iPSC-derived skeletal muscle cells connected via a collagen gel bridge to ALS patient-derived MN spheroids that were opto-genetically engineered to allow for optically stimulated muscle contraction with optical stimulation (Fig. C) (Osaki et al. ) Their model, measured via micropillar displacement, exhibited ALS pathological features with reduced muscle contraction force (Fig. D), that improved upon treatment with ALS drug candidates rapamycin and bosutinib, thus creating a platform for ALS drug screening and disease modeling. Another ALS-on-a-chip platform with iPSC-derived MNs from ALS patients was used by Guo et al. to investigate NMJ functionality in ALS lines with mutated genes (Guo et al. ). Using the same opto-genetic system as Osaki et al. Vila et al. developed an in vitro microfluidic model to characterize impaired NMJ in MG by co-culturing human skeletal myoblasts and optogenetically engineered neurospheres derived from the same donor, creating the first patient-specific human NMJ (hNMJ). The group recapitulated diseased NMJ pathology by subjecting their NMJ model to serum derived from MG patients and observing diminished NMJ function, as skeletal muscle cells failed to contract upon optical stimulation of MNs. Building upon this platform, the same group incorporated automated video-processing algorithms to create a diagnostic tool that automatically quantifies NMJ function and MG severity in a high-throughput manner based on sera from MG donors (Vila et al. ). Another opto-genetically engineered NMJ model was recently developed by Solomon et al. to test the functionality of NMJ in response to vecuronium, a competitive inhibitor of acetylcholine receptors (AChR) (Solomon et al. ). The first mature hNMJ model was observed by Bakooshli where primary human myoblasts and human pluripotent stem cell (hPSC)-derived MNs were directed to self-assemble in 3D co-culture, modeling NMJ function in healthy versus MG-afflicted conditions. Mature functional NMJs were observed generating calcium transients in response to glutamate stimuli with and without MG patient-derived IgG treatments. Brack et al.’s model was the first in vitro hNMJ model to demonstrate the upregulated expression of adult AChR epsilon in muscle fibers, thus creating a mature hNMJ platform to study pathology and development of diseases that affect adult NMJs. A recent study led by Faustino Martins et al. created a breakthrough in generating self-assembled 3D human neuromuscular organoids (NMOs) containing supporting terminal Schwann cells and functional NMJs capable of modeling MG pathology through IgG treatment from MG patients. The 3D NMOs were developed from hiPSC-derived neuro-mesodermal progenitors and were capable of being used for long-term culture studies, with the NMJs reported to being stable up to 150 days (Faustino Martins et al. ). In the context of personalized disease modeling, Maffioletti et al. developed an in vitro humanized muscular dystrophy model by generating iPSC-differentiated skeletal muscles derived from patients with DMD, limb-girdle muscular dystrophy, and skeletal laminopathies. Using this 3D platform, abnormal phenotypes of muscular dystrophies were observed, thus providing a platform to study the development of various muscular disorders and potential treatment regimen (Maffioletti et al. ). Recently, Mondrinos et al. developed an in vitro muscle model where MSC-derived muscle cells were allowed to “sculpt” themselves into muscle tissue via anisotropic contraction post differentiation with functionalized nodes in the PDMS mold serving as “muscle anchors” to prevent detachment. This proof-of-concept model was used to mimic oxidative injury through hydrogen peroxide treatment, and the therapeutic effect of the drugOlaparib and a combination of retinoic acid (RA) and omega-3 fatty acid eicosapentaenoic acid (EPA) pre-treatments was observed to rescue citrate synthase (CS) activity. By adding a tumor compartment containing A549 spheroids, lung fibroblasts, and macrophages into a separate microfluidic chamber, a model of lung cancer cachexia was developed based on this aforementioned oxidative injury model (Mondrinos et al. ). The utilization of 3D nozzle-based bioprinting technology has also been very recently applied to the fabrication of NMJ to recapitulate the complex 3D NMJ structure more closely by spatial cell patterning (Kong et al. ; Sanz et al. ); however, 3D in vitro NMJ models generated from microfluidic devices still remain a more widely used alternative to accommodate for the different media requirements of muscle and neuron cells. The applications of 3D printing technology coupled with micro-fluidics to generate 3D in vitro muscle models, as demonstrated by previously mentioned examples, possess significant potential for the future of muscle development studies, disease modeling, and drug screening compared to 2D culture models. However, the ability to create a reproducible, high throughput in vitro muscle model for disease modeling and drug screening remains a huge challenge.
Cancer remains a significant public health issue worldwide due to its high occurrence, mortality, and economic impact. 3D bioprinting offers an opportunity to advance the in vitro modeling of various cancers types, such as brain cancer (Tang et al. , ), pancreatic cancer (Hakobyan et al. ), liver cancer (Ma et al. , ), lung cancer, colorectal cancer, ovarian cancer (Xu et al. ), breast cancer (Hribar et al. ; Zhu et al. ), and metastatic models (Meng et al. ; Zhu et al. ), owing to its ability to recapitulate the complex cellular and material heterogeneities. Brain cancer Tang et al. developed multicellular glioblastoma (GBM) models using DLP-based bioprinting (Tang et al. ). Patient-derived GBM stem cells (GSCs), macrophages, neural progenitor cells, and astrocytes were fabricated into a defined spatial organization to form a brain tumor within brain architecture, recapitulating immune interactions and functional dependencies in 3D microenvironment (Fig. A). Tumor cells demonstrated higher drug resistance and invasion capacity with the inclusion of macrophages. Extrusion-based bioprinting was also used to generate GBM models composed of GSCs, patient-derived GBM-associated stromal cells, and microglia in an alginate-based hydrogel (Hermida et al. ). Alginate hydrogels were modified with RGDs for better cell attachment. The 3D GBM models demonstrated enhanced resistance to cisplatin which failed in many clinical trials but showed promising efficacy in 2D cell cultures. 3D models have a potential application for more reliable efficacy evaluations. Pancreatic cancer Hakobyan et al. used laser-assisted bioprinting (LAB) for generation of pancreatic ductal adenocarcinoma (PDAC) spheroid arrays consisted of both acinar and ductal cells in GelMA (Hakobyan et al. ). These models were used for interrogation of different factors that contribute to the precursor PDAC lesions at the early stage PDAC onset and progression. Xu et al. developed PDAC models with biomimetic materials consisted of surface-engineered cellulose nanofibrils (CNFs) and photo-crosslinkable galactoglucomannan methacrylates (GGMMAs), and a UV-assisted extrusion-based printing techniques. The bioinks demonstrated promising biocompatibility and supported pancreatic cancer cell and dermal fibroblast proliferation. Utama et al. reported a drop-on-demand method to rapidly form PDAC models in 96-well format (Utama et al. ). Tunable biological and mechanical properties were enabled by the 4-arm PEG-based polymers that can form hydrogels within seconds. Lung cancer Mondal et al. utilized extrusion-based printing and a sodium alginate–gelatin hydrogel to develop non-small cell lung cancer (NSCLC) co-culture models with patient-derived xenograft cells and cancer-associated fibroblasts (Mondal et al. ). The methods demonstrated high printability and good cell viability. After two weeks of in vitro culture, the NSCLC spheroids could reach a diameter ranging from 50 to 1,100 µm, creating hypoxic cores within the spheroids for further research. Cellular crosstalk created by the co-culture system was confirmed by upregulation of specific genes, such as vimentin and α-SMA. Wang et al. reported a method that combined low-temperature molding and 3D bioprinting technique to fabricate a lung cancer model (Wang et al. ). A biomimetic 3D hydrogel grid scaffold was generated with gelatin, sodium alginate, and lung cancer cells A549/95-D. Cell proliferation plateaued after two weeks of culture and had a viability over 90%. Colorectal cancer Chen et al. reported a 3D printed colorectal cancer (CRC) model that closely mimicked the physiological functions and cellular crosstalk between the tumor cells and tumor-associated stromal cells (Chen et al. ). Co-culture of colorectal cancer cells, cancer-associated fibroblasts, and tumor-associated endothelial cells on bio-printed scaffolds reprogrammed normal stromal cells into tumor-associated phenotypes. Cellular processes and vascularization were observed, and could help elucidate oncogenesis factors and evaluate the efficiency of potential drugs. Tariq et al. utilized 3D magnetic bioprinted CRC models to investigate P-glycoprotein associated multidrug resistance (MDR) in cancer treatment (Tariq et al. ). The authors delivered siRNA, designed against MDR1 gene to silence the gene in Caco-2 cells and studied the role of MDR-1 gene in both 2D and 3D culture conditions. The 3D model was compared to 2D culture and demonstrated that the knockdown of MDR1 gene in colorectal carcinoma cells can significantly reduce the tumor cell migration in both 2D cell culture and 3D bioprinted models. Breast cancer Langer et al. reported generation of a multicellular scaffold-free tumor tissue representing subtypes of breast cancer and pancreatic cancer using 3D printing. The multiple cell types within the printed structure could self-organize into biomimetic morphologies and secreted their own ECMs to reform the tissues. Incorporation of patient-derived cells into the models offeres a translational tool for investigating the therapeutic responses, potential oncogenic endpoints, and crosstalk between different cell types relevant to individual patients. Vinson et al. investigated epithelial-adipose interactions in breast cancer using a 3D-printed breast cancer model. Patient-derived breast cancer cells MCF-7 and MDA-MB-231 and differentiated adipocytes were spatially patterned by laser direct-write bioprinting technique. Investigations of early onset of cancer cell invasion through cellular and tissue-level interactions in the adipose tissue were enabled by the 3D models. Duchamp et al. established a sacrificial bioprinting strategy to generate biomimetic mammary duct cancer models to study the oncogenesis and invasion processes of breast cancer. The models were first generated with GelMA into duct-like structures, and the channels were then populated with MCF-7 cells, which was reported to have relatively low invasiveness. Agarose was used as a sacrificial material for convenient extraction. The breast cancer model could be cultured over 24 days and outward invasion of cancer cells into the duct-like matrix was observed. This proof-of-concept model demonstrates the potential value of 3D printed models in studying the mechanism of oncogenesis of breast cancer.
Tang et al. developed multicellular glioblastoma (GBM) models using DLP-based bioprinting (Tang et al. ). Patient-derived GBM stem cells (GSCs), macrophages, neural progenitor cells, and astrocytes were fabricated into a defined spatial organization to form a brain tumor within brain architecture, recapitulating immune interactions and functional dependencies in 3D microenvironment (Fig. A). Tumor cells demonstrated higher drug resistance and invasion capacity with the inclusion of macrophages. Extrusion-based bioprinting was also used to generate GBM models composed of GSCs, patient-derived GBM-associated stromal cells, and microglia in an alginate-based hydrogel (Hermida et al. ). Alginate hydrogels were modified with RGDs for better cell attachment. The 3D GBM models demonstrated enhanced resistance to cisplatin which failed in many clinical trials but showed promising efficacy in 2D cell cultures. 3D models have a potential application for more reliable efficacy evaluations.
Hakobyan et al. used laser-assisted bioprinting (LAB) for generation of pancreatic ductal adenocarcinoma (PDAC) spheroid arrays consisted of both acinar and ductal cells in GelMA (Hakobyan et al. ). These models were used for interrogation of different factors that contribute to the precursor PDAC lesions at the early stage PDAC onset and progression. Xu et al. developed PDAC models with biomimetic materials consisted of surface-engineered cellulose nanofibrils (CNFs) and photo-crosslinkable galactoglucomannan methacrylates (GGMMAs), and a UV-assisted extrusion-based printing techniques. The bioinks demonstrated promising biocompatibility and supported pancreatic cancer cell and dermal fibroblast proliferation. Utama et al. reported a drop-on-demand method to rapidly form PDAC models in 96-well format (Utama et al. ). Tunable biological and mechanical properties were enabled by the 4-arm PEG-based polymers that can form hydrogels within seconds.
Mondal et al. utilized extrusion-based printing and a sodium alginate–gelatin hydrogel to develop non-small cell lung cancer (NSCLC) co-culture models with patient-derived xenograft cells and cancer-associated fibroblasts (Mondal et al. ). The methods demonstrated high printability and good cell viability. After two weeks of in vitro culture, the NSCLC spheroids could reach a diameter ranging from 50 to 1,100 µm, creating hypoxic cores within the spheroids for further research. Cellular crosstalk created by the co-culture system was confirmed by upregulation of specific genes, such as vimentin and α-SMA. Wang et al. reported a method that combined low-temperature molding and 3D bioprinting technique to fabricate a lung cancer model (Wang et al. ). A biomimetic 3D hydrogel grid scaffold was generated with gelatin, sodium alginate, and lung cancer cells A549/95-D. Cell proliferation plateaued after two weeks of culture and had a viability over 90%.
Chen et al. reported a 3D printed colorectal cancer (CRC) model that closely mimicked the physiological functions and cellular crosstalk between the tumor cells and tumor-associated stromal cells (Chen et al. ). Co-culture of colorectal cancer cells, cancer-associated fibroblasts, and tumor-associated endothelial cells on bio-printed scaffolds reprogrammed normal stromal cells into tumor-associated phenotypes. Cellular processes and vascularization were observed, and could help elucidate oncogenesis factors and evaluate the efficiency of potential drugs. Tariq et al. utilized 3D magnetic bioprinted CRC models to investigate P-glycoprotein associated multidrug resistance (MDR) in cancer treatment (Tariq et al. ). The authors delivered siRNA, designed against MDR1 gene to silence the gene in Caco-2 cells and studied the role of MDR-1 gene in both 2D and 3D culture conditions. The 3D model was compared to 2D culture and demonstrated that the knockdown of MDR1 gene in colorectal carcinoma cells can significantly reduce the tumor cell migration in both 2D cell culture and 3D bioprinted models.
Langer et al. reported generation of a multicellular scaffold-free tumor tissue representing subtypes of breast cancer and pancreatic cancer using 3D printing. The multiple cell types within the printed structure could self-organize into biomimetic morphologies and secreted their own ECMs to reform the tissues. Incorporation of patient-derived cells into the models offeres a translational tool for investigating the therapeutic responses, potential oncogenic endpoints, and crosstalk between different cell types relevant to individual patients. Vinson et al. investigated epithelial-adipose interactions in breast cancer using a 3D-printed breast cancer model. Patient-derived breast cancer cells MCF-7 and MDA-MB-231 and differentiated adipocytes were spatially patterned by laser direct-write bioprinting technique. Investigations of early onset of cancer cell invasion through cellular and tissue-level interactions in the adipose tissue were enabled by the 3D models. Duchamp et al. established a sacrificial bioprinting strategy to generate biomimetic mammary duct cancer models to study the oncogenesis and invasion processes of breast cancer. The models were first generated with GelMA into duct-like structures, and the channels were then populated with MCF-7 cells, which was reported to have relatively low invasiveness. Agarose was used as a sacrificial material for convenient extraction. The breast cancer model could be cultured over 24 days and outward invasion of cancer cells into the duct-like matrix was observed. This proof-of-concept model demonstrates the potential value of 3D printed models in studying the mechanism of oncogenesis of breast cancer.
According to a study by the American Heart Association , cardiovascular disease (CVD) affects nearly 50% of the US population and accounted for more than 360 billion dollars in consumer costs from 2016 to 2017, thus making CVD of significant medical, scientific, and economic importance. To help delve into and improve research surrounding CVD, significant effort has been invested into state-of-the-art solutions, from 3D-printed cardiac patches for damaged hearts to engineered heart tissues (EHTs) for evaluating drug efficacy and toxicity (Dvir et al. ). When evaluating a drug, researchers have commonly used traditional 2D cultures and/or animal models. Due to the lack of chemical and biophysical cues a 2D culture receives from its environment; specifically the cell–extracellular matrix, cell–cell, and tissue-level interactions; these cells are unable to properly recapitulate the response of a mature adult heart (Veldhuizen et al. ; Zuppinger ). On the other end of the spectrum, murine models are appealing as they can capture these environmental cues. However, inter-species differences in ion channels, biological pathways and pharmacokinetic properties negatively impact the predictive ability of these models for human hearts (Mathur et al. ). The need for a better predictive model of drug toxicity is paramount, as evidenced by the fact that 45% of post-approval drug withdrawal from the market is related to cardiovascular system issues (Ferri et al. ). To address these issues, researchers have developed various cardiac models and EHTs. Using a variety of methods, researchers have formed tissues with measurable functionality (beating frequency and force) against drugs or toxins (Mathur et al. ; Nam et al. ; Veldhuizen et al. ). One such EHT is the flexible cardiac thin film, developed by the Parker group (Grosberg et al. ). The model was created by seeding cardiac cells onto a thin sheet of fibronectin-stamped polydimethylsiloxane (PDMS), with the stamped fibronectin encouraging an anisotropic cardiac orientation, which is important for optimal force generation. The contractility of the tissue was then evaluated by measuring the “curl” of the thin film that occurred during tissue contraction. After developing this initial model, the group then expanded on the original study with multiple derivations and improvements of the original model (Ahn et al. ; Lind et al. ; McCain et al. ; Wang et al. ). In one compelling study, the group investigated Barth syndrome, a cardiomyopathic disease caused by mutated TAZ (the gene Tafazzin), in Cas9-edited induced pluripotent stem cell cardiomyocytes (iPSC-CMs). Using the in vitro model to analyze sarcomere assembly, contractile stress generation, and ROS (reactive oxygen species) production differences in the modified iPSC-CM compared to the wild type, the researchers were able to successfully show that both the reintroduction of wild-type TAZ and that suppression of ROS by the mitochondria reversed cardiomyopathic symptoms (Wang et al. ). However, many researchers have created much thicker tissues, as opposed to thin films, to better recapitulate a mature heart. One of the most common EHTs used in the field for thick tissues is a pillar design, where cardiac cells are attached to two anchor points, either pillars or wires (Hinson et al. ; Liu et al. ; Ma et al. ; Miller et al. ; Ronaldson-Bouchard et al. ; Tiburcy et al. ; Williams et al. ). These anchors are commonly formed from molded PDMS, which is then immersed in a suspension of cells and extracellular matrix (ECM), naturally forming a 3D tissue over multiple days of culture. Like the thin films, these models have also been specialized for disease types. For example, Hinson et al. investigated the effect of different titin protein mutations, an important protein for sarcomere functionality. Using the 3D pillar model, the researchers were able to examine the impact of different titin variants on tissue contractility (Hinson et al. ). In another study, Williams et al. developed an arrhythmic cardiac model by dosing culture models with methyl-beta cyclodextrin (MBCD). The MBCD induced arrhythmic behavior in the tissue, which subsequently lost calcium handling ability and exhibited fibrotic activity. This impacted the function of the tissue even after removal of MBCD from culture (Williams et al. ). However, these thicker EHTs rely on passive tension for the cardiac cells to self-organize into an anisotropic orientation. To directly address this, some researchers have incorporated 3D bioprinting into the model by directly printing encapsulated cardiac cells into small lines stretching between two pillars (Fig. B) (Liu et al. ; Miller et al. ). This is significant, as alignment has been shown to increase the maturity of the iPSC-CMs, which thereby increases the ability of the cells to recapitulate the adult heart during drug and toxin testing (Guo and Pu ; Hirt et al. ). Moreover, since the cells are directly printed, the tissues can be aligned in a small spatial footprint, enabling high-throughput testing on a 96-well plate, as opposed to the 24- or 48-well format used by other pillar models (Miller et al. ). Applications of 3D printing have also expanded into full heart and chamber models (Lee et al. ; Wang et al. ). However, the field still has a long way to go before regularly using these models for in vitro testing. In particular, the immaturity of iPSC-CMs, which are the preferred cardiomyocyte source, is a persistent issue (Hirt et al. ; Ronaldson-Bouchard et al. ). To fully repair or even recapitulate an adult heart, we need to have a cell source that is robust and mature. Nevertheless, 3D models have undoubtedly advanced the field of human cardiac research and will continue to do so as new techniques are developed.
The liver functions natively as the primary site for metabolism and detoxification in the body, thus making it a popular tissue for in vitro modeling of screening drugs, mechanistic studies, and liver regeneration. A wide range of biomaterials, such as gelatin, alginate, GelMA, dECM, hyaluronan, and collagen in different combinations, as well as different cell sources, such as primary hepatocytes, liver cancer cell lines, and stem cell-derived hepatocytes, have been utilized for creating biomimetic structures of liver (Ma et al. ). Kang et al. developed a vascularized liver lobule array model with hepatic cells and endothelial cells using preset extrusion bioprinting (Kang et al. ). Briefly, a preset cartridge with hepatic, endothelial, and lumen regions was fabricated and injected with bioink with hepatic cells, endothelial cells, and sacrificial materials, respectively. Spatially patterned models of different cell types demonstrated improved functional properties of liver, including higher albumin secretion and urea production, compared to mixed cell types with no spatial organization. Endothelial cells provided structural integrity of the model after culturing for a week. Grix et al. reported a perfusion-enabled liver model with twelve micro-channels open at both sides in a hexagonal structure (Grix et al. ). HepaRG cells and human stellate cells were patterned using stereolithography. Stable expression of tight junctions and metabolism markers was observed in the model. The channels within the bioprinted liver were shown to be perfusable. Efforts have been made on developing more biomimetic bioink for liver tissue engineering. Several groups reported the benefits of using liver dECM for generating liver models, in terms of improved printability, mechanical properties, as well as biological properties (Kim et al. ; Lee et al. ; Yu et al. developed photo-crosslinkable liver-specific dECM bioinks to generate complex patient-specific liver models. HiPSC-derived hepatocytes were patterned into hexagonal microscale architectures and demonstrated high cell viability and improved maturation in dECM bioinks compared to collagen bioink (Fig. E–G). Mao et al. also reported using DLP-based bioprinting and GelMA/dECM to generate a micro-liver tissue with improved hepatic function restoration. Human-induced hepatocytes demonstrated improved viability and functionality in bioink with dECM compared to bioink with only GelMA. Mazzocchi et al. improved the printability of collagen I by mixing it with thiolated hyaluronic acid at various ratios. Primary hepatocytes and stellate cells were printed using the composite bioink which remained viable for two weeks. Gori et al. developed a thermo-responsive hydrogel with alginate and sacrificial Pluronic materials for culturing hepatic cells in 3D constructs. The Pluronic materials improved the diffusion properties of the hydrogel and supported better cell viability. Goulart et al. compared using single cell dispersion, 2D HiPSC-hepatocytes, and HiPSC-hepatocyte spheroids for 3D printing with non-parenchymal cells. Single cell format had the worst performance, demonstrating reduced viability and hepatic functions after 18-day cultivation period. Loss of hepatic phenotype was also observed in single cell models. In contrast, spheroid-based models demonstrated improved functionality and stability. Engineered human liver models have a steadily increased use in the pharmaceutical industry due to their improved functionality, maturation, and steady metabolism compared to 2D-cultured cells (Underhill and Khetani ). The drug acetaminophen demonstrated significantly increased sensitivity in 3D-printed liver models than their 2D counterparts (Gori et al. ). The liver models can also serve as tools for viral study and infectious viruses. Bioprinted liver models using optimized bioink consisting of alginate, gelatin, and dECM were efficiently transduced with adeno-associated virus (AAV) and supported adenovirus replication (Hiller et al. ).
Skeletal muscle is the most abundant tissue in the human body and is innervated by motor neurons through neuromuscular junctions (NMJs), crucial for both locomotion and the coordination of tasks through directional force generation via contraction and relaxation of myofibers (Grefte et al. ). Healthy skeletal muscle tissue possesses the ability to regenerate upon injury through the migration, proliferation, and differentiation of nearby satellite cells into mature functional myofibers (Greising et al. ), but can lose this regenerative capacity due to a number of factors, including but not limited to traumatic injury, aging, or diseases, such as amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), spinal muscular atrophy, and myasthenia gravis (MG), which can lead to a reduction in quality of life (Larkindale et al. , Cappello and Francolini ). To understand the underlying mechanisms of muscular disorders to come up with novel therapeutic intervention strategies, research efforts have been made toward developing physiologically relevant in vitro skeletal muscle models to overcome the limitations imposed by current animal models, such as species–species pathological differences and response. Microfluidic device serves as suitable candidates for developing in vitro NMJ platforms, where muscle and motor neuron cells can be cultured in separate chambers with their own media reservoirs, but have a bridge connecting them, allowing for axonal sprouting to recapitulate a functional NMJ. Santhanam et al. developed a microfluidic in vitro NMJ model for drug toxicity testing where human skeletal myoblasts were co-cultured with human motoneurons (MNs) in separate PDMS chambers with a microtunnel array connecting the two chambers as described, allowing for axonal outgrowth. The MNs were electrically stimulated via electrodes, with the resulting myotube contractions exhibiting dose-dependent responses to the toxins—Bungarotoxin, BOTOX ® , and curare (Santhanam et al. ). Osaki et al. utilized a similar setup to develop the first ALS-on-a-chip model by co-culturing iPSC-derived skeletal muscle cells connected via a collagen gel bridge to ALS patient-derived MN spheroids that were opto-genetically engineered to allow for optically stimulated muscle contraction with optical stimulation (Fig. C) (Osaki et al. ) Their model, measured via micropillar displacement, exhibited ALS pathological features with reduced muscle contraction force (Fig. D), that improved upon treatment with ALS drug candidates rapamycin and bosutinib, thus creating a platform for ALS drug screening and disease modeling. Another ALS-on-a-chip platform with iPSC-derived MNs from ALS patients was used by Guo et al. to investigate NMJ functionality in ALS lines with mutated genes (Guo et al. ). Using the same opto-genetic system as Osaki et al. Vila et al. developed an in vitro microfluidic model to characterize impaired NMJ in MG by co-culturing human skeletal myoblasts and optogenetically engineered neurospheres derived from the same donor, creating the first patient-specific human NMJ (hNMJ). The group recapitulated diseased NMJ pathology by subjecting their NMJ model to serum derived from MG patients and observing diminished NMJ function, as skeletal muscle cells failed to contract upon optical stimulation of MNs. Building upon this platform, the same group incorporated automated video-processing algorithms to create a diagnostic tool that automatically quantifies NMJ function and MG severity in a high-throughput manner based on sera from MG donors (Vila et al. ). Another opto-genetically engineered NMJ model was recently developed by Solomon et al. to test the functionality of NMJ in response to vecuronium, a competitive inhibitor of acetylcholine receptors (AChR) (Solomon et al. ). The first mature hNMJ model was observed by Bakooshli where primary human myoblasts and human pluripotent stem cell (hPSC)-derived MNs were directed to self-assemble in 3D co-culture, modeling NMJ function in healthy versus MG-afflicted conditions. Mature functional NMJs were observed generating calcium transients in response to glutamate stimuli with and without MG patient-derived IgG treatments. Brack et al.’s model was the first in vitro hNMJ model to demonstrate the upregulated expression of adult AChR epsilon in muscle fibers, thus creating a mature hNMJ platform to study pathology and development of diseases that affect adult NMJs. A recent study led by Faustino Martins et al. created a breakthrough in generating self-assembled 3D human neuromuscular organoids (NMOs) containing supporting terminal Schwann cells and functional NMJs capable of modeling MG pathology through IgG treatment from MG patients. The 3D NMOs were developed from hiPSC-derived neuro-mesodermal progenitors and were capable of being used for long-term culture studies, with the NMJs reported to being stable up to 150 days (Faustino Martins et al. ). In the context of personalized disease modeling, Maffioletti et al. developed an in vitro humanized muscular dystrophy model by generating iPSC-differentiated skeletal muscles derived from patients with DMD, limb-girdle muscular dystrophy, and skeletal laminopathies. Using this 3D platform, abnormal phenotypes of muscular dystrophies were observed, thus providing a platform to study the development of various muscular disorders and potential treatment regimen (Maffioletti et al. ). Recently, Mondrinos et al. developed an in vitro muscle model where MSC-derived muscle cells were allowed to “sculpt” themselves into muscle tissue via anisotropic contraction post differentiation with functionalized nodes in the PDMS mold serving as “muscle anchors” to prevent detachment. This proof-of-concept model was used to mimic oxidative injury through hydrogen peroxide treatment, and the therapeutic effect of the drugOlaparib and a combination of retinoic acid (RA) and omega-3 fatty acid eicosapentaenoic acid (EPA) pre-treatments was observed to rescue citrate synthase (CS) activity. By adding a tumor compartment containing A549 spheroids, lung fibroblasts, and macrophages into a separate microfluidic chamber, a model of lung cancer cachexia was developed based on this aforementioned oxidative injury model (Mondrinos et al. ). The utilization of 3D nozzle-based bioprinting technology has also been very recently applied to the fabrication of NMJ to recapitulate the complex 3D NMJ structure more closely by spatial cell patterning (Kong et al. ; Sanz et al. ); however, 3D in vitro NMJ models generated from microfluidic devices still remain a more widely used alternative to accommodate for the different media requirements of muscle and neuron cells. The applications of 3D printing technology coupled with micro-fluidics to generate 3D in vitro muscle models, as demonstrated by previously mentioned examples, possess significant potential for the future of muscle development studies, disease modeling, and drug screening compared to 2D culture models. However, the ability to create a reproducible, high throughput in vitro muscle model for disease modeling and drug screening remains a huge challenge.
With the development of both 3D culture and bio-fabrication technologies, 3D in vitro tissue models have been recognized as an advanced approach for toxicity studies as they provide controllable variations to identify mechanisms of treatment response; close imitation of the microenvironment and physiology of the native tissue; and high efficiency and throughput of model establishment. In an early study, HepaRG and HepG2 spheroids (Fig. A, B) were fabricated with a hanging drop system and tested for toxicity response against aflatoxin B1, amiodarone, valproic and chlorpromazine. Compared to the 2D culture, the spheroids showed increased metabolic activities as well as increased gluconeogenesis, cell polarization, and diffusion barrier effect of ECM with the drugs of interest. These distinctions in the drug response between 2D and 3D models demonstrate the importance of the in vivo environment in evaluating toxicity (Mueller et al. ). Parallelly, Ramaiahgari et al. used Matrigel to 3D culture HepG2 cells. The size of the spheroids was regulated by the bottom surface area of the well plate and the amounts of cells. Increased cell polarity, functionality and thereby the sensitivity to the hepatotoxic drugs were also observed. For better control over the remodeling and architecture recapture of the complex tissue, 3D printing has been involved in the production of the in vitro models, resulting in an improved mimicry of the biochemical, histological and functional recapitulation, as well as addressing the different study purposes. For example, micro-extrusion-based printing was employed to optimize a 3D liver in vitro model. Alginate, gelatin and Matrigel were blended at an optimized ratio for printability, cell viability, metabolism and long-term stability of the scaffold. The viability and secretion of albumin were maintained up to 21 days, indicating the potential of the model for chronic toxicity testing (Schmidt et al. ). On the other hand, the rapid yet well-controlled production of the liver spheroid by 3D printing enabled the quantitative evaluation of drug-induced toxicity in an in situ manner (Fig. C, D), which can play an accelerating role in the long and expensive drug development process (Hong and Song ). One of the important complexed features of the native tissues, which should be taken into consideration for toxicity assessment, is that they are highly vascularized (Fig. E). Utilizing extrusion-based 3D printing, Massa et al. fabricated 3D vascularized liver model with perfusable channels (Fig. F–H) involving an endothelial layer that barriers the diffusion of molecules. The model was tested for the toxicity study of acetaminophen, which has been revealed to damage the endothelial cells in the liver sinusoid. In this model, the toxicity effect of the drug was tested on the endothelial layer and the HepG2/C3A cell protected by the endothelial layer, better mimicking the in vivo exposure scenario (Massa et al. ). While 3D bio-printing offers many advantages, there are still challenges and limitations to overcome, as well as room to improve. To successfully replicate complicated tissue in vitro, a model should not only reproduce its histological and biochemical characteristics, but also its functions, such as metabolite exchange, nutrient transportation and contraction. Ahn et al., for example, constructed a 3D heart cantilever based on rat cardiomyocytes, in which a piece of cardiac tissue was remodeled, and the contraction function was recapitulated and evaluated. Dosed with titanium dioxide at 100 μg/mL, they observed a gradual decrease in the contractile force, and the spontaneous beating ceased in 24 h (Ahn et al. ). The result did not align with a previous study with a 2D culture model, where the spontaneous beating of the rat cardiomyocytes was maintained despite the reduced beating rate and amplitude (Jawad et al. ). There are a few hypotheses as to why the 3D heart tissue stopped beating, including: (1) cardiomyocytes are more resistant to the titanium dioxide toxicity in 2D than in 3D culture; (2) the remaining contractility of the cardiomyocytes after dosing was enough to be observed on the single cell level, but not enough to support the beating function; and (3) the toxicity toward the tissue ECM was reflected in the 3D model but neglected in 2D culture. In any case, the difference between the two studies emphasizes the importance of function recapitulation in the in vitro modeling. However, the gap between the exquisite native tissue and our limited understanding of the in vivo conditions and appropriate fabrication technology remains a key difficulty and hot topic in ongoing research. Tissue complexity The complexity of the tissue is one of the challenges in reproducing a native tissue. Regardless of the biomaterial sources available, it remains a challenge to fully represent the biophysical and biochemical features of the native ECM in an artificial bio-microenvironment. One solution for the dilemma is to incorporate dECM in the biomaterial applied in the 3D printing. As reported by Ma et al. , the photo-patterning of cells and stiffness of the scaffold were precisely regulated via 3D printing of the dECM-blended bioink to provide biomimetic physical signals to the cells, while the biochemical cues embedded in the native liver ECM were simultaneously delivered. Similar results were achieved in a later study by another group (Mao et al. ). For control of the biochemical cues individually in the scaffold, highly engineered synthetic material customized with biomolecules of interest has been developed to facilitate the establishment of 3D culture systems. In an early collective study conducted by Taubenberger et al., PEG was decorated with biochemical mediators, such as metalloproteinase-cleavage spot, ECM mimicking cell adhesion peptides and a set of growth factors. The highly programmable platform they developed was able to create an in vitro bio-microenvironment with multiplexed biochemical cues and controlled mechanical properties of the matrix (Taubenberger et al. ). Another challenge is that the ECM is highly dynamic. Material properties must be carefully tuned to match the progression of the in vitro model and physiological process. For example, in the cardiac micro-tissue fabricated via DLP printing, the crosslinking density of the scaffold made of GelMA was carefully tuned, so that the degradation rate of the scaffold matched the ECM deposition of human cardiac fibroblasts (HCF), which essentially supported the maturation and contraction phenotype of the artificial tissue over 7 days (Miller et al. ). To improve the mimicry, a dynamic culture system could be combined with the artificial tissue. For example, a microfluidic chip with pressure channels was designed for colon tumor organoid culture to mimic the peristalsis, which is an important feature of their native microenvironment. The proliferation and organoid size were significantly increased compared to static culture as the media flowed through the pressure channels and mechanical stimulus was delivered. The peristalsis-stimulated organoid also showed decreased uptake and response to ellipticine-laden micelle dosing, implying that genuine recapitulation of ECM dynamics may have a significant impact on the in vitro model's drug/toxin response (Fang et al. ). On the other hand, native tissue has a complex cell composition, whereas much of the earlier research used only a few cell lines. It is an effective approach to simplify the model for certain scientific problems, but it hinders the recapitulation of tissue integrity, functionality, and response to drug/toxin doses. Organoid and in situ differentiation offer significant advantages in breaking down this barrier. Gu et al. created 3D neuron constructs from human neuron stem cells using extrusion-based 3D printing. After in situ differentiation of the stem cells, a neuron micro-tissue composed of functional neurons and supporting neuroglia was obtained (Gu et al. ). A follow-up study using iPSCs was conducted by the same group, demonstrating the strategy's potential for application in a variety of tissues (Gu et al. ). This method is particularly useful for models that require difficult-to-obtain cell sources. For example, because of the non-proliferating nature of cardiomyocytes and the requirement for high cardiomyocyte density, it has been difficult to establish an in vitro cardiac model in a large dimension and with integrated anatomy structures. Kupfer et al. used in situ differentiation of 3D printing iPSCs to create a cardiac organoid with highly biomimetic chamber structure and pumping activities on a 1.3 cm scale. While the organoid technology and in situ differentiation provide a versatile strategy to achieve multicellular construct, it is difficult to control the ratio of the cellular component. For designated cell composition, 3D printing could also be exploited as it provides high resolution for cell deposition. Focusing on the subject has resulted in significant progress. Because of their high yet biocompatible printing resolution, light-assisted printing processes, such as stereolithography and DLP printing, are intrinsically advantageous for multicellular printing. Furthermore, multiplexed printing systems have been developed to aid the facile multicellular printing; for example, multi-head and core–shell structured nozzles for extrusion and inkjet-based bioprinting have been developed. In place of the distinct flow mechanics at the micro-scale, microfluidic-based strategies can also be incorporated to the conventional 3D printing processes for precise control on the heterogeneity of the bioink. Ashammakhi et al. have provided a more in-depth review on the topic. Systematic recapitulation Another gap to be filled is the lack of mimicry on the systematic level. Many pharmacological and toxicological studies are hampered by poor in vitro–in vivo correlation because the response is usually generated by organ-organ interaction rather than a single organ or tissue. To address this concern, multi-organ-on-a-chip models incorporating multiple tissue models linked to a shared medium circulation system have been developed. Microfluidic devices have emerged as a powerful platform with individual chambers for the culture of each tissue and controlled flow of media enabled by designated channels, and extensive work has been devoted to such culture systems, resulting in improved drug response prediction outcomes (Sung ). Further improvement could be achieved by incorporating the evolving in vitro tissue models as discussed above as well as more customized and tailored design in the microfluidic devices for better mimicry of the flow dynamics and substance exchange (Goldstein et al. ). In addition, it is necessary for systematic modeling to involve the in vitro recapitulation of the immune system, which plays a critical role in many disease progression and pharmacological/toxicological responses, and has become increasingly important in the development of therapeutics for many conditions. Keeping this in mind, recent studies have focused on incorporating immune components into in vitro models and have achieved effective results. iPSC and THP-1-derived macrophages, for example, were incorporated into the in vitro glioblastoma microenvironment model to mimic the infiltration of macrophage alongside microglia via DLP printing. The engagement of the macrophages resulted in a significantly more authentic recapitulation of the original tissue, which had a substantial impact on the drug response of the model (Tang et al. ). THP-1 was also used to investigate the general pro-inflammatory response in a human-based multi-organ-on-a-chip model which involved hepatocyte, cardiomyocyte and skeletal muscle myoblasts (Sasserath et al. ). However, most of the work has been devoted to the innate immune system, while the in vitro modeling of the adaptive immune system remains to be explored. In fact, the state-of-the-art in vitro modeling of the immune system is still in its early stages. A more in-depth and comprehensive review regarding the topic has been provided by Polini et al. . Microarchitecture Furthermore, due to the limited resolution of bioprinting and cell deposition, it is difficult to recreate the microarchitecture and hierarchical nature of the tissue, which is particularly important in the integrated functionality of certain organs, such as the lungs, kidneys, and livers. The alveoli in the lungs, for example, are physically air sacs held and entangled by a network of capillaries (Fig. A). This extremely sophisticated structure is necessary for the exchange of oxygen and carbon dioxide between the pulmonary and circulatory systems, but it also adds a significant challenge to the fabrication process, particularly with soft materials. Earlier studies focused on multilayer 3D culture integrated on a PDMS chamber to capture the air–liquid interface in the alveolus for disease modeling and drug testing (Fig. B) (Benam et al. ; Huh et al. ). While the biochemical and histological characteristics of the tissue were successfully replicated to some extent, the membrane thickness of the blood vessels and the cell-liquid ratio were not fully recapitulated, resulting in inaccurate capture of tissue metabolism and biochemical signaling (Shrestha et al. ). The advanced 3D printing technique can improve the recapitulation of the alveolar microarchitecture. Innovatively applying food dye as a photo-absorber, Grigoryan et al. enhanced the printing resolution of soft hydrogel, PEGDA ( M w = 6 kDa), in DLP-based 3D printing (Fig. C). This allowed the printing of the complexed microstructure of alveolar with a biocompatible bioink, in which oxygenation and flow of human red blood cells was supported and vascularization of human umbilical vein endothelial cells (HUVECs) was achieved (Grigoryan et al. ). One limitation of this study is that the native alveolar cell composition was not included or tested, which limits the model's application in drug and toxin studies. Nonetheless, it is stated that the recapitulation of the exquisite native tissue's microarchitecture is within reach owing to the rising resolution of 3D printing techniques and a wider range of biomaterials. Vascularization As the nature route of nutrient and oxygen transportation and an essential part in the induction of organogenesis (Ding et al. ), vasculatures remain a stumbling block in both the maturation of organoids (Vargas-Valderrama et al. ) and the 3D fabrication of larger in vitro tissues. Currently, the major approach to introduce the vascularization to an in vitro model is to involve a sacrificial template. For example, Skylar-Scott et al. proposed a sacrificial writing into functional tissue (SWIFT) method, in which they compact a large volume of organoids in a thermal-gelling ECM and directly write the vascular channels with gelatin ink in an embedding manner at 0–4 °C. After printing, the gelatin was removed by perfusion as the construction was warmed up to 37 °C for future culture. This novel strategy enabled addition of vasculature and large-dimension culture of organoids (Skylar-Scott et al. ). The sacrificial gelatin ink could also facilitate direct printing of vasculatures in a co-axial extrusion-based 3D printing set-up. As reported by Shao et al. perfusable core–shell constructs with a scale larger than 1 cm have been printed with HUVECs mixed in the inner gelatin phase and tissue cells (human breast cancer cell MDA-MB-231 and mouse osteoblast MC3T3-E1) laden in the outer GelMA phase. After removal of gelatin and auto-seeding of HUVECs, the construct remodeled into a vasculature and was cultured for over 20 days (Shao et al. ). The phase-transition removal of gelatin is more biocompatible to avoid unwanted chemical stimulation compared to alginate-Ca and Pluronic, which are commonly used in earlier work, but the temperature shock involved in this process should be taken into consideration for specific cells and models (Lindquist and Mertens ). Moreover, the process to remove the template still hinders the efficient fabrication of the in vitro model. In contrast, DLP printing remains advantageous to fabricate vasculatures directly and rapidly. Zhu et al. reported the DLP-based continuous bioprinting of a pre-vascularized in vitro tissue with gradient channel diameter and biomimetic branching configuration. Facilitated with a motorized syringe pump, each construct was printed within 1 min; lumen-like structure and tight junctions of the endothelium was developed after culture for 1 week (Zhu et al. ). Despite the evolving techniques to produce vascularized perfusable channels, most work was conducted with primary HUVECs, which are isolated from an identical organ and stage of human life (Cao et al. ). However, the endothelium is highly heterogeneous throughout the different organs in terms of histological features, metabolism, angiogenesis and involvement in the immune response (Przysinda et al. ). It is noteworthy that in the development of an organ-specific vasculature, the diverse phenotypes of the endothelium should be recognized and well characterized. High throughput High-throughput screening (HTS) systems are widely used by the pharmaceutical industry as an efficient method to process large numbers of compounds and molecular targets in a rapid manner. As such, pharmaceutical companies are turning to the use of 3D biomimetic human tissues in HTS format for preclinical toxicity testing of potential drug candidates. 3D bioprinting approaches, such as inkjet and micro-extrusion-based processes, are limited in scalability, resolution, patterning flexibility, and/or speed, which makes them unsuitable for high-throughput fabrication of complex cell-laden 3D microstructures within multi-well plates commonly used in HTS systems for drug screening and assay development. These shortcomings severely limit the widespread adoption of 3D printed cell culture platforms since researchers often rely on products configured to interface with commonly used lab instruments and equipment. Recently, DLP printing has made HTS possible. With rational integration of the micro-continuous projection printing and the automated well plate registration, Hwang et al. managed to fabricate complex 3D bio-constructs directly in a well plate rapidly and consistently. Depending on the complexity of the tissue construct, the total fabrication time for a fully populated 96-well plate typically ranges from 20 to 40 min (Hwang et al. ). Future work for printing in a 384-well plate or more wells is yet to be developed for HTS applications. Concluding remarks 3D bioprinting has made significant progress in in vitro recapitulation of complex tissues. By applying an appropriate printing technique and biomaterial, accurate recapture of the ECM composition, cell population, bio-microenvironment, organ microarchitecture, and tissue functionality has been achieved in a variety of tissues, such as cancer, heart, muscle, liver, kidney, and will continue to be broadened and enhanced as the studies go on. Despite the encouraging results being achieved, it is noteworthy that they are usually labor and technique intensive. To bridge the gap between fundamental research and pragmatic applications, the scalability and reproducibility of the modeling strategies must also be addressed. In addition, with the arising need in the evaluation of therapeutic and substance in subdivided population and in the point-of-care scenario, the required time of production of personalized in vitro model must also be improved.
The complexity of the tissue is one of the challenges in reproducing a native tissue. Regardless of the biomaterial sources available, it remains a challenge to fully represent the biophysical and biochemical features of the native ECM in an artificial bio-microenvironment. One solution for the dilemma is to incorporate dECM in the biomaterial applied in the 3D printing. As reported by Ma et al. , the photo-patterning of cells and stiffness of the scaffold were precisely regulated via 3D printing of the dECM-blended bioink to provide biomimetic physical signals to the cells, while the biochemical cues embedded in the native liver ECM were simultaneously delivered. Similar results were achieved in a later study by another group (Mao et al. ). For control of the biochemical cues individually in the scaffold, highly engineered synthetic material customized with biomolecules of interest has been developed to facilitate the establishment of 3D culture systems. In an early collective study conducted by Taubenberger et al., PEG was decorated with biochemical mediators, such as metalloproteinase-cleavage spot, ECM mimicking cell adhesion peptides and a set of growth factors. The highly programmable platform they developed was able to create an in vitro bio-microenvironment with multiplexed biochemical cues and controlled mechanical properties of the matrix (Taubenberger et al. ). Another challenge is that the ECM is highly dynamic. Material properties must be carefully tuned to match the progression of the in vitro model and physiological process. For example, in the cardiac micro-tissue fabricated via DLP printing, the crosslinking density of the scaffold made of GelMA was carefully tuned, so that the degradation rate of the scaffold matched the ECM deposition of human cardiac fibroblasts (HCF), which essentially supported the maturation and contraction phenotype of the artificial tissue over 7 days (Miller et al. ). To improve the mimicry, a dynamic culture system could be combined with the artificial tissue. For example, a microfluidic chip with pressure channels was designed for colon tumor organoid culture to mimic the peristalsis, which is an important feature of their native microenvironment. The proliferation and organoid size were significantly increased compared to static culture as the media flowed through the pressure channels and mechanical stimulus was delivered. The peristalsis-stimulated organoid also showed decreased uptake and response to ellipticine-laden micelle dosing, implying that genuine recapitulation of ECM dynamics may have a significant impact on the in vitro model's drug/toxin response (Fang et al. ). On the other hand, native tissue has a complex cell composition, whereas much of the earlier research used only a few cell lines. It is an effective approach to simplify the model for certain scientific problems, but it hinders the recapitulation of tissue integrity, functionality, and response to drug/toxin doses. Organoid and in situ differentiation offer significant advantages in breaking down this barrier. Gu et al. created 3D neuron constructs from human neuron stem cells using extrusion-based 3D printing. After in situ differentiation of the stem cells, a neuron micro-tissue composed of functional neurons and supporting neuroglia was obtained (Gu et al. ). A follow-up study using iPSCs was conducted by the same group, demonstrating the strategy's potential for application in a variety of tissues (Gu et al. ). This method is particularly useful for models that require difficult-to-obtain cell sources. For example, because of the non-proliferating nature of cardiomyocytes and the requirement for high cardiomyocyte density, it has been difficult to establish an in vitro cardiac model in a large dimension and with integrated anatomy structures. Kupfer et al. used in situ differentiation of 3D printing iPSCs to create a cardiac organoid with highly biomimetic chamber structure and pumping activities on a 1.3 cm scale. While the organoid technology and in situ differentiation provide a versatile strategy to achieve multicellular construct, it is difficult to control the ratio of the cellular component. For designated cell composition, 3D printing could also be exploited as it provides high resolution for cell deposition. Focusing on the subject has resulted in significant progress. Because of their high yet biocompatible printing resolution, light-assisted printing processes, such as stereolithography and DLP printing, are intrinsically advantageous for multicellular printing. Furthermore, multiplexed printing systems have been developed to aid the facile multicellular printing; for example, multi-head and core–shell structured nozzles for extrusion and inkjet-based bioprinting have been developed. In place of the distinct flow mechanics at the micro-scale, microfluidic-based strategies can also be incorporated to the conventional 3D printing processes for precise control on the heterogeneity of the bioink. Ashammakhi et al. have provided a more in-depth review on the topic.
Another gap to be filled is the lack of mimicry on the systematic level. Many pharmacological and toxicological studies are hampered by poor in vitro–in vivo correlation because the response is usually generated by organ-organ interaction rather than a single organ or tissue. To address this concern, multi-organ-on-a-chip models incorporating multiple tissue models linked to a shared medium circulation system have been developed. Microfluidic devices have emerged as a powerful platform with individual chambers for the culture of each tissue and controlled flow of media enabled by designated channels, and extensive work has been devoted to such culture systems, resulting in improved drug response prediction outcomes (Sung ). Further improvement could be achieved by incorporating the evolving in vitro tissue models as discussed above as well as more customized and tailored design in the microfluidic devices for better mimicry of the flow dynamics and substance exchange (Goldstein et al. ). In addition, it is necessary for systematic modeling to involve the in vitro recapitulation of the immune system, which plays a critical role in many disease progression and pharmacological/toxicological responses, and has become increasingly important in the development of therapeutics for many conditions. Keeping this in mind, recent studies have focused on incorporating immune components into in vitro models and have achieved effective results. iPSC and THP-1-derived macrophages, for example, were incorporated into the in vitro glioblastoma microenvironment model to mimic the infiltration of macrophage alongside microglia via DLP printing. The engagement of the macrophages resulted in a significantly more authentic recapitulation of the original tissue, which had a substantial impact on the drug response of the model (Tang et al. ). THP-1 was also used to investigate the general pro-inflammatory response in a human-based multi-organ-on-a-chip model which involved hepatocyte, cardiomyocyte and skeletal muscle myoblasts (Sasserath et al. ). However, most of the work has been devoted to the innate immune system, while the in vitro modeling of the adaptive immune system remains to be explored. In fact, the state-of-the-art in vitro modeling of the immune system is still in its early stages. A more in-depth and comprehensive review regarding the topic has been provided by Polini et al. .
Furthermore, due to the limited resolution of bioprinting and cell deposition, it is difficult to recreate the microarchitecture and hierarchical nature of the tissue, which is particularly important in the integrated functionality of certain organs, such as the lungs, kidneys, and livers. The alveoli in the lungs, for example, are physically air sacs held and entangled by a network of capillaries (Fig. A). This extremely sophisticated structure is necessary for the exchange of oxygen and carbon dioxide between the pulmonary and circulatory systems, but it also adds a significant challenge to the fabrication process, particularly with soft materials. Earlier studies focused on multilayer 3D culture integrated on a PDMS chamber to capture the air–liquid interface in the alveolus for disease modeling and drug testing (Fig. B) (Benam et al. ; Huh et al. ). While the biochemical and histological characteristics of the tissue were successfully replicated to some extent, the membrane thickness of the blood vessels and the cell-liquid ratio were not fully recapitulated, resulting in inaccurate capture of tissue metabolism and biochemical signaling (Shrestha et al. ). The advanced 3D printing technique can improve the recapitulation of the alveolar microarchitecture. Innovatively applying food dye as a photo-absorber, Grigoryan et al. enhanced the printing resolution of soft hydrogel, PEGDA ( M w = 6 kDa), in DLP-based 3D printing (Fig. C). This allowed the printing of the complexed microstructure of alveolar with a biocompatible bioink, in which oxygenation and flow of human red blood cells was supported and vascularization of human umbilical vein endothelial cells (HUVECs) was achieved (Grigoryan et al. ). One limitation of this study is that the native alveolar cell composition was not included or tested, which limits the model's application in drug and toxin studies. Nonetheless, it is stated that the recapitulation of the exquisite native tissue's microarchitecture is within reach owing to the rising resolution of 3D printing techniques and a wider range of biomaterials.
As the nature route of nutrient and oxygen transportation and an essential part in the induction of organogenesis (Ding et al. ), vasculatures remain a stumbling block in both the maturation of organoids (Vargas-Valderrama et al. ) and the 3D fabrication of larger in vitro tissues. Currently, the major approach to introduce the vascularization to an in vitro model is to involve a sacrificial template. For example, Skylar-Scott et al. proposed a sacrificial writing into functional tissue (SWIFT) method, in which they compact a large volume of organoids in a thermal-gelling ECM and directly write the vascular channels with gelatin ink in an embedding manner at 0–4 °C. After printing, the gelatin was removed by perfusion as the construction was warmed up to 37 °C for future culture. This novel strategy enabled addition of vasculature and large-dimension culture of organoids (Skylar-Scott et al. ). The sacrificial gelatin ink could also facilitate direct printing of vasculatures in a co-axial extrusion-based 3D printing set-up. As reported by Shao et al. perfusable core–shell constructs with a scale larger than 1 cm have been printed with HUVECs mixed in the inner gelatin phase and tissue cells (human breast cancer cell MDA-MB-231 and mouse osteoblast MC3T3-E1) laden in the outer GelMA phase. After removal of gelatin and auto-seeding of HUVECs, the construct remodeled into a vasculature and was cultured for over 20 days (Shao et al. ). The phase-transition removal of gelatin is more biocompatible to avoid unwanted chemical stimulation compared to alginate-Ca and Pluronic, which are commonly used in earlier work, but the temperature shock involved in this process should be taken into consideration for specific cells and models (Lindquist and Mertens ). Moreover, the process to remove the template still hinders the efficient fabrication of the in vitro model. In contrast, DLP printing remains advantageous to fabricate vasculatures directly and rapidly. Zhu et al. reported the DLP-based continuous bioprinting of a pre-vascularized in vitro tissue with gradient channel diameter and biomimetic branching configuration. Facilitated with a motorized syringe pump, each construct was printed within 1 min; lumen-like structure and tight junctions of the endothelium was developed after culture for 1 week (Zhu et al. ). Despite the evolving techniques to produce vascularized perfusable channels, most work was conducted with primary HUVECs, which are isolated from an identical organ and stage of human life (Cao et al. ). However, the endothelium is highly heterogeneous throughout the different organs in terms of histological features, metabolism, angiogenesis and involvement in the immune response (Przysinda et al. ). It is noteworthy that in the development of an organ-specific vasculature, the diverse phenotypes of the endothelium should be recognized and well characterized.
High-throughput screening (HTS) systems are widely used by the pharmaceutical industry as an efficient method to process large numbers of compounds and molecular targets in a rapid manner. As such, pharmaceutical companies are turning to the use of 3D biomimetic human tissues in HTS format for preclinical toxicity testing of potential drug candidates. 3D bioprinting approaches, such as inkjet and micro-extrusion-based processes, are limited in scalability, resolution, patterning flexibility, and/or speed, which makes them unsuitable for high-throughput fabrication of complex cell-laden 3D microstructures within multi-well plates commonly used in HTS systems for drug screening and assay development. These shortcomings severely limit the widespread adoption of 3D printed cell culture platforms since researchers often rely on products configured to interface with commonly used lab instruments and equipment. Recently, DLP printing has made HTS possible. With rational integration of the micro-continuous projection printing and the automated well plate registration, Hwang et al. managed to fabricate complex 3D bio-constructs directly in a well plate rapidly and consistently. Depending on the complexity of the tissue construct, the total fabrication time for a fully populated 96-well plate typically ranges from 20 to 40 min (Hwang et al. ). Future work for printing in a 384-well plate or more wells is yet to be developed for HTS applications.
3D bioprinting has made significant progress in in vitro recapitulation of complex tissues. By applying an appropriate printing technique and biomaterial, accurate recapture of the ECM composition, cell population, bio-microenvironment, organ microarchitecture, and tissue functionality has been achieved in a variety of tissues, such as cancer, heart, muscle, liver, kidney, and will continue to be broadened and enhanced as the studies go on. Despite the encouraging results being achieved, it is noteworthy that they are usually labor and technique intensive. To bridge the gap between fundamental research and pragmatic applications, the scalability and reproducibility of the modeling strategies must also be addressed. In addition, with the arising need in the evaluation of therapeutic and substance in subdivided population and in the point-of-care scenario, the required time of production of personalized in vitro model must also be improved.
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Selecting Targets for Molecular Imaging of Gastric Cancer: An Immunohistochemical Evaluation | a5269e04-9773-4e11-a8aa-ba62e4885204 | 11860997 | Biochemistry[mh] | Gastric cancer is the fifth most common malignancy, with a worldwide incidence of more than 1 million cases per year. Despite recent therapeutic advances, prognosis remains poor, with a 5-year survival of approximately 40%, resulting in more than 700,000 deaths worldwide annually . Achieving local control through subtotal or total gastrectomy combined with lymphadenectomy remains the cornerstone of multidisciplinary gastric cancer treatment . Preoperatively, adequate disease staging is pivotal for patient-tailored treatment selection and maximizing its efficacy. Preoperative work-up of gastric cancers is comprised of endoscopy, computed tomography (CT) imaging, 18 F-fluorodeoxyglucose positron emission tomography ( 18 F-FDG PET) and/or diagnostic laparoscopy in clinically curable locally advanced disease (>cT3 and/or N+, M0) . However, each modality has its limitations for accurate tumor detection, potentially leading to erroneous tumor staging and, consequently, unnecessary tumor resections, futile biopsies, extra imaging procedures and/or unnecessary administration of systemic therapy. For example, CT imaging provides accurate T-staging (sensitivity 83–100% for tumors with serosal involvement), while sensitivity for N-staging is lower at approximately 60% . Importantly, sensitivity for small-sized distant metastases and peritoneal metastases is limited at 23–76%. Also, a significant proportion of gastric cancers have absent 18 F-FDG PET avidity (≈ 20%), and non-specific uptake in the stomach wall can also mask tumor presence . The use of 18 F-FDG PET for nodal and distant metastasis staging is also unsatisfactory, with sensitivity of 49% and 33–56%, respectively . To improve the accuracy of gastric cancer staging, diagnostic laparoscopy with or without peritoneal cytology is frequently performed . A systematic review highlighted that 9–60% of patients who were preoperatively staged as M0 had irresectable disease intraoperatively . Recently, the PLASTIC trial reported the limited added value of 18 F-FDG PET and showed the superiority of diagnostic laparoscopy in accurate staging of locally advanced gastric cancer . Limitations of laparoscopy, however, include its invasiveness, inability to accurately identify non-superficial liver metastases, lymph node metastases or extraperitoneal lesions, and the absence of tactile feedback for identifying malignant tissue . Besides tumor staging, an intraoperative challenge is encountered when radical resection is considered feasible. Studies showed that the presence of microscopically tumor-positive resection margins (i.e., R1 resection) is still observed in approximately 7% of gastric cancer patients, which has been associated with higher peritoneal recurrence rates and poorer survival . To address these challenges, tumor-targeted positron emission tomography (PET) and real-time fluorescence-guided surgery (FGS) using near-infrared light have emerged as valuable tools to enhance tumor imaging, respectively, by providing high-contrast visualization of malignant tissue . These molecular imaging technologies could improve assessment of tumor localization, potentially avoiding resection for irresectable disease, as well as assisting surgeons in radical tumor resection. However, the success of molecular imaging hinges on the adequate selection of tumor-specific targets. An ideal molecular imaging target is abundantly and homogenously expressed on the tumor cell membrane across all patients, while expression in healthy surrounding tissue is absent . Preferably, the target-of-interest is also present on lymph node and distant metastases and its expression remains present in microscopic residual disease after neoadjuvant therapy (NAT). However, governed by tumor heterogeneity, among others, a universal molecular imaging target for gastric cancer has still not been identified. Over the last years, several targets were recognized as promising for molecular imaging of gastrointestinal cancers, including integrin α v β 6 , carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor-2 (HER2) . Consequently, tracers targeting some of these biomarkers were evaluated in clinical trials for various gastrointestinal tumor types . However, their potential for molecular imaging of gastric cancer has been underexplored. This study, therefore, provides the first crucial step towards application of these tracers in gastric cancer, by evaluating α v β 6 , CEACAM5, EGFR, EpCAM and HER2 as molecular imaging targets for gastric cancer and its metastases. To accomplish this, biomarker expression was evaluated within the full anatomical context of gastric cancer. Biomarker expression was, therefore, determined using immunohistochemistry on human tissue specimens of primary tumors, healthy surrounding stomach tissue, but also on esophageal and duodenal tissue, and LN + and LN − , and distant metastases. Patient and Tissue Specimen Selection Pathology reports of patients who underwent resection for gastric adenocarcinoma at the Leiden University Medical Center (LUMC) from 2013 to 2020 were retrospectively reviewed. Representative formalin-fixed paraffin-embedded (FFPE) tissue blocks and hematoxylin and eosin (HE) slides of 87 patients, containing primary gastric tumor, healthy stomach, esophageal, duodenal and/or (metastatic) lymph node tissue, were selected and obtained from the biobank at the LUMC. To allow proper subgroup analyses, the cohort was constituted to contain approximately a 1:1 ratio of patients with diffuse and intestinal type tumors according to the Laurén classification. Patients with mixed type Laurén classification were excluded. Selection of FFPE tissue blocks was performed by a gastrointestinal pathologist (ASLPC) based on the HE slides. Tissue specimens containing gastric adenocarcinoma metastases biopsy tissue were also obtained from 19 patients. Clinicopathological data were obtained from patients’ medical records. Pathological tumor (pT) and pathological lymph node (pN) stages were defined according to the 8 th edition of the American Joint Committee on Cancer and Union for International Cancer Control (AJCC/UICC) TNM staging system for gastric cancer. The study protocol was approved by both the Gastroenterology Biobank Review Committee (protocol reference: 2020-16) as well as the local medical ethical review committee (protocol reference: B20.052). This study was conducted in compliance with the Dutch code of conduct for responsible use of human tissue in medical research. Tissue specimens and clinicopathological data were handled in an anonymized manner and in compliance with the Declaration of Helsinki (1964). Immunohistochemistry Tissue sections μm thick and cut from FFPE tissues were mounted on glass slides. Sections underwent deparaffinization in xylene for 15 min, followed by rehydration through sequential ethanol concentrations (100%, 50% and 25%). Subsequently, endogenous peroxidase was blocked using a 0.3% hydrogen peroxide solution. Antigen retrieval was tailored to the primary antibody employed, as outlined in Supplementary Table 1 in the electronic supplementary material (ESM). Post-antigen retrieval, slides were thoroughly rinsed in phosphate-buffered saline (PBS, pH 7.4). Primary antibodies (see Supplementary Table 1 in the ESM) targeting α v β 6 , CEACAM5, EGFR, EpCAM or HER2 were applied to the tissue sections, which were subsequently left to incubate overnight at room temperature in a humid incubator. After overnight incubation, slides were rinsed in PBS to remove any residual primary antibodies. Anti-mouse horseradish peroxidase (HRP) or anti-rabbit HRP secondary antibodies (Envision, Dako, Glostrup, Denmark) were subsequently applied for 30 min at room temperature in a humid incubator for 30 min. Secondary antibodies were then removed by thorough PBS rinsing. Visualization of antibody binding was achieved using a 3,3-diaminobenzidine (DAB) tetrahydrochloride solution (K3468, Agilent Technologies, Inc., Santa Clara, CA, USA) for 10 min at room temperature. Finally, slides were counterstained with Mayer’s hematoxylin (Klinipath B.V., Olen, Belgium), dehydrated in a dry incubator for 2 h and mounted using Pertex (Leica Microsystems, Wetzlar, Germany). Evaluation of Immunohistochemical Staining Whole slide images of the stained tissue slides were captured using the PANNORAMIC ® 250 Flash III DX scanner (3DHISTECH Ltd, Budapest, Hungary). DAB staining was quantified using the total immunostaining score (TIS), which was computed by multiplying the staining proportion (0 = ≤ 9%, 1 = 10–25%, 2 = 26–50%, 3 = 51–75%, 4 = ≥ 76%) by the staining intensity (0 = none, 1 = weak, 2 = moderate, 3 = strong). Staining based on the TIS was categorized as follows: 0 = negative; 1, 2, 3, 4 = weak expression; 6, 8 = moderate expression; 9, 12 = strong expression. A panel of three independent observers (RDH, MvD, ASLPC) conducted the scoring. Instances of disagreement were discussed in a consensus meeting, during which the final score was conclusively determined. Statistical Analysis For categorial data, groups at baseline were compared using a Chi-square test. An independent samples t test was used to compare continuous variables of patient characteristics. TIS values between tumor, healthy surrounding stomach, esophageal and duodenal tissue were compared using Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Biomarker expression subgroup analyses were performed using a Mann-Whitney test. IBM SPSS statistics version 29 (IBM Corporation, Armonk, NY, USA) was used for all statistical analyses of patient characteristics. Graphs and statistical analyses for biomarker expression were created and performed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA). Differences with a p value < 0.05 were considered significant. Pathology reports of patients who underwent resection for gastric adenocarcinoma at the Leiden University Medical Center (LUMC) from 2013 to 2020 were retrospectively reviewed. Representative formalin-fixed paraffin-embedded (FFPE) tissue blocks and hematoxylin and eosin (HE) slides of 87 patients, containing primary gastric tumor, healthy stomach, esophageal, duodenal and/or (metastatic) lymph node tissue, were selected and obtained from the biobank at the LUMC. To allow proper subgroup analyses, the cohort was constituted to contain approximately a 1:1 ratio of patients with diffuse and intestinal type tumors according to the Laurén classification. Patients with mixed type Laurén classification were excluded. Selection of FFPE tissue blocks was performed by a gastrointestinal pathologist (ASLPC) based on the HE slides. Tissue specimens containing gastric adenocarcinoma metastases biopsy tissue were also obtained from 19 patients. Clinicopathological data were obtained from patients’ medical records. Pathological tumor (pT) and pathological lymph node (pN) stages were defined according to the 8 th edition of the American Joint Committee on Cancer and Union for International Cancer Control (AJCC/UICC) TNM staging system for gastric cancer. The study protocol was approved by both the Gastroenterology Biobank Review Committee (protocol reference: 2020-16) as well as the local medical ethical review committee (protocol reference: B20.052). This study was conducted in compliance with the Dutch code of conduct for responsible use of human tissue in medical research. Tissue specimens and clinicopathological data were handled in an anonymized manner and in compliance with the Declaration of Helsinki (1964). Tissue sections μm thick and cut from FFPE tissues were mounted on glass slides. Sections underwent deparaffinization in xylene for 15 min, followed by rehydration through sequential ethanol concentrations (100%, 50% and 25%). Subsequently, endogenous peroxidase was blocked using a 0.3% hydrogen peroxide solution. Antigen retrieval was tailored to the primary antibody employed, as outlined in Supplementary Table 1 in the electronic supplementary material (ESM). Post-antigen retrieval, slides were thoroughly rinsed in phosphate-buffered saline (PBS, pH 7.4). Primary antibodies (see Supplementary Table 1 in the ESM) targeting α v β 6 , CEACAM5, EGFR, EpCAM or HER2 were applied to the tissue sections, which were subsequently left to incubate overnight at room temperature in a humid incubator. After overnight incubation, slides were rinsed in PBS to remove any residual primary antibodies. Anti-mouse horseradish peroxidase (HRP) or anti-rabbit HRP secondary antibodies (Envision, Dako, Glostrup, Denmark) were subsequently applied for 30 min at room temperature in a humid incubator for 30 min. Secondary antibodies were then removed by thorough PBS rinsing. Visualization of antibody binding was achieved using a 3,3-diaminobenzidine (DAB) tetrahydrochloride solution (K3468, Agilent Technologies, Inc., Santa Clara, CA, USA) for 10 min at room temperature. Finally, slides were counterstained with Mayer’s hematoxylin (Klinipath B.V., Olen, Belgium), dehydrated in a dry incubator for 2 h and mounted using Pertex (Leica Microsystems, Wetzlar, Germany). Whole slide images of the stained tissue slides were captured using the PANNORAMIC ® 250 Flash III DX scanner (3DHISTECH Ltd, Budapest, Hungary). DAB staining was quantified using the total immunostaining score (TIS), which was computed by multiplying the staining proportion (0 = ≤ 9%, 1 = 10–25%, 2 = 26–50%, 3 = 51–75%, 4 = ≥ 76%) by the staining intensity (0 = none, 1 = weak, 2 = moderate, 3 = strong). Staining based on the TIS was categorized as follows: 0 = negative; 1, 2, 3, 4 = weak expression; 6, 8 = moderate expression; 9, 12 = strong expression. A panel of three independent observers (RDH, MvD, ASLPC) conducted the scoring. Instances of disagreement were discussed in a consensus meeting, during which the final score was conclusively determined. For categorial data, groups at baseline were compared using a Chi-square test. An independent samples t test was used to compare continuous variables of patient characteristics. TIS values between tumor, healthy surrounding stomach, esophageal and duodenal tissue were compared using Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Biomarker expression subgroup analyses were performed using a Mann-Whitney test. IBM SPSS statistics version 29 (IBM Corporation, Armonk, NY, USA) was used for all statistical analyses of patient characteristics. Graphs and statistical analyses for biomarker expression were created and performed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA). Differences with a p value < 0.05 were considered significant. Patient Characteristics Eighty-seven patients diagnosed with gastric adenocarcinoma were included, of which 45 (52%) had diffuse type disease and 42 (48%) had intestinal type disease. Clinicopathological characteristics are summarized in Table . In the intestinal type group, 17 patients (40%) had well–moderately differentiated tumors, compared to 0 (0%) in the diffuse type group ( p < 0.001). NAT consisted of chemotherapy, while one patient received chemoradiotherapy. Albeit not statistically significant, there was a small difference in the number of patients who received NAT in both groups (diffuse type: 32 [71%]; intestinal type: 22 [52%]; p = 0.097). Other baseline characteristics did not differ between both groups. Biomarker Expression in Primary Gastric Cancer Tissue Specimens Tissue slides were stained for α v β 6 , CEACAM5, EGFR, EpCAM and HER2 expression, and expression was quantified using the TIS. Representative examples of these stainings are shown in Fig. . Positive expression (TIS values ≥ 1) on primary gastric tumors was found in 86% of the tumors for α v β 6 , 72% for CEACAM5, 77% for EGFR, 93% for EpCAM and 71% for HER2 (Table ). Categorized staining intensities are depicted in Table . All biomarkers showed a membranous staining pattern, with α v β 6 and EpCAM showing a mostly homogenous staining pattern, while staining was slightly more heterogeneous for CEACAM5, EGFR and HER2 (Fig. ). Additionally, biomarker co-expression in primary gastric tumors was analyzed (Table ). The highest co-expressing biomarker combination was α v β 6 and EpCAM, which were simultaneously expressed in 84% for primary gastric tumors. The remaining biomarker combinations were always expressed in more than 55% of cases, indicating moderate co-expression. Additionally, for all biomarker combinations, 88–98% of primary tumors were positive for at least one of the two biomarkers (≥ 1). Subgroup Analyses of Biomarker Expression in Primary Gastric Cancer Tissue Specimens Subgroup analyses revealed that median expression between diffuse and intestinal type tumors did not differ for all biomarkers except for HER2, which showed a lower median TIS on diffuse type tumors (median TIS 4.0 vs. 2.0, p = 0.0004; also see Supplementary Table 2 in the ESM). Moreover, subgroup analyses of biomarker expression in primary tumor tissues between patients who did not receive NAT and those who received NAT revealed that the median TIS for CEACAM5 and EGFR was lower on tumor specimens derived from patients who received NAT (CEACAM5: median TIS 9.0 vs. 3.5, p = 0.0215; EGFR: median TIS 6.0 vs. 3.0, p = 0.0072; also see Supplementary Table 3 in the ESM). For the remaining biomarkers, primary tumor expression was similar in patients who received NAT and patients who did not receive NAT. Biomarker Expression in Primary Gastric Cancer Versus Healthy Surrounding Stomach, Esophageal and Duodenal Tissue Specimens Images of sequential tumor sections showing biomarker expression in primary gastric cancer and healthy surrounding stomach, esophageal and duodenal tissue specimens are shown in Fig. . Quantified TIS values representing expression of α v β 6 , CEACAM5, EGFR, EpCAM and HER2 as well as the statistical comparison of biomarker expression is depicted in Fig. and Supplementary Table 4 in the ESM. For α v β 6 , median expression in primary gastric cancer tissue was lower compared to healthy surrounding stomach (median TIS 6.0 vs. 9.0, p < 0.0001) and duodenal tissue (median TIS 6.0 vs. 9.0, p = 0.0427), and similar to expression in esophageal tissue (median TIS 6.0 vs. 8.5, p > 0.9999). For CEACAM5, expression in primary tumor tissue was higher compared to healthy surrounding stomach (median TIS 4.0 vs. 0.0, p < 0.0001) and duodenal tissue (median TIS 4.0 vs. 0.0, p = 0.0003), but comparable to esophageal tissue (median TIS 4.0 vs. 4.0, p > 0.9999). EGFR expression in primary tumor tissue was higher compared to healthy surrounding stomach tissue (median TIS 4.0 vs. 2.0, p = 0.0023) but similar to esophageal (median TIS 4.0 vs. 6.0, p = 0.2235) and duodenal tissue (median TIS 4.0 vs. 3.0, p > 0.9999). EpCAM expression in tumor tissue was higher compared to healthy surround stomach (median TIS 9.0 vs. 0.0, p < 0.0001) and esophageal tissue (median TIS 9.0 vs. 0.0, p < 0.0001), but comparable to expression in duodenal tissue (median TIS 9.0 vs. 6.0, p = 0.7003). Lastly, HER2 expression in primary tumor tissue was not different from healthy surrounding stomach (median TIS 2.0 vs. 2.5, p > 0.9999) and esophageal tissue (median TIS 2.0 vs. 5.0, p = 0.1454) and lower than duodenal tissue (median TIS 2.0 vs. 8.0, p = 0.0152). Expression of Biomarkers in Tumor-Positive and Tumor-Negative Lymph Node Tissue Specimens Biomarker expression was evaluated on metastatic lymph nodes (LN + ) and tumor-negative lymph nodes (LN − ). Representative immunohistochemical (IHC) images showing expression of α v β 6 , CEACAM5, EGFR, EpCAM and HER2 on LN + tissue are shown in Fig. . Sensitivity, specificity, positive predictive value, negative predictive value and area under the curve were calculated based on dichotomous (positive/negative) biomarker expression and are depicted in Table . Although sensitivity for LN + versus LN − differentiation was moderate for HER2 and CEACAM5 (both 56%), no false-positive staining was observed. For the remaining biomarkers, higher sensitivity (range 72–82%) and 100% specificity for differentiation between LN + and LN − were observed, indicating their potential to serve as targets for imaging of metastatic lymph nodes. Accuracy for identifying LN + and LN − ranged between 82% and 93% for all biomarkers. Expression of Selected Biomarkers in Metastatic Gastric Cancer Tissue Specimens Based on their tumor-specific expression pattern and accurate LN + detection potential, CEACAM5, EGFR and EpCAM were selected for further analysis of their expression in metastatic gastric cancer tissue specimens derived from 19 patients. Patient characteristics of this cohort are described in Supplementary Table 5 in the ESM. Tissue specimens were derived from various locations, with the most common locations including the abdominal wall (4/19, 21%), peritoneum (3/19, 16%) and large/small intestine (both 2/19, 11%). Representative IHC images of HE, CEACAM5, EGFR and EpCAM staining are depicted in Fig. . Positive biomarker expression in metastatic gastric cancer tissue specimens was observed in 94% for CEACAM5, 88% for EGFR and 95% for EpCAM (Table ). As can be derived from Table , CEACAM5 and EpCAM staining was predominantly strong, while EGFR staining was somewhat weaker. Eighty-seven patients diagnosed with gastric adenocarcinoma were included, of which 45 (52%) had diffuse type disease and 42 (48%) had intestinal type disease. Clinicopathological characteristics are summarized in Table . In the intestinal type group, 17 patients (40%) had well–moderately differentiated tumors, compared to 0 (0%) in the diffuse type group ( p < 0.001). NAT consisted of chemotherapy, while one patient received chemoradiotherapy. Albeit not statistically significant, there was a small difference in the number of patients who received NAT in both groups (diffuse type: 32 [71%]; intestinal type: 22 [52%]; p = 0.097). Other baseline characteristics did not differ between both groups. Tissue slides were stained for α v β 6 , CEACAM5, EGFR, EpCAM and HER2 expression, and expression was quantified using the TIS. Representative examples of these stainings are shown in Fig. . Positive expression (TIS values ≥ 1) on primary gastric tumors was found in 86% of the tumors for α v β 6 , 72% for CEACAM5, 77% for EGFR, 93% for EpCAM and 71% for HER2 (Table ). Categorized staining intensities are depicted in Table . All biomarkers showed a membranous staining pattern, with α v β 6 and EpCAM showing a mostly homogenous staining pattern, while staining was slightly more heterogeneous for CEACAM5, EGFR and HER2 (Fig. ). Additionally, biomarker co-expression in primary gastric tumors was analyzed (Table ). The highest co-expressing biomarker combination was α v β 6 and EpCAM, which were simultaneously expressed in 84% for primary gastric tumors. The remaining biomarker combinations were always expressed in more than 55% of cases, indicating moderate co-expression. Additionally, for all biomarker combinations, 88–98% of primary tumors were positive for at least one of the two biomarkers (≥ 1). Subgroup analyses revealed that median expression between diffuse and intestinal type tumors did not differ for all biomarkers except for HER2, which showed a lower median TIS on diffuse type tumors (median TIS 4.0 vs. 2.0, p = 0.0004; also see Supplementary Table 2 in the ESM). Moreover, subgroup analyses of biomarker expression in primary tumor tissues between patients who did not receive NAT and those who received NAT revealed that the median TIS for CEACAM5 and EGFR was lower on tumor specimens derived from patients who received NAT (CEACAM5: median TIS 9.0 vs. 3.5, p = 0.0215; EGFR: median TIS 6.0 vs. 3.0, p = 0.0072; also see Supplementary Table 3 in the ESM). For the remaining biomarkers, primary tumor expression was similar in patients who received NAT and patients who did not receive NAT. Images of sequential tumor sections showing biomarker expression in primary gastric cancer and healthy surrounding stomach, esophageal and duodenal tissue specimens are shown in Fig. . Quantified TIS values representing expression of α v β 6 , CEACAM5, EGFR, EpCAM and HER2 as well as the statistical comparison of biomarker expression is depicted in Fig. and Supplementary Table 4 in the ESM. For α v β 6 , median expression in primary gastric cancer tissue was lower compared to healthy surrounding stomach (median TIS 6.0 vs. 9.0, p < 0.0001) and duodenal tissue (median TIS 6.0 vs. 9.0, p = 0.0427), and similar to expression in esophageal tissue (median TIS 6.0 vs. 8.5, p > 0.9999). For CEACAM5, expression in primary tumor tissue was higher compared to healthy surrounding stomach (median TIS 4.0 vs. 0.0, p < 0.0001) and duodenal tissue (median TIS 4.0 vs. 0.0, p = 0.0003), but comparable to esophageal tissue (median TIS 4.0 vs. 4.0, p > 0.9999). EGFR expression in primary tumor tissue was higher compared to healthy surrounding stomach tissue (median TIS 4.0 vs. 2.0, p = 0.0023) but similar to esophageal (median TIS 4.0 vs. 6.0, p = 0.2235) and duodenal tissue (median TIS 4.0 vs. 3.0, p > 0.9999). EpCAM expression in tumor tissue was higher compared to healthy surround stomach (median TIS 9.0 vs. 0.0, p < 0.0001) and esophageal tissue (median TIS 9.0 vs. 0.0, p < 0.0001), but comparable to expression in duodenal tissue (median TIS 9.0 vs. 6.0, p = 0.7003). Lastly, HER2 expression in primary tumor tissue was not different from healthy surrounding stomach (median TIS 2.0 vs. 2.5, p > 0.9999) and esophageal tissue (median TIS 2.0 vs. 5.0, p = 0.1454) and lower than duodenal tissue (median TIS 2.0 vs. 8.0, p = 0.0152). Biomarker expression was evaluated on metastatic lymph nodes (LN + ) and tumor-negative lymph nodes (LN − ). Representative immunohistochemical (IHC) images showing expression of α v β 6 , CEACAM5, EGFR, EpCAM and HER2 on LN + tissue are shown in Fig. . Sensitivity, specificity, positive predictive value, negative predictive value and area under the curve were calculated based on dichotomous (positive/negative) biomarker expression and are depicted in Table . Although sensitivity for LN + versus LN − differentiation was moderate for HER2 and CEACAM5 (both 56%), no false-positive staining was observed. For the remaining biomarkers, higher sensitivity (range 72–82%) and 100% specificity for differentiation between LN + and LN − were observed, indicating their potential to serve as targets for imaging of metastatic lymph nodes. Accuracy for identifying LN + and LN − ranged between 82% and 93% for all biomarkers. Based on their tumor-specific expression pattern and accurate LN + detection potential, CEACAM5, EGFR and EpCAM were selected for further analysis of their expression in metastatic gastric cancer tissue specimens derived from 19 patients. Patient characteristics of this cohort are described in Supplementary Table 5 in the ESM. Tissue specimens were derived from various locations, with the most common locations including the abdominal wall (4/19, 21%), peritoneum (3/19, 16%) and large/small intestine (both 2/19, 11%). Representative IHC images of HE, CEACAM5, EGFR and EpCAM staining are depicted in Fig. . Positive biomarker expression in metastatic gastric cancer tissue specimens was observed in 94% for CEACAM5, 88% for EGFR and 95% for EpCAM (Table ). As can be derived from Table , CEACAM5 and EpCAM staining was predominantly strong, while EGFR staining was somewhat weaker. Molecular imaging through tumor-targeted PET and FGS can address current limitations in pre- and intraoperative staging as well as resection margin assessment of gastric cancer. Adequate selection and application of molecular imaging targets is the main prerequisite for adequate tumor visualization using these techniques. The current study showed that α v β 6 , CEACAM5, EGFR, EpCAM and HER2, all promising tumor-specific targets for gastrointestinal cancers, were abundantly expressed in primary gastric tumor tissue specimens, with positive expression ranging from 71% to 93%. Regarding biomarker co-expression, 88–98% of primary gastric tumors showed positive expression of at least one of two biomarkers for all possible biomarker combinations, indicating the potential added value of bispecific tracers to increase the number of patients eligible for molecular imaging. Additionally, CEACAM5, EGFR and EpCAM showed higher expression in tumor tissue compared to healthy surrounding stomach tissue, classifying these targets as suitable for primary gastric cancer imaging. As α v β 6 and HER2 expression in healthy surrounding stomach tissue was higher or did not differ from primary tumor expression, respectively, we consider these targets not suitable for molecular imaging of primary gastric cancer. Despite moderate sensitivity for LN + detection observed for CEACAM5 and HER2 (both 56%), all biomarkers could distinguish LN + and LN − with high accuracy, indicating the potential of these targets for pre- and intraoperative N-staging. Lastly, EGFR, EpCAM and CEACAM5 showed moderate to strong expression in virtually all distant (peritoneal) metastases, highlighting their potential as targets for M-staging. Our study therefore demonstrated the feasibility of EGFR, EpCAM and CEACAM5 as molecular imaging targets for gastric cancer in a clinically relevant context. The abundant tumor expression of the biomarkers reported herein is largely in line with previous studies, albeit we reported higher percentages of positive IHC staining compared to previous research, particularly for α v β 6 , EGFR and HER2 . This could, among others, have been caused by the use of different scoring systems, primary antibodies or antigen retrieval techniques during IHC staining, as well as inter- and intratumoral heterogeneity, and the relatively small sample sizes of previous IHC studies . Confirmation of our results in a different or larger cohort of gastric cancer patients could verify validity of the results observed herein, as well as elucidate underlying mechanisms contributing to these discrepancies. A strong methodological point of this study is the additional evaluation of biomarker expression in healthy surrounding esophageal and duodenal tissue specimens. Similar CEACAM5 and EpCAM expression levels were found on healthy esophageal and duodenal tissue compared to tumor expression, respectively, while EGFR expression in both tissue types did not differ from tumor expression. Consistent with our findings, expression of CEACAM5 and EGFR has been identified in healthy esophageal tissue, while EGFR and EpCAM expression in, respectively, duodenal mucosa and epithelia of both the small and large intestines was also reported . Although previous literature described that EpCAM is overexpressed in gastrointestinal tumors compared to healthy surrounding tissue, it should be noted that EpCAM’s presence in the small and large intestine might impact the detection of peritoneal metastases of gastric cancer using EpCAM-targeted molecular imaging tracers . Taken further, the absence of EpCAM in esophageal tissue makes EpCAM a more suitable target for delineation of proximal gastric cancers located near the esophageal-gastric junction (EGJ), while absence of CEACAM5 on duodenum epithelium makes this target appropriate for assessing resection margins of distal gastric cancers invading the duodenum. Of note, EGJ and duodenal invasion are frequently present in (sub)cardia (33–50%) and distal gastric cancer (14–33%), respectively . Moreover, considering the increased R1 resection rate and reduced patient survival in these locally advanced cancers, adequate intraoperative tumor delineation may be a valuable tool to improve adequate resection margin assessment and, potentially, patient outcomes . In addition, the inclusion of patients with diffuse and intestinal type adenocarcinomas, as well as patients who received NAT, allowed subgroup analyses to study potential effect of these clinicopathological factors on the biomarkers’ expression level. Interestingly, we found similar biomarker expression in diffuse and intestinal type adenocarcinomas for all biomarkers, except for HER2, which showed lower TIS values on intestinal type tumor tissue specimens. This makes the remaining biomarkers broadly applicable as molecular imaging markers in gastric cancer patients. Moreover, this finding is particularly promising for molecular imaging of diffuse type gastric cancers, given the lower 18 F-FDG PET avidity, more frequent underestimation of the proximal margin length and increased irradical resection rate in this histological subtype . Additionally, subgroup analyses revealed that CEACAM5 and EGFR expression was lower in patients who received NAT. Consequently, care should be taken when targeting CEACAM5 and EGFR for molecular imaging of primary gastric tumors after NAT. Preoperatively, several targeted PET tracers have aimed to address current limitations in staging of gastric cancer in both the preclinical and clinical setting, with a strong focus on fibroblast activation protein (FAP)-targeted agents . FAP is expressed in 55–75% of gastric carcinomas and is associated with increased migration, invasion and reduced survival, while expression in healthy surrounding tissues is virtually absent . A recent meta-analysis showed that FAPI PET outperformed conventional 18 F-FDG PET sensitivity for primary tumor, lymph node metastasis and peritoneal dissemination of gastric cancer, thereby indicating the potential of both FAPI PET and targeted PET in general . However, the overexpression of FAP in tissue during instances of tissue remodeling, such as wound healing or chronic inflammation, could pose a threat for its potential to delineate benign from malignant tissue . Nevertheless, although our study intended to focus on tumor cell-specific molecular imaging targets, additional evaluation and comparison of FAP expression in our cohort would be an interesting continuation of this study. Of the targets evaluated herein, only HER2 has been clinically targeted for PET imaging in gastric cancer. Using 89 Zr-trastuzumab, O'Donoghue et al. observed tumor accumulation in 80% of patients with positive HER2 status; however, not all known lesions could be visualized in these patients . Interestingly, the authors did not observe significant stomach uptake as one would expect based on our observation of similar HER2 expression in primary tumors and healthy surrounding stomach tissue. It should be noted that, although positive biomarker expression remains a fundamental criterion for successful molecular imaging, it does not invariably correlate with positive tumor uptake in the clinical setting, underscoring the importance of both tumor heterogeneity and extensive clinical validation of molecular imaging tracers. Noteworthy, significant stomach wall and intestine uptake is commonly reported for α v β 6 -targeting PET tracers, thus reflecting our findings of high α v β 6 expression in these tissue types . FGS-related research in gastric cancer has particularly focused on fluorescence-guided lymphadenectomy, as opposed to primary tumor imaging or intraoperative tumor staging. For instance, Chen et al. randomized gastric cancer patients between indocyanine green tracer-guided (ICG) lymphadenectomy using submucosal injection 1 day preoperatively and conventional laparoscopic gastrectomy . The authors showed that ICG lymphadenectomy yielded more lymph nodes compared to the non-ICG group, leading to less unremoved lymph node stations, while complication rates between both groups were similar. However, sensitivity for metastatic lymph node detection was moderate at 56%. Considering these and previously outlined constraints in accurate intraoperative staging of gastric cancer staging, redirecting focus in FGS-related research towards tumor-targeted imaging could pave the way for novel tracers that address these limitations. This study has some limitations. For instance, the relatively small sample size may have reduced the robustness of our subgroup analyses. Therefore, the findings of the subgroup analyses reported herein, although relevant for the assessment of a molecular imaging target’s suitability, should be interpreted with caution. Secondly, due to the presence of staining artefacts, some slides were not suitable for scoring. Nevertheless, as the amount of excluded tissue slides per marker was limited (maximum 3/87 primary tumor specimens), we do not anticipate this influenced the findings of our study and the reproducibility thereof. Future research into molecular imaging targets for gastric cancer could focus on their expression in premalignant tissue, such as chronic gastritis, intestinal metaplasia or dysplasia, thereby establishing the targets’ potential for differentiation between malignant and benign tissue . Also, as molecular targets are not expressed in all patients, preoperative screening for positive biomarker expression could be performed, followed by application of the most suitable molecular imaging tracer. When feasible, such a strategy would form a robust and efficient way of patient-tailored employment of molecular imaging tracers in gastric cancer, maximizing its potential to improve pre- and intraoperative staging as well as resection margin assessment. This may be performed using biopsy material of primary gastric tumors or metastases, which is routinely obtained for histological diagnosis. Moreover, the predictive value of biomarker expression in tumor biopsies for primary gastric tumor expression remains to be elucidated. Our findings show that CEACAM5, EGFR and EpCAM are promising targets for molecular imaging of gastric cancer, as well as lymph node and distant metastases. By improving pre- and intraoperative identification of tumor tissue, targeted PET and FGS could enhance gastric cancer staging and resection, ultimately leading to improved patient outcomes. Further clinical evaluation of PET and FGS tracers targeting these antigens is warranted. Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 28 KB) |
Time-resolved proteomic profiling of | f6a0d4fd-2fc1-4824-9704-912e12e19065 | 11886801 | Biochemistry[mh] | The use of copper-based antimicrobials is a re-emerging strategy for decontamination purposes in agriculture, food industry, health settings, and even spaceflight applications . Before implementation, the bacteriostatic effect of Cu ions needs to be thoroughly tested. It has been shown that sublethal concentrations of Cu ions can induce the so-called viable-but-nonculturable (VBNC) cell state in several bacterial species . This VBNC state is thought to be a cellular survival strategy with the purpose of ensuring viability under harsh environmental conditions such as nutrient depletion, drastic temperature shifts, or exposure to metals, among others . A central feature of VBNC cells is their inability to grow on regular microbiological media. This is a concern when relying on culture-based testing for bacterial diagnostics and monitoring as it could lead to false negative results . Under more favorable conditions, VBNC cells can restore culturability, a phenomenon called resuscitation. During resuscitation, vital cellular functions are recovered, including the virulence of pathogenic bacteria that might pose a risk in settings such as public health . Resuscitation is usually (but not necessarily) triggered by external factors reverting the unfavorable conditions, such as nutrient supplementation, metal ion chelation, but also biological stimulation . Conversely, spontaneous resuscitation based on the activation of intrinsic factors is rarely described. Recently, Maertens et al . found that water-borne cells of Cupriavidus metallidurans enter the VBNC state upon exposure to low levels of Cu ions, and spontaneously resuscitate after 7 days of incubation . Cupriavidus metallidurans , a soil bacterium from the Burkholderiaceae family, is a model organism for the study of bacterial metal resistance. Type strain CH34 features a remarkable amount of metal resistance genes, many of which are located on one of its two megaplasmids . In the aforementioned study, resuscitation was only possible when these plasmid-encoded metal resistance determinants were present . This finding highlighted an interesting association between the VBNC state and bacterial Cu resistance. In the present study, we are further investigating this association at a proteomic level. In recent years, efforts to elucidate cellular mechanisms controlling the VBNC state have increasingly focused on the application of omics techniques . However, only a few studies explored the Cu-induced VBNC state . Here, we analyzed whole proteome adaptations of C. metallidurans CH34 during a Cu-induced VBNC state and, notably, during spontaneous resuscitation. Rather than comparing only two different cell populations (e.g. before and after VBNC induction), our aim was to capture time-resolved proteomic changes through sampling at multiple time points. This approach allowed us to gain valuable insights into the dynamics of the VBNC state in C. metallidurans CH34, serving as a stepping stone toward the unravelling of the enigmatic VBNC state which can eventually help to improve countermeasures against bacterial contaminations.
Bacterial strains and culture conditions Cupriavidus metallidurans strain CH34 was routinely grown in Tris-buffered minimal medium (MM284) with 0.2% Na-gluconate (VWR chemicals, Leuven, Belgium) at 30°C. Liquid cultures were grown in a shaking incubator at 180 rpm. Agar plates were prepared by adding 2% bacteriological agar (Oxoid, Hampshire, UK). For motility assays, 0.4% agar plates were prepared. VBNC induction, resuscitation, and motility assays Fully grown cultures were washed twice with filter-sterilized (0.2 µm filter) bottled mineral water (Ordal, Belgium and Evian, France; additional information on their composition can be found in ) and diluted to a starting inoculum of 10 8 cells/ml in the same mineral water. To induce the VBNC state, 10 µM CuSO 4 (Merck KGaA, Darmstadt, Germany) was added, while no CuSO 4 was added to the control condition, and samples were incubated in a shaking incubator at 30°C. Where applicable, 5.6 mM thiamine hydrochloride (Sigma-Aldrich, Saint Louis, Missouri, USA) was added. Samples were drawn after 0, 1, 2, 3, 4, 5, 24, 48, 72, 96, and 120 h of incubation, and the total viable count was determined by plating 100 µl of a serial 10-fold dilution (in sterile PBS) on MM284 agar and counting after 3 days at 30°C. To test swarming potential, 10 µl of cell suspension from samples at time points 48, 72, 96, and 120 h was plated onto MM284-gluconate plates containing 0.4% bacterial agar. After 3 days of incubation at 30°C, the visible swarming diameter was measured with a ruler and divided by corresponding CFU counts to yield the normalized swarming range. All experimental conditions were carried out in biological triplicates. Statistical analysis was performed using unpaired t -tests implemented in GraphPad Prism 10. Cell counting, viability determination, and PHB quantification via flow cytometry All flow cytometry assays were performed on a NovoCyte Quanteon flow cytometer (Agilent, Santa Clara, California, USA). Similar to the viable count, samples were drawn after 0, 1, 2, 3, 4, 5, 24, 48, 72, 96, and 120 h of incubation. Viability was assessed with a SYBR Green/PI assay. Briefly, cell suspensions were 100-fold diluted in filtered mineral water and SYBR Green (Life technologies, Carlsbad, California, USA) and propidium iodide (Merck KGaA, Darmstadt, Germany) were added to a final concentration of 1X (starting from a 10 000X commercial stock solution) and 200 µM, respectively. Suspensions were incubated in the dark at 37°C for 20 min to allow complete binding of the dyes. Stained cells (including autoclaved cells as a control for dead cells) were analyzed in “fast” flow mode and fluorescence was acquired using the FITC and PI detection channels. Additionally, total cell numbers in unstained cell suspensions were enumerated based on manual forward vs side scatter (FSC vs SSC) gating, excluding background events. To quantify intracellular polyhydroxybutyrate (PHB) levels, the protocol described in was adapted to flow cytometry: Nile Red stock solution (1 mg/ml) was prepared by dissolving Nile Red solid dye (Sigma-Aldrich, Saint Louis, Missouri, USA) in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint Louis, Missouri, USA) and filter-sterilizing the solution through a 0.45 µm DMSO-proof filter. C. metallidurans cells were diluted to approximately 10 6 cells/ml in filtered mineral water and stained by adding Nile Red stock solution to a final dye concentration of 1 µg/ml, followed by a 30-min incubation step at 30°C. Stained and unstained samples (including a water blank) were analyzed in “fast” flow mode and fluorescence was acquired using the PE-Cy5 detection channel. Data analysis was carried out using the provided NovoExpress software (Agilent, Santa Clara, California, USA) and GraphPad Prism 10. For PHB quantification, median fluorescence intensities were compared using Mood's median test and effect sizes were calculated using Cramér's V . Protein extraction Samples for protein extraction were taken every 60 min until 5 h of incubation, as well as 24, 48, 72, 96, and 120 h after the start of the experiment. Samples were pelleted and washed twice with ice-cold PBS (Life technologies, Carlsbad, California, USA). Pellets were then resuspended in lysis buffer [2% sodium dodecyl sulfate (Life technologies, Carlsbad, California, USA) in 50 mM ammonium bicarbonate aqueous solution (Sigma-Aldrich, Santa Clara, California, USA)], vortexed for 30 s and subsequently incubated at 95°C for 5 minutes. After cooling on ice, lysis was completed through sonication using a UP50H ultrasonic processor (Hielscher, Teltow, Germany). The lysates were centrifuged at 14 000 g and 4°C for 20 min and supernatants were stored at −80°C for further processing. Protein concentrations in the crude extracts were determined using the bicinchoninic acid kit for protein determination (Sigma-Aldrich, Santa Clara, California, USA) following the manufacturer's instructions. Protein sample processing Extracted proteins were further processed using the suspension trapping method, S-trap (Bioconnect, Huissen, The Netherlands), following the manufacturer's instructions. The LC-MS/MS analysis was performed using a nanoElute UHPLC (Bruker Daltonics, Bremen, Germany) connected to a QTOF-MS instrument (Impact II, Bruker Daltonics, Germany) via a CaptiveSpray nanoflow electrospray source (Bruker Daltonics, Bremen, Germany). Samples were run in a random order in order to identify a potential batch effect. In total, 2 µg of tryptic digest (in solvent A) was injected onto a trapping column setup (300 µm x 5 mm, C18 PepMap 300, 5 µm, 100 Å; Bruker Daltonics, Bremen, Germany). Subsequently, peptides were separated using a C18 Reprosil AQ, 1.9 µm, 120 Å, 0.075 × 150 mm column operated at 40°C (Bruker Daltonics, Bremen, Germany) at a flow rate of 0.2 µl/min. Gradient conditions were: 2%–35% solvent B for 100 min; 35%–95% solvent B for 10 min; and 95% solvent B held for 10 min (solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile). Drying gas flow and temperature of the CaptiveSpray were set to 4 l/min and 180°C, respectively, and nebulizer gas pressure was set to 0.4 bar. The MS acquisition rate was set to 2 Hz and data have been acquired over a 150–2200 m/z mass range. In all the full-scan measurements, a lock-mass (m/z 1221.9906, Hexakis (1H, 1H, 4H-hexafluorobutyloxy)phosphazine) (Bruker Daltonics, Bremen, Germany) was used as an internal calibrator. The Instant Expertise method (Compass otofSeries 4.1, Bruker Daltonics, Bremen, Germany) was used to select as many as possible of the most intense ions per cycle of 3 s MS/MS accumulation depending on the MS1 level. The threshold (per 1000 summation) absolute was 2500 cts (spectral rate of 2 Hz). Peptide fragmentation was performed with nitrogen gas on the most abundant and at least doubly charged to five charged ions detected in the initial MS scan. Active exclusion was performed after 1 spectrum for 0.50 min unless the intensity of the precursor ions was more than 3 times higher than in the previous scan. Proteomics data analysis All raw mass spectrometry spectra files were processed using MaxQuant software version 2.0.1.0 and proteins were identified with the built-in Andromeda search engine . In total, 60 raw files, with three replicates of each 20 conditions (control and Cu-induced at 10 different time points) were processed in parallel. The MS/MS spectra searches were performed with a database containing all C. metallidurans CH34 (Taxonomy ID: 266 264) UniProt protein sequences (downloaded from ftp.uniprot.org on 2022-03-30). Searches were performed with default MaxQuant parameter settings with cysteine carbamidomethylation as fixed modification, and methionine oxidation and Protein N-terminal acetylation as variable modification. False-discovery rate cutoffs were set to 1% on peptide, protein, and site decoy level, trypsin as a digestion enzyme and seven amino acids as minimum peptide length. The resulting data from MaxQuant with minimum two unique peptides was retained, processed to remove reverse hits and contaminants, and Log 2 transformed for further analysis. The technical variation among replicates was removed by median normalization on Log 2 -transformed intensities. Missing values in the data were replaced by values from a normal distribution with settings width = 0.3 and downshift = 1.8 . Sample distribution was examined via density and box plots, as well as PCA analysis . Protein differential expression was calculated using the LIMMA R package , based on the empirical Bayes moderated test-statistics. Differential expression was considered significant at a q -value < 0.05 and FC ≤ 0.67 or ≥ 1.5 (|Log 2 -FC| ≥ 0.5849).
Cupriavidus metallidurans strain CH34 was routinely grown in Tris-buffered minimal medium (MM284) with 0.2% Na-gluconate (VWR chemicals, Leuven, Belgium) at 30°C. Liquid cultures were grown in a shaking incubator at 180 rpm. Agar plates were prepared by adding 2% bacteriological agar (Oxoid, Hampshire, UK). For motility assays, 0.4% agar plates were prepared.
Fully grown cultures were washed twice with filter-sterilized (0.2 µm filter) bottled mineral water (Ordal, Belgium and Evian, France; additional information on their composition can be found in ) and diluted to a starting inoculum of 10 8 cells/ml in the same mineral water. To induce the VBNC state, 10 µM CuSO 4 (Merck KGaA, Darmstadt, Germany) was added, while no CuSO 4 was added to the control condition, and samples were incubated in a shaking incubator at 30°C. Where applicable, 5.6 mM thiamine hydrochloride (Sigma-Aldrich, Saint Louis, Missouri, USA) was added. Samples were drawn after 0, 1, 2, 3, 4, 5, 24, 48, 72, 96, and 120 h of incubation, and the total viable count was determined by plating 100 µl of a serial 10-fold dilution (in sterile PBS) on MM284 agar and counting after 3 days at 30°C. To test swarming potential, 10 µl of cell suspension from samples at time points 48, 72, 96, and 120 h was plated onto MM284-gluconate plates containing 0.4% bacterial agar. After 3 days of incubation at 30°C, the visible swarming diameter was measured with a ruler and divided by corresponding CFU counts to yield the normalized swarming range. All experimental conditions were carried out in biological triplicates. Statistical analysis was performed using unpaired t -tests implemented in GraphPad Prism 10.
All flow cytometry assays were performed on a NovoCyte Quanteon flow cytometer (Agilent, Santa Clara, California, USA). Similar to the viable count, samples were drawn after 0, 1, 2, 3, 4, 5, 24, 48, 72, 96, and 120 h of incubation. Viability was assessed with a SYBR Green/PI assay. Briefly, cell suspensions were 100-fold diluted in filtered mineral water and SYBR Green (Life technologies, Carlsbad, California, USA) and propidium iodide (Merck KGaA, Darmstadt, Germany) were added to a final concentration of 1X (starting from a 10 000X commercial stock solution) and 200 µM, respectively. Suspensions were incubated in the dark at 37°C for 20 min to allow complete binding of the dyes. Stained cells (including autoclaved cells as a control for dead cells) were analyzed in “fast” flow mode and fluorescence was acquired using the FITC and PI detection channels. Additionally, total cell numbers in unstained cell suspensions were enumerated based on manual forward vs side scatter (FSC vs SSC) gating, excluding background events. To quantify intracellular polyhydroxybutyrate (PHB) levels, the protocol described in was adapted to flow cytometry: Nile Red stock solution (1 mg/ml) was prepared by dissolving Nile Red solid dye (Sigma-Aldrich, Saint Louis, Missouri, USA) in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint Louis, Missouri, USA) and filter-sterilizing the solution through a 0.45 µm DMSO-proof filter. C. metallidurans cells were diluted to approximately 10 6 cells/ml in filtered mineral water and stained by adding Nile Red stock solution to a final dye concentration of 1 µg/ml, followed by a 30-min incubation step at 30°C. Stained and unstained samples (including a water blank) were analyzed in “fast” flow mode and fluorescence was acquired using the PE-Cy5 detection channel. Data analysis was carried out using the provided NovoExpress software (Agilent, Santa Clara, California, USA) and GraphPad Prism 10. For PHB quantification, median fluorescence intensities were compared using Mood's median test and effect sizes were calculated using Cramér's V .
Samples for protein extraction were taken every 60 min until 5 h of incubation, as well as 24, 48, 72, 96, and 120 h after the start of the experiment. Samples were pelleted and washed twice with ice-cold PBS (Life technologies, Carlsbad, California, USA). Pellets were then resuspended in lysis buffer [2% sodium dodecyl sulfate (Life technologies, Carlsbad, California, USA) in 50 mM ammonium bicarbonate aqueous solution (Sigma-Aldrich, Santa Clara, California, USA)], vortexed for 30 s and subsequently incubated at 95°C for 5 minutes. After cooling on ice, lysis was completed through sonication using a UP50H ultrasonic processor (Hielscher, Teltow, Germany). The lysates were centrifuged at 14 000 g and 4°C for 20 min and supernatants were stored at −80°C for further processing. Protein concentrations in the crude extracts were determined using the bicinchoninic acid kit for protein determination (Sigma-Aldrich, Santa Clara, California, USA) following the manufacturer's instructions.
Extracted proteins were further processed using the suspension trapping method, S-trap (Bioconnect, Huissen, The Netherlands), following the manufacturer's instructions. The LC-MS/MS analysis was performed using a nanoElute UHPLC (Bruker Daltonics, Bremen, Germany) connected to a QTOF-MS instrument (Impact II, Bruker Daltonics, Germany) via a CaptiveSpray nanoflow electrospray source (Bruker Daltonics, Bremen, Germany). Samples were run in a random order in order to identify a potential batch effect. In total, 2 µg of tryptic digest (in solvent A) was injected onto a trapping column setup (300 µm x 5 mm, C18 PepMap 300, 5 µm, 100 Å; Bruker Daltonics, Bremen, Germany). Subsequently, peptides were separated using a C18 Reprosil AQ, 1.9 µm, 120 Å, 0.075 × 150 mm column operated at 40°C (Bruker Daltonics, Bremen, Germany) at a flow rate of 0.2 µl/min. Gradient conditions were: 2%–35% solvent B for 100 min; 35%–95% solvent B for 10 min; and 95% solvent B held for 10 min (solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile). Drying gas flow and temperature of the CaptiveSpray were set to 4 l/min and 180°C, respectively, and nebulizer gas pressure was set to 0.4 bar. The MS acquisition rate was set to 2 Hz and data have been acquired over a 150–2200 m/z mass range. In all the full-scan measurements, a lock-mass (m/z 1221.9906, Hexakis (1H, 1H, 4H-hexafluorobutyloxy)phosphazine) (Bruker Daltonics, Bremen, Germany) was used as an internal calibrator. The Instant Expertise method (Compass otofSeries 4.1, Bruker Daltonics, Bremen, Germany) was used to select as many as possible of the most intense ions per cycle of 3 s MS/MS accumulation depending on the MS1 level. The threshold (per 1000 summation) absolute was 2500 cts (spectral rate of 2 Hz). Peptide fragmentation was performed with nitrogen gas on the most abundant and at least doubly charged to five charged ions detected in the initial MS scan. Active exclusion was performed after 1 spectrum for 0.50 min unless the intensity of the precursor ions was more than 3 times higher than in the previous scan.
All raw mass spectrometry spectra files were processed using MaxQuant software version 2.0.1.0 and proteins were identified with the built-in Andromeda search engine . In total, 60 raw files, with three replicates of each 20 conditions (control and Cu-induced at 10 different time points) were processed in parallel. The MS/MS spectra searches were performed with a database containing all C. metallidurans CH34 (Taxonomy ID: 266 264) UniProt protein sequences (downloaded from ftp.uniprot.org on 2022-03-30). Searches were performed with default MaxQuant parameter settings with cysteine carbamidomethylation as fixed modification, and methionine oxidation and Protein N-terminal acetylation as variable modification. False-discovery rate cutoffs were set to 1% on peptide, protein, and site decoy level, trypsin as a digestion enzyme and seven amino acids as minimum peptide length. The resulting data from MaxQuant with minimum two unique peptides was retained, processed to remove reverse hits and contaminants, and Log 2 transformed for further analysis. The technical variation among replicates was removed by median normalization on Log 2 -transformed intensities. Missing values in the data were replaced by values from a normal distribution with settings width = 0.3 and downshift = 1.8 . Sample distribution was examined via density and box plots, as well as PCA analysis . Protein differential expression was calculated using the LIMMA R package , based on the empirical Bayes moderated test-statistics. Differential expression was considered significant at a q -value < 0.05 and FC ≤ 0.67 or ≥ 1.5 (|Log 2 -FC| ≥ 0.5849).
Cupriavidus metallidurans enters the VBNC state upon Cu stress and resuscitates spontaneously Maertens et al . identified the transition into a VBNC state when C. metallidurans cells are exposed to elevated Cu 2+ levels in drinking water, showing spontaneous resuscitation within several days of incubation . In a first step, we corroborated this observation and added sampling time points as well as statistical analyses. Mineral water (Ordal) was inoculated with cells of type strain CH34 to a starting cell number of approximately 10 8 cells/ml and supplemented with 10 µM CuSO 4 (no CuSO 4 in control condition). Colony forming units were assessed during the first 5 h and every 24 h after starting the experiment (Fig. ). In agreement with Maertens et al ., the number of culturable cells decreased by 4 Log during the first hours of Cu treatment. Already after 1 h, Cu-treated cells showed a significant reduction in culturability ( P < .001). From 24 h (1 day) after the start of Cu treatment, a gradual increase in CFU counts could be observed, while cell numbers in the control condition were staying relatively constant. Until 96 h (4 days), the reduction in culturability was still significant ( P < .05). From 120 h (5 days) onwards, no statistical difference between Cu and control condition could be observed, corresponding to the complete resuscitation (Fig. ). Throughout the experiment, the total cell numbers as assessed by flow cytometry were not significantly different between the Cu-treated and control conditions . In addition, cell viability was also assessed with a SYBR Green/PI assay that monitors viability based on membrane integrity, which indicated that 10%–20% of Cu-treated cells remained viable between 24 and 120 h of exposure . Finally, to verify that VBNC induction and resuscitation was not unique to the used mineral water, we tested an additional mineral water (Evian; ), which showed the same phenomena. VBNC and resuscitating cells show dynamic proteomes To investigate the proteomic state of VBNC cells and during resuscitation, cells were pelleted for protein extraction at the following sample time points: 1, 2, 3, 4, 5, 24, 48, 72, 96, and 120 h of incubation (Cu and control condition). Proteome analyses were performed via protein extraction and subsequent LC-MS/MS analysis. All detected proteins, including data from single replicates, are listed in . The number of identified proteins for each time point ranged between 2195 and 2570 (Table , Fig. ). In general, a good coverage of CH34’s annotated proteome could be reached, with an average of 2373 identified proteins, which amounts to 36.6% of the 6488 annotated coding sequences in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database . For each time point, DE proteins were determined by comparing their abundance in Cu-treated and control samples. Proteins with a FC ≤0.67 or ≥1.5 (|Log2-FC| ≥0.5849) and q -values <0.05 were considered DE (Table ). The number of DE proteins was the lowest at 5 h with 32 DE proteins (Fig. ). Afterward, the number of DE proteins gradually increased to reach a maximum at 120 h (1038 DE proteins). Identified proteins were categorized based on Clusters of Orthologous Genes (COGs) . For each COG class, the number of up-/downregulated proteins among the total number of proteins linked to the COG class was determined and represented as a ratio. Repeating this step for every time point, we obtained an overview of proteomic adaptations in VBNC and resuscitating cells (Fig. ). At most time points, high numbers of upregulated proteins (ratios up to 0.5) were observed in the classes “Translation, ribosomal structure and biogenesis,” “Cell motility,” and “Intracellular trafficking, secretion, and vesicular transport.” These classes coincidently showed low numbers of downregulated proteins (ratios partly below 0.1). In contrast, many proteins were continuously downregulated in e.g. “Energy production and conversion,” “Transcription,” “Replication, recombination, and repair” and “Secondary metabolites' biosynthesis, transport and catabolism.” For other classes, the ratios fluctuated more strongly at different phases of incubation. “Cell cycle control, cell division, chromosome partitioning” as well as “Lipid transport and metabolism” (among others) showed high numbers of upregulated proteins between 48 and 120 h (2–5 days, resuscitation phase), but not in the early incubation stage (1–4 h, VBNC induction phase). Conversely, the class “Cell wall/membrane/envelope biogenesis” had many upregulated proteins only in the early incubation stage. Based on COG analysis, we can assume that cells going through the VBNC state and resuscitation are characterized by highly dynamic proteomic profiles. Next, we showcased in detail the similarities and differences between the proteomes of these two cell states. Motility is activated by Cu treatment A noticeable feature of Cu-treated CH34 cells was the strong expression of proteins linked to cell motility, regardless of incubation time (Fig. ). While the chemotaxis protein CheA was upregulated only at day 5, CheB1, CheD, and CheY were upregulated at almost all time points. The CheY-like response regulator Rmet_3680 was also found upregulated at several time points (2–4 h, 24 h, and 5 days). Interestingly, the flagellin protein FliC2 was downregulated in the first 4 h of incubation, but strongly upregulated in later time points, showing Log 2 -FC value of 3.25 (24 h) and 3.48 (5 days). During the VBNC phase, several negative but nonsignificant Log 2 -FC values were obtained for certain proteins of the Pil family (e.g. PilL2, PilM, PilN, PilO). In general, however, the data points toward higher abundance of Pil proteins in Cu-treated samples, especially during the resuscitation phase (e.g. PilJ: Log 2 -FC > 5.0 for days 1–4). To evaluate whether this proteomic response translated to a phenotypic change, we tested cellular motility by plating cell suspensions on 0.4% agar plates. The swarming diameter after a 3-day incubation period was measured for time points 48, 72, 96, and 120 h and normalized by the corresponding CFU counts. Samples from time points before 48 h were not included in this assay since an accurate measurement of the swarming range was impeded by the lack of culturable cells. The average swarming range was significantly higher ( P <0.001) in all Cu-treated samples compared to the control condition (Fig. ). This result complies with our proteomics data, indicating increased motility in cells experiencing and resuscitating from a Cu-induced VBNC state. Ribosomal proteins are upregulated throughout Cu exposure Proteomic profiling revealed that many proteins linked to translation were upregulated in both VBNC and resuscitated cells (Fig. ). In fact, a strikingly high number of significantly upregulated ribosomal proteins (RPs) was found at most time points (Fig. ). Remarkably, after 1 h of Cu-treatment, already 41% (22 out of 54) of the identified RPs were upregulated. Strikingly, at 5 h, none of the 53 identified RPs were DE. However, 23% (12 out of 52) of the identified RPs were again upregulated at 24 h, which increased to a maximum of 93% (50 out of 54) at 96 h. Between days 2 and 5 of incubation (48–120 h), at least 80% of the identified RPs were upregulated, suggesting a need for enhanced translation and/or the preparation for cell division during the resuscitation phase. Cell division proteins are upregulated during resuscitation Several proteins linked to cell division were upregulated in Cu-treated cells between days 2 and 5 (48–120 h, Fig. ) during which resuscitation took place. The cell division topological specificity factor MinE was first downregulated after 1 h of Cu treatment and upregulated from day 2 onward. The cell shape determining protein MreB was slightly upregulated between 3 and 5 days. Upregulation from 1 day onward was observed for the Z-ring protein FtsZ, with especially high Log 2 -FC on days 3 and 4 (Log 2 -FC of 3.98 and 3.62). The corresponding DNA segregation, ATPase FtsK, was significantly upregulated between 2 and 5 days of incubation. Another DNA segregation ATPase, Smc, was slightly upregulated at several time points of the resuscitation phase. The glucose-inhibited cell-division protein GidA was downregulated until 24 h of incubation but was upregulated during the resuscitation phase (days 2–5). Interestingly, a strong upregulation was observed from 2 days onward for the ParA and ParB partitioning proteins encoded by the megaplasmid pMOL30 as well as for the replication protein RepA at day 5 . Such an upregulation was not observed for megaplasmid pMOL28 . Metal stress response is active during VBNC induction and resuscitation Cupriavidus metallidurans CH34 features a multitude of metal resistance determinants that are induced by Cu stress . However, it is yet to be investigated whether the transition into a nonculturable state and its regulation can be linked to the activation of such cellular metal detoxification systems . Differential expression was observed for several Cu detoxification systems at different time points of incubation. Their expression profiles, genomic locations and (putative) functions are summarized in Fig. . Firstly, the periplasmic copper detoxification system CopA 1 B 1 C 1 D 1 (Cop1), encoded by pMOL30, as well as the chromosomally encoded homolog CopA 2 B 2 C 2 D 2 (Cop2) were upregulated mostly at later stages of incubation (2–5 days) and non-DE or even downregulated at the beginning of Cu treatment (1–24 h). In fact, the multicopper oxidase CopA1 was one of the most induced proteins during resuscitation (Log 2 -FC of 5.90 at 5 days). The P IB1 -type ATPase CopF and the putative cupredoxin-like copper-binding protein CopI, both encoded by pMOL30, were also upregulated at later stages (2–5 days) and strongly downregulated at early stages of Cu treatment (1–24 h). CopH and CopK, both periplasmic Cu-binding proteins also encoded by pMOL30, were strongly induced during resuscitation (e.g. CopH: Log 2 -FC of 7.92 at 4 days) but were not identified before 2 days. In addition, the three component cation efflux system SilABC, encoded by pMOL30 in proximity to Cop1, showed remarkable downregulation during the first few hours of incubation (nonsignificant for SilA) and moderate upregulation in the resuscitation phase (SilC non-DE). A dissimilar expression profile was found for the Cu chaperone CupC (upregulated until 4 h, afterward mostly non-DE), while the corresponding Cu-transporting ATPase CupA showed no significant differential expression over all time points except slight upregulation at 4 days. We also observed sporadic induction of other metal resistance determinants in Cu-treated cells (Fig. ). Most notably, CzcE was strongly upregulated from day 2 onward, with Log 2 -FC ranging from 3.74 (2 days) to 7.29 (5 days) but was not identified in samples before day 2. Interestingly, other proteins of the Czc system, which are involved in Cd(II), Zn(II), Co(II) resistance, were non-DE at most time points or even downregulated (e.g. CzcC at 1, 2, 4 h and 3 days). ZniA and ZniC, parts of a tricomponent metal cation efflux system, were upregulated at 5 days while showing no significant differential expression at other time points. Moreover, HmzRS, a two-component metal response regulatory system, was upregulated at many time points, with Log 2 -FC values between 0.83 (3 h) and 5.12 (4 days). The heavy metal cation tricomponent efflux protein HmvB was slightly upregulated at 1 and 4 h of incubation (Log 2 -FC of 0.72; 0.64). Its analogue HmyB was strongly upregulated from 2 days onward but was not identified before that time point. Another heavy metal cation tricomponent efflux protein, NimB, was slightly upregulated during the first 4 h of incubation and non-DE after that (but again upregulated at 5 days). Interestingly, the cation-transporting ATPase CtpF was downregulated at almost all time points. Other stress response systems affected by Cu treatment Differential expression of several systems related to general and oxidative stress response was observed (Fig. ). Firstly, the superoxide dismutases SodB and SodC together with the transcriptional activator for superoxide response SoxR were upregulated at several time points between 1 and 24 h of Cu exposure. Interestingly, during resuscitation (2–5 days), these proteins were non-DE or even downregulated. Conversely, many proteins of the universal stress protein UspA family (e.g. UspA5, UspA10, UspA14) were downregulated during VBNC induction (1–5 h) and were later upregulated during resuscitation. Different subunits of the ATP-dependent Clp protease were upregulated at several time points (1, 3, 5 days), and the 10- and 60 kDa chaperonines GroES and GroEL were upregulated until 4 h (GroEL even until day 5). These results could hint at the potential activation of cellular systems counteracting oxidative stress (e.g. protein modulation) imposed by Cu treatment. Terpenoid and cell wall biogenesis are involved in VBNC entry and exit As Cu ions exhibit detrimental effects on bacterial cell walls, it was not surprising that several proteins linked to the COG class “Cell wall/membrane/envelope biogenesis” were DE. The outer membrane proteins Rmet_0712, Rmet_4834 and Rmet_3234 as well as the membrane-bound transglycosylases MltA and MltB were upregulated at several time points during the early incubation phase. OmpP2, a porin involved in diffusion of small hydrophilic molecules, showed strong upregulation at most time points, especially during the resuscitation phase (Log 2 -FC of 5.75 at 5 days). Notably, OmpP2 is encoded between the cop and sil clusters on pMOL30, which could indicate a possible link with Cu detoxification. The outer membrane lipoprotein Pal and the glycosyltransferase Rmet_0756 were upregulated at 1 h of Cu treatment but were downregulated between 1 and 5 days. Interestingly, the lipoprotein carrier protein LolA followed a similar trend (upregulation until 4 h, downregulation from day 2 onward). The opposite expression profile (downregulation in the VBNC induction phase and upregulation during resuscitation) could be observed for the bifunctional protein GlmU and the phospholipid synthase Cfa. Following these results, we can consider that cell wall modulation might be involved in the regulation of the Cu-induced VBNC state. In addition, most enzymes involved in the 2-C-Methylerythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway for the biosynthesis of isopentenyl diphosphate (IPP) were DE at different incubation phases (Fig. ). Dxs and Dxr, catalyzing the initial reaction steps, were slightly downregulated in the first hours of incubation. Conversely, IspD, IspE, and IspF were slightly upregulated at various time points of Cu treatment. IspG was highly upregulated starting from 24 h (non-DE before that). We identified two analogues of IspH (Rmet_4169 and Rmet_2868), an enzyme catalyzing the final step of IPP biosynthesis. Interestingly, the two analogues showed diverging expression profiles during the VBNC induction phase, either strong downregulation or moderate upregulation (1–5 h). However, both were upregulated at later time points (4–5 days). IPP can be processed to different types of terpenoids via conversion to farnesyl diphosphate catalyzed by IspA (non-DE). We noticed that two enzymes of opposing downstream metabolic routes, namely the octaprenyl diphosphate synthase IspB and the squalene synthase (Rmet_5617), were showing contrasting expression profiles depending on incubation time. During the VBNC induction phase (1–4 h), IspB was downregulated while the squalene synthase was highly upregulated. During the resuscitation phase, both enzymes were mostly non-DE, however IspB was slightly upregulated at day 3, while the squalene synthase was downregulated at day 5. Moreover, other enzymes using farnesyl as substrate (UppS and Ste24 endopeptidase) were also upregulated between days 1 and 5. We can thus assume that during VBNC induction, terpenoid biosynthesis is activated and steered toward squalene formation, whereas this effect might be less relevant during resuscitation. Induction of the VBNC state instigates reorganization of carbon metabolism Polyhydroxybutyrate metabolism Since stationary-phase cells were suspended in mineral water without additional nutrients, the question arose what kind of energy resources are being used to establish (partially) strong differential protein expression over the course of several days. Subsequently, we further investigated whether transition into the VBNC state also correlated with changes in the central carbon and energy metabolism. Differential expression of proteins linked to PHB metabolism, used as an energy reserve during carbon-limiting conditions , was found during the resuscitation phase (Fig. ). The PHA-granule associated protein Phasin was strongly upregulated from 1 day onward. Phasins participate in PHB synthesis and degradation through activation of both PHB polymerases and depolymerases . Accordingly, we monitored upregulation of both, polymerase PhaC3 and depolymerase PhaZ1 between 1 and 5 days (PhaC1 was non-DE). Both proteins were non-DE during the VBNC induction phase. It was shown for Cupriavidus necator that phosphorylation of PHB poly- and depolymerases is involved in their regulation . It is likely that a similar effect is present here to prevent simultaneous build-up and break-down of polymers. The 3-hydroxybutyrate oligomer hydrolase PhaY and the D-beta-hydroxybutyrate dehydrogenase BdhA, involved in subsequent catabolic reaction steps resulting in the formation of acetoacetate, were slightly upregulated (BdhA only during resuscitation), hinting at a net-flux toward PHB degradation. From here, two different metabolic routes are possible. Firstly, acetoacetate can be further processed to two equivalents of Acetyl-CoA (AcCoA) through the AcCoA synthetase (AcsA) and the AcCoA acetyltransferase (BktB), both of which were upregulated between 1 and 5 days (AcsA was downregulated in the first hours of incubation). The intermediate product acetoacetyl-CoA could also be fed back toward PHB synthesis through the enzyme PhaB, which however was not differentially expressed (DE). Net-flux will therefore end up in the formation of AcCoA, to deliver a metabolically pluripotent compound, as well as to avoid a futile cycle of PHB synthesis and degradation. Secondly, acetoacetate can be converted to acetone via the acetoacetate decarboxylase, however this enzyme was not found in our data, nor is it mapped for C. metallidurans in the KEGG database. Decarboxylation could also theoretically happen through the reverse reaction catalyzed by the acetone carboxylase with concomitant ATP formation. Notably, the acetone carboxylase subunits AcxAB were found highly upregulated throughout most sample time points (downregulated only at 5 and 24 h), hinting at a potential involvement of the enzyme in this pathway. Subunit AcxC and the corresponding regulator AcxR were only identified at 5 and 24 h. Deletion of acxR (which resulted in the loss of acetone carboxylase production and growth on acetone or isopropanol ; ) resulted in a higher proportion of cells entering the VBNC state compared to the parental strain but did not affect the overall resuscitation behavior. It is therefore possible that besides AcCoA production, acetoacetate is used for the formation of ATP during increased energy requirements of cells facing Cu stress. However, literature is not clear about whether this reaction is favorable in physiological conditions, and acetoacetate decarboxylation was even reported to happen spontaneously in aqueous solution . Nonetheless, following these results, we can contemplate that through degradation of PHB granules, a pool of AcCoA is generated that might be used to reactivate the central carbon metabolism of dormant cells. This consideration prompted us to investigate whether intracellular PHB levels are different before and after resuscitation. To this end, the PHB content of cells was quantified via a Nile red assay . The median RFU (mRFU), which correlates with intracellular PHB levels and can thus be used for relative quantification , was 1170 for the starting condition. In addition, control cells had relatively constant fluorescence over all time points (mRFU between 758 and 878). Using Mood's median test, we could verify that mRFU values were significantly higher for Cu-treated cells at all time points (Table ). However, due to the very large number of recorded events, we resorted to the estimation of effect sizes in the Cu-treated condition using Cramér's V . A high effect size was detected by comparing the 5-h Cu sample to the starting condition (Fig. ), suggesting a relative change in PHB levels. This result was surprising as our proteomics data showed no considerable upregulation of PHB metabolizing enzymes in the first few hours of Cu treatment. Mostly, low effect sizes were detected when comparing Cu-treated samples at adjacent time points (Table , Cu T vs Cu T+1 ), indicating only small correlations between incubation time and changes in PHB levels. One single medium effect size between 24 and 48 h of Cu treatment could however corroborate the increased expression of PHB metabolizing enzymes starting at these time points (Fig. ). While the log-transformed RFU of cells at 5 h of Cu treatment seemed to follow a normal density function, transition to a bimodal distribution could be observed starting from 24 h, with skew toward lower RFU values (Fig. and ). Cu-treated cells clearly show a more heterogenous composition at 120 h than at 5 h, suggesting that a subpopulation with lower PHB content is present (Fig. ). This could indicate the emergence of a subpopulation that more actively metabolizes PHB, corroborating the strong upregulation of PHB degrading enzymes observed during resuscitation. Interestingly, a subpopulation with higher RFU was detected at 120 hours in the control condition, with an inverted proportion of cellular events compared to the Cu sample (Fig. ). Therefore, PHB synthesis in nontreated cells could be active to a similar extent as degradation in treated cells after several days of incubation in mineral water. Glyoxylate cycle and gluconeogenesis Cells in the resuscitation phase showed high upregulation of both isocitrate lyase homologues AceA and AceA2 (Fig. , particularly high AceA2 expression e.g. 6.53 Log 2 -FC at 4 days). Upregulation of this enzyme is an indicator for the glyoxylate cycle, which bypasses the two decarboxylation steps of the citric acid cycle to activate carbohydrate synthesis . Precursors of the glyoxylate cycle are oxaloacetate and AcCoA, the latter can be supplied from PHB degradation. Condensation of these two molecules catalyzed by the citrate synthase (GltA, non-DE) yields citrate (identical reaction to the citric acid cycle). Citrate can be converted to isocitrate by the aconitase Acn. AcnB was upregulated from day 2 onward (AcnA was non-DE). The highly upregulated isocitrate lyase cleaves isocitrate into succinate and glyoxylate. The second enzyme that is characteristic of the glyoxylate cycle, the malate synthase (AceB), uses glyoxylate and another equivalent of AcCoA to form malate. Even though AceB was non-DE, the bypass is driven forward since this reaction step is thought to be irreversible . The malate dehydrogenase Mdh (slightly upregulated at 4 and 24 h, else non-DE) closes the cycle by converting malate to oxaloacetate. The formed succinate is simultaneously converted to another equivalent of oxalacetate . Subsequently, it can be further processed to phosphoenolpyruvate through the phosphoenolpyruvate carboxykinase PckG, which was upregulated between days 2 and 5 (non-DE at time points before day 2). This marks the first step of gluconeogenesis. Catalyzing the rate-determining step of gluconeogenesis, the fructose-1,6-bisphosphatase Fbp2 was also upregulated from day 2 to 5. In addition, many other gluconeogenetic enzymes (e.g. GpmB, Pgk, CbbG, CbbA; ) followed the same expression profile (upregulation during resuscitation, non-DE during VBNC induction). Meanwhile, the competing isocitrate dehydrogenase (Icd), feeding into the citric acid cycle, was mostly non-DE. Interestingly, the corresponding kinase AceK was upregulated at days 4 and 5 (not detected or no significant differential expression before that). These results suggest that the glyoxylate shunt and subsequent gluconeogenesis are active during resuscitation. Pyruvate metabolism Many pyruvate-metabolizing enzymes were significantly downregulated in VBNC and resuscitating cells (Fig. ). The pyruvate kinase PykA2, converting phosphoenolpyruvate to pyruvate, was distinctly downregulated until 5 h of Cu treatment (however, PykA1 and Pps were non-DE). Notably, we also observed decreased expression of enzymes involved in the interconversion of pyruvate and AcCoA: AceE (downregulated from 24 h onward) and LpdA2 (downregulated at most time points). Downregulation of these enzymes could be an important mechanism to avoid interference with the glyoxylate cycle. In addition, Ldh and Dld, enzymes involved in pyruvate-lactate conversions, as well as the malate dehydrogenase MaeB were downregulated at several time points (MaeB1 was non-DE). The aspartate aminotransferase AatA, catalyzing alanine-pyruvate conversion, was downregulated during the first few hours of incubation. Conversely, several enzymes of the Ilv and Leu families involved in the synthesis of branched amino acids were upregulated throughout Cu treatment, suggesting that adaptations in amino acid availability might be required at different stages of the Cu-induced VBNC state. Vitamin biosynthesis—thiamine supplementation prevents VBNC formation In our proteomic analysis, proteins involved in thiamine biosynthesis were found upregulated especially during the resuscitation phase (Fig. ). Most notably, ThiC and ThiO were upregulated between 1 and 5 days of Cu exposure (ThiO also between 1 and 4 h), and ThiE was slightly upregulated at these time points. The thiazole synthase ThiG was upregulated at 5 days but was not identified or non-DE in samples before that. ThiD was downregulated in the VBNC induction phase (between 1 and 4 h) and slightly upregulated afterward at 1 day of incubation (non-DE during the later phase). Other enzymes in the thiamine biosynthetic pathway (Dxs, IscS) were slightly upregulated only at 5 days (Dxs was also downregulated during the VBNC induction phase). The pathway leads to the formation of thiamine monophosphate, which can be further processed to thiamine pyrophosphate by the thiamine monophosphate kinase ThiL. ThiL was upregulated between 2 and 4 days, while the competing phosphatases PhoA and RsgA were non-DE or slightly downregulated. Interestingly, PhoA was upregulated at several time points until 1 day. In addition, many enzymes of the biotin synthesis pathway (BioABCDF, FabGI) were upregulated between 2 and 5 days but non-DE or downregulated at earlier time points, hinting at a potential role of this vitamin in resuscitation. Thiamine had previously been tested as a compound with potential resuscitation-promoting properties . Interestingly, supplementing mineral water with 5.6 mM thiamine at the start of incubation impeded loss of culturability in CH34 cells upon Cu treatment . This result suggests that abundant thiamine might prevent CH34 from entering the VBNC state. It is possible that this effect is caused by thiamine-mediated sequestration of Cu ions. However, when fully grown CH34 was challenged with a lethal dose of 5 mM CuSO 4 for 2 h, thiamine addition (equimolar) did not lower the toxic effect .
enters the VBNC state upon Cu stress and resuscitates spontaneously Maertens et al . identified the transition into a VBNC state when C. metallidurans cells are exposed to elevated Cu 2+ levels in drinking water, showing spontaneous resuscitation within several days of incubation . In a first step, we corroborated this observation and added sampling time points as well as statistical analyses. Mineral water (Ordal) was inoculated with cells of type strain CH34 to a starting cell number of approximately 10 8 cells/ml and supplemented with 10 µM CuSO 4 (no CuSO 4 in control condition). Colony forming units were assessed during the first 5 h and every 24 h after starting the experiment (Fig. ). In agreement with Maertens et al ., the number of culturable cells decreased by 4 Log during the first hours of Cu treatment. Already after 1 h, Cu-treated cells showed a significant reduction in culturability ( P < .001). From 24 h (1 day) after the start of Cu treatment, a gradual increase in CFU counts could be observed, while cell numbers in the control condition were staying relatively constant. Until 96 h (4 days), the reduction in culturability was still significant ( P < .05). From 120 h (5 days) onwards, no statistical difference between Cu and control condition could be observed, corresponding to the complete resuscitation (Fig. ). Throughout the experiment, the total cell numbers as assessed by flow cytometry were not significantly different between the Cu-treated and control conditions . In addition, cell viability was also assessed with a SYBR Green/PI assay that monitors viability based on membrane integrity, which indicated that 10%–20% of Cu-treated cells remained viable between 24 and 120 h of exposure . Finally, to verify that VBNC induction and resuscitation was not unique to the used mineral water, we tested an additional mineral water (Evian; ), which showed the same phenomena.
To investigate the proteomic state of VBNC cells and during resuscitation, cells were pelleted for protein extraction at the following sample time points: 1, 2, 3, 4, 5, 24, 48, 72, 96, and 120 h of incubation (Cu and control condition). Proteome analyses were performed via protein extraction and subsequent LC-MS/MS analysis. All detected proteins, including data from single replicates, are listed in . The number of identified proteins for each time point ranged between 2195 and 2570 (Table , Fig. ). In general, a good coverage of CH34’s annotated proteome could be reached, with an average of 2373 identified proteins, which amounts to 36.6% of the 6488 annotated coding sequences in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database . For each time point, DE proteins were determined by comparing their abundance in Cu-treated and control samples. Proteins with a FC ≤0.67 or ≥1.5 (|Log2-FC| ≥0.5849) and q -values <0.05 were considered DE (Table ). The number of DE proteins was the lowest at 5 h with 32 DE proteins (Fig. ). Afterward, the number of DE proteins gradually increased to reach a maximum at 120 h (1038 DE proteins). Identified proteins were categorized based on Clusters of Orthologous Genes (COGs) . For each COG class, the number of up-/downregulated proteins among the total number of proteins linked to the COG class was determined and represented as a ratio. Repeating this step for every time point, we obtained an overview of proteomic adaptations in VBNC and resuscitating cells (Fig. ). At most time points, high numbers of upregulated proteins (ratios up to 0.5) were observed in the classes “Translation, ribosomal structure and biogenesis,” “Cell motility,” and “Intracellular trafficking, secretion, and vesicular transport.” These classes coincidently showed low numbers of downregulated proteins (ratios partly below 0.1). In contrast, many proteins were continuously downregulated in e.g. “Energy production and conversion,” “Transcription,” “Replication, recombination, and repair” and “Secondary metabolites' biosynthesis, transport and catabolism.” For other classes, the ratios fluctuated more strongly at different phases of incubation. “Cell cycle control, cell division, chromosome partitioning” as well as “Lipid transport and metabolism” (among others) showed high numbers of upregulated proteins between 48 and 120 h (2–5 days, resuscitation phase), but not in the early incubation stage (1–4 h, VBNC induction phase). Conversely, the class “Cell wall/membrane/envelope biogenesis” had many upregulated proteins only in the early incubation stage. Based on COG analysis, we can assume that cells going through the VBNC state and resuscitation are characterized by highly dynamic proteomic profiles. Next, we showcased in detail the similarities and differences between the proteomes of these two cell states.
A noticeable feature of Cu-treated CH34 cells was the strong expression of proteins linked to cell motility, regardless of incubation time (Fig. ). While the chemotaxis protein CheA was upregulated only at day 5, CheB1, CheD, and CheY were upregulated at almost all time points. The CheY-like response regulator Rmet_3680 was also found upregulated at several time points (2–4 h, 24 h, and 5 days). Interestingly, the flagellin protein FliC2 was downregulated in the first 4 h of incubation, but strongly upregulated in later time points, showing Log 2 -FC value of 3.25 (24 h) and 3.48 (5 days). During the VBNC phase, several negative but nonsignificant Log 2 -FC values were obtained for certain proteins of the Pil family (e.g. PilL2, PilM, PilN, PilO). In general, however, the data points toward higher abundance of Pil proteins in Cu-treated samples, especially during the resuscitation phase (e.g. PilJ: Log 2 -FC > 5.0 for days 1–4). To evaluate whether this proteomic response translated to a phenotypic change, we tested cellular motility by plating cell suspensions on 0.4% agar plates. The swarming diameter after a 3-day incubation period was measured for time points 48, 72, 96, and 120 h and normalized by the corresponding CFU counts. Samples from time points before 48 h were not included in this assay since an accurate measurement of the swarming range was impeded by the lack of culturable cells. The average swarming range was significantly higher ( P <0.001) in all Cu-treated samples compared to the control condition (Fig. ). This result complies with our proteomics data, indicating increased motility in cells experiencing and resuscitating from a Cu-induced VBNC state.
Proteomic profiling revealed that many proteins linked to translation were upregulated in both VBNC and resuscitated cells (Fig. ). In fact, a strikingly high number of significantly upregulated ribosomal proteins (RPs) was found at most time points (Fig. ). Remarkably, after 1 h of Cu-treatment, already 41% (22 out of 54) of the identified RPs were upregulated. Strikingly, at 5 h, none of the 53 identified RPs were DE. However, 23% (12 out of 52) of the identified RPs were again upregulated at 24 h, which increased to a maximum of 93% (50 out of 54) at 96 h. Between days 2 and 5 of incubation (48–120 h), at least 80% of the identified RPs were upregulated, suggesting a need for enhanced translation and/or the preparation for cell division during the resuscitation phase.
Several proteins linked to cell division were upregulated in Cu-treated cells between days 2 and 5 (48–120 h, Fig. ) during which resuscitation took place. The cell division topological specificity factor MinE was first downregulated after 1 h of Cu treatment and upregulated from day 2 onward. The cell shape determining protein MreB was slightly upregulated between 3 and 5 days. Upregulation from 1 day onward was observed for the Z-ring protein FtsZ, with especially high Log 2 -FC on days 3 and 4 (Log 2 -FC of 3.98 and 3.62). The corresponding DNA segregation, ATPase FtsK, was significantly upregulated between 2 and 5 days of incubation. Another DNA segregation ATPase, Smc, was slightly upregulated at several time points of the resuscitation phase. The glucose-inhibited cell-division protein GidA was downregulated until 24 h of incubation but was upregulated during the resuscitation phase (days 2–5). Interestingly, a strong upregulation was observed from 2 days onward for the ParA and ParB partitioning proteins encoded by the megaplasmid pMOL30 as well as for the replication protein RepA at day 5 . Such an upregulation was not observed for megaplasmid pMOL28 .
Cupriavidus metallidurans CH34 features a multitude of metal resistance determinants that are induced by Cu stress . However, it is yet to be investigated whether the transition into a nonculturable state and its regulation can be linked to the activation of such cellular metal detoxification systems . Differential expression was observed for several Cu detoxification systems at different time points of incubation. Their expression profiles, genomic locations and (putative) functions are summarized in Fig. . Firstly, the periplasmic copper detoxification system CopA 1 B 1 C 1 D 1 (Cop1), encoded by pMOL30, as well as the chromosomally encoded homolog CopA 2 B 2 C 2 D 2 (Cop2) were upregulated mostly at later stages of incubation (2–5 days) and non-DE or even downregulated at the beginning of Cu treatment (1–24 h). In fact, the multicopper oxidase CopA1 was one of the most induced proteins during resuscitation (Log 2 -FC of 5.90 at 5 days). The P IB1 -type ATPase CopF and the putative cupredoxin-like copper-binding protein CopI, both encoded by pMOL30, were also upregulated at later stages (2–5 days) and strongly downregulated at early stages of Cu treatment (1–24 h). CopH and CopK, both periplasmic Cu-binding proteins also encoded by pMOL30, were strongly induced during resuscitation (e.g. CopH: Log 2 -FC of 7.92 at 4 days) but were not identified before 2 days. In addition, the three component cation efflux system SilABC, encoded by pMOL30 in proximity to Cop1, showed remarkable downregulation during the first few hours of incubation (nonsignificant for SilA) and moderate upregulation in the resuscitation phase (SilC non-DE). A dissimilar expression profile was found for the Cu chaperone CupC (upregulated until 4 h, afterward mostly non-DE), while the corresponding Cu-transporting ATPase CupA showed no significant differential expression over all time points except slight upregulation at 4 days. We also observed sporadic induction of other metal resistance determinants in Cu-treated cells (Fig. ). Most notably, CzcE was strongly upregulated from day 2 onward, with Log 2 -FC ranging from 3.74 (2 days) to 7.29 (5 days) but was not identified in samples before day 2. Interestingly, other proteins of the Czc system, which are involved in Cd(II), Zn(II), Co(II) resistance, were non-DE at most time points or even downregulated (e.g. CzcC at 1, 2, 4 h and 3 days). ZniA and ZniC, parts of a tricomponent metal cation efflux system, were upregulated at 5 days while showing no significant differential expression at other time points. Moreover, HmzRS, a two-component metal response regulatory system, was upregulated at many time points, with Log 2 -FC values between 0.83 (3 h) and 5.12 (4 days). The heavy metal cation tricomponent efflux protein HmvB was slightly upregulated at 1 and 4 h of incubation (Log 2 -FC of 0.72; 0.64). Its analogue HmyB was strongly upregulated from 2 days onward but was not identified before that time point. Another heavy metal cation tricomponent efflux protein, NimB, was slightly upregulated during the first 4 h of incubation and non-DE after that (but again upregulated at 5 days). Interestingly, the cation-transporting ATPase CtpF was downregulated at almost all time points.
Differential expression of several systems related to general and oxidative stress response was observed (Fig. ). Firstly, the superoxide dismutases SodB and SodC together with the transcriptional activator for superoxide response SoxR were upregulated at several time points between 1 and 24 h of Cu exposure. Interestingly, during resuscitation (2–5 days), these proteins were non-DE or even downregulated. Conversely, many proteins of the universal stress protein UspA family (e.g. UspA5, UspA10, UspA14) were downregulated during VBNC induction (1–5 h) and were later upregulated during resuscitation. Different subunits of the ATP-dependent Clp protease were upregulated at several time points (1, 3, 5 days), and the 10- and 60 kDa chaperonines GroES and GroEL were upregulated until 4 h (GroEL even until day 5). These results could hint at the potential activation of cellular systems counteracting oxidative stress (e.g. protein modulation) imposed by Cu treatment.
As Cu ions exhibit detrimental effects on bacterial cell walls, it was not surprising that several proteins linked to the COG class “Cell wall/membrane/envelope biogenesis” were DE. The outer membrane proteins Rmet_0712, Rmet_4834 and Rmet_3234 as well as the membrane-bound transglycosylases MltA and MltB were upregulated at several time points during the early incubation phase. OmpP2, a porin involved in diffusion of small hydrophilic molecules, showed strong upregulation at most time points, especially during the resuscitation phase (Log 2 -FC of 5.75 at 5 days). Notably, OmpP2 is encoded between the cop and sil clusters on pMOL30, which could indicate a possible link with Cu detoxification. The outer membrane lipoprotein Pal and the glycosyltransferase Rmet_0756 were upregulated at 1 h of Cu treatment but were downregulated between 1 and 5 days. Interestingly, the lipoprotein carrier protein LolA followed a similar trend (upregulation until 4 h, downregulation from day 2 onward). The opposite expression profile (downregulation in the VBNC induction phase and upregulation during resuscitation) could be observed for the bifunctional protein GlmU and the phospholipid synthase Cfa. Following these results, we can consider that cell wall modulation might be involved in the regulation of the Cu-induced VBNC state. In addition, most enzymes involved in the 2-C-Methylerythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway for the biosynthesis of isopentenyl diphosphate (IPP) were DE at different incubation phases (Fig. ). Dxs and Dxr, catalyzing the initial reaction steps, were slightly downregulated in the first hours of incubation. Conversely, IspD, IspE, and IspF were slightly upregulated at various time points of Cu treatment. IspG was highly upregulated starting from 24 h (non-DE before that). We identified two analogues of IspH (Rmet_4169 and Rmet_2868), an enzyme catalyzing the final step of IPP biosynthesis. Interestingly, the two analogues showed diverging expression profiles during the VBNC induction phase, either strong downregulation or moderate upregulation (1–5 h). However, both were upregulated at later time points (4–5 days). IPP can be processed to different types of terpenoids via conversion to farnesyl diphosphate catalyzed by IspA (non-DE). We noticed that two enzymes of opposing downstream metabolic routes, namely the octaprenyl diphosphate synthase IspB and the squalene synthase (Rmet_5617), were showing contrasting expression profiles depending on incubation time. During the VBNC induction phase (1–4 h), IspB was downregulated while the squalene synthase was highly upregulated. During the resuscitation phase, both enzymes were mostly non-DE, however IspB was slightly upregulated at day 3, while the squalene synthase was downregulated at day 5. Moreover, other enzymes using farnesyl as substrate (UppS and Ste24 endopeptidase) were also upregulated between days 1 and 5. We can thus assume that during VBNC induction, terpenoid biosynthesis is activated and steered toward squalene formation, whereas this effect might be less relevant during resuscitation.
Polyhydroxybutyrate metabolism Since stationary-phase cells were suspended in mineral water without additional nutrients, the question arose what kind of energy resources are being used to establish (partially) strong differential protein expression over the course of several days. Subsequently, we further investigated whether transition into the VBNC state also correlated with changes in the central carbon and energy metabolism. Differential expression of proteins linked to PHB metabolism, used as an energy reserve during carbon-limiting conditions , was found during the resuscitation phase (Fig. ). The PHA-granule associated protein Phasin was strongly upregulated from 1 day onward. Phasins participate in PHB synthesis and degradation through activation of both PHB polymerases and depolymerases . Accordingly, we monitored upregulation of both, polymerase PhaC3 and depolymerase PhaZ1 between 1 and 5 days (PhaC1 was non-DE). Both proteins were non-DE during the VBNC induction phase. It was shown for Cupriavidus necator that phosphorylation of PHB poly- and depolymerases is involved in their regulation . It is likely that a similar effect is present here to prevent simultaneous build-up and break-down of polymers. The 3-hydroxybutyrate oligomer hydrolase PhaY and the D-beta-hydroxybutyrate dehydrogenase BdhA, involved in subsequent catabolic reaction steps resulting in the formation of acetoacetate, were slightly upregulated (BdhA only during resuscitation), hinting at a net-flux toward PHB degradation. From here, two different metabolic routes are possible. Firstly, acetoacetate can be further processed to two equivalents of Acetyl-CoA (AcCoA) through the AcCoA synthetase (AcsA) and the AcCoA acetyltransferase (BktB), both of which were upregulated between 1 and 5 days (AcsA was downregulated in the first hours of incubation). The intermediate product acetoacetyl-CoA could also be fed back toward PHB synthesis through the enzyme PhaB, which however was not differentially expressed (DE). Net-flux will therefore end up in the formation of AcCoA, to deliver a metabolically pluripotent compound, as well as to avoid a futile cycle of PHB synthesis and degradation. Secondly, acetoacetate can be converted to acetone via the acetoacetate decarboxylase, however this enzyme was not found in our data, nor is it mapped for C. metallidurans in the KEGG database. Decarboxylation could also theoretically happen through the reverse reaction catalyzed by the acetone carboxylase with concomitant ATP formation. Notably, the acetone carboxylase subunits AcxAB were found highly upregulated throughout most sample time points (downregulated only at 5 and 24 h), hinting at a potential involvement of the enzyme in this pathway. Subunit AcxC and the corresponding regulator AcxR were only identified at 5 and 24 h. Deletion of acxR (which resulted in the loss of acetone carboxylase production and growth on acetone or isopropanol ; ) resulted in a higher proportion of cells entering the VBNC state compared to the parental strain but did not affect the overall resuscitation behavior. It is therefore possible that besides AcCoA production, acetoacetate is used for the formation of ATP during increased energy requirements of cells facing Cu stress. However, literature is not clear about whether this reaction is favorable in physiological conditions, and acetoacetate decarboxylation was even reported to happen spontaneously in aqueous solution . Nonetheless, following these results, we can contemplate that through degradation of PHB granules, a pool of AcCoA is generated that might be used to reactivate the central carbon metabolism of dormant cells. This consideration prompted us to investigate whether intracellular PHB levels are different before and after resuscitation. To this end, the PHB content of cells was quantified via a Nile red assay . The median RFU (mRFU), which correlates with intracellular PHB levels and can thus be used for relative quantification , was 1170 for the starting condition. In addition, control cells had relatively constant fluorescence over all time points (mRFU between 758 and 878). Using Mood's median test, we could verify that mRFU values were significantly higher for Cu-treated cells at all time points (Table ). However, due to the very large number of recorded events, we resorted to the estimation of effect sizes in the Cu-treated condition using Cramér's V . A high effect size was detected by comparing the 5-h Cu sample to the starting condition (Fig. ), suggesting a relative change in PHB levels. This result was surprising as our proteomics data showed no considerable upregulation of PHB metabolizing enzymes in the first few hours of Cu treatment. Mostly, low effect sizes were detected when comparing Cu-treated samples at adjacent time points (Table , Cu T vs Cu T+1 ), indicating only small correlations between incubation time and changes in PHB levels. One single medium effect size between 24 and 48 h of Cu treatment could however corroborate the increased expression of PHB metabolizing enzymes starting at these time points (Fig. ). While the log-transformed RFU of cells at 5 h of Cu treatment seemed to follow a normal density function, transition to a bimodal distribution could be observed starting from 24 h, with skew toward lower RFU values (Fig. and ). Cu-treated cells clearly show a more heterogenous composition at 120 h than at 5 h, suggesting that a subpopulation with lower PHB content is present (Fig. ). This could indicate the emergence of a subpopulation that more actively metabolizes PHB, corroborating the strong upregulation of PHB degrading enzymes observed during resuscitation. Interestingly, a subpopulation with higher RFU was detected at 120 hours in the control condition, with an inverted proportion of cellular events compared to the Cu sample (Fig. ). Therefore, PHB synthesis in nontreated cells could be active to a similar extent as degradation in treated cells after several days of incubation in mineral water. Glyoxylate cycle and gluconeogenesis Cells in the resuscitation phase showed high upregulation of both isocitrate lyase homologues AceA and AceA2 (Fig. , particularly high AceA2 expression e.g. 6.53 Log 2 -FC at 4 days). Upregulation of this enzyme is an indicator for the glyoxylate cycle, which bypasses the two decarboxylation steps of the citric acid cycle to activate carbohydrate synthesis . Precursors of the glyoxylate cycle are oxaloacetate and AcCoA, the latter can be supplied from PHB degradation. Condensation of these two molecules catalyzed by the citrate synthase (GltA, non-DE) yields citrate (identical reaction to the citric acid cycle). Citrate can be converted to isocitrate by the aconitase Acn. AcnB was upregulated from day 2 onward (AcnA was non-DE). The highly upregulated isocitrate lyase cleaves isocitrate into succinate and glyoxylate. The second enzyme that is characteristic of the glyoxylate cycle, the malate synthase (AceB), uses glyoxylate and another equivalent of AcCoA to form malate. Even though AceB was non-DE, the bypass is driven forward since this reaction step is thought to be irreversible . The malate dehydrogenase Mdh (slightly upregulated at 4 and 24 h, else non-DE) closes the cycle by converting malate to oxaloacetate. The formed succinate is simultaneously converted to another equivalent of oxalacetate . Subsequently, it can be further processed to phosphoenolpyruvate through the phosphoenolpyruvate carboxykinase PckG, which was upregulated between days 2 and 5 (non-DE at time points before day 2). This marks the first step of gluconeogenesis. Catalyzing the rate-determining step of gluconeogenesis, the fructose-1,6-bisphosphatase Fbp2 was also upregulated from day 2 to 5. In addition, many other gluconeogenetic enzymes (e.g. GpmB, Pgk, CbbG, CbbA; ) followed the same expression profile (upregulation during resuscitation, non-DE during VBNC induction). Meanwhile, the competing isocitrate dehydrogenase (Icd), feeding into the citric acid cycle, was mostly non-DE. Interestingly, the corresponding kinase AceK was upregulated at days 4 and 5 (not detected or no significant differential expression before that). These results suggest that the glyoxylate shunt and subsequent gluconeogenesis are active during resuscitation. Pyruvate metabolism Many pyruvate-metabolizing enzymes were significantly downregulated in VBNC and resuscitating cells (Fig. ). The pyruvate kinase PykA2, converting phosphoenolpyruvate to pyruvate, was distinctly downregulated until 5 h of Cu treatment (however, PykA1 and Pps were non-DE). Notably, we also observed decreased expression of enzymes involved in the interconversion of pyruvate and AcCoA: AceE (downregulated from 24 h onward) and LpdA2 (downregulated at most time points). Downregulation of these enzymes could be an important mechanism to avoid interference with the glyoxylate cycle. In addition, Ldh and Dld, enzymes involved in pyruvate-lactate conversions, as well as the malate dehydrogenase MaeB were downregulated at several time points (MaeB1 was non-DE). The aspartate aminotransferase AatA, catalyzing alanine-pyruvate conversion, was downregulated during the first few hours of incubation. Conversely, several enzymes of the Ilv and Leu families involved in the synthesis of branched amino acids were upregulated throughout Cu treatment, suggesting that adaptations in amino acid availability might be required at different stages of the Cu-induced VBNC state. Vitamin biosynthesis—thiamine supplementation prevents VBNC formation In our proteomic analysis, proteins involved in thiamine biosynthesis were found upregulated especially during the resuscitation phase (Fig. ). Most notably, ThiC and ThiO were upregulated between 1 and 5 days of Cu exposure (ThiO also between 1 and 4 h), and ThiE was slightly upregulated at these time points. The thiazole synthase ThiG was upregulated at 5 days but was not identified or non-DE in samples before that. ThiD was downregulated in the VBNC induction phase (between 1 and 4 h) and slightly upregulated afterward at 1 day of incubation (non-DE during the later phase). Other enzymes in the thiamine biosynthetic pathway (Dxs, IscS) were slightly upregulated only at 5 days (Dxs was also downregulated during the VBNC induction phase). The pathway leads to the formation of thiamine monophosphate, which can be further processed to thiamine pyrophosphate by the thiamine monophosphate kinase ThiL. ThiL was upregulated between 2 and 4 days, while the competing phosphatases PhoA and RsgA were non-DE or slightly downregulated. Interestingly, PhoA was upregulated at several time points until 1 day. In addition, many enzymes of the biotin synthesis pathway (BioABCDF, FabGI) were upregulated between 2 and 5 days but non-DE or downregulated at earlier time points, hinting at a potential role of this vitamin in resuscitation. Thiamine had previously been tested as a compound with potential resuscitation-promoting properties . Interestingly, supplementing mineral water with 5.6 mM thiamine at the start of incubation impeded loss of culturability in CH34 cells upon Cu treatment . This result suggests that abundant thiamine might prevent CH34 from entering the VBNC state. It is possible that this effect is caused by thiamine-mediated sequestration of Cu ions. However, when fully grown CH34 was challenged with a lethal dose of 5 mM CuSO 4 for 2 h, thiamine addition (equimolar) did not lower the toxic effect .
Since stationary-phase cells were suspended in mineral water without additional nutrients, the question arose what kind of energy resources are being used to establish (partially) strong differential protein expression over the course of several days. Subsequently, we further investigated whether transition into the VBNC state also correlated with changes in the central carbon and energy metabolism. Differential expression of proteins linked to PHB metabolism, used as an energy reserve during carbon-limiting conditions , was found during the resuscitation phase (Fig. ). The PHA-granule associated protein Phasin was strongly upregulated from 1 day onward. Phasins participate in PHB synthesis and degradation through activation of both PHB polymerases and depolymerases . Accordingly, we monitored upregulation of both, polymerase PhaC3 and depolymerase PhaZ1 between 1 and 5 days (PhaC1 was non-DE). Both proteins were non-DE during the VBNC induction phase. It was shown for Cupriavidus necator that phosphorylation of PHB poly- and depolymerases is involved in their regulation . It is likely that a similar effect is present here to prevent simultaneous build-up and break-down of polymers. The 3-hydroxybutyrate oligomer hydrolase PhaY and the D-beta-hydroxybutyrate dehydrogenase BdhA, involved in subsequent catabolic reaction steps resulting in the formation of acetoacetate, were slightly upregulated (BdhA only during resuscitation), hinting at a net-flux toward PHB degradation. From here, two different metabolic routes are possible. Firstly, acetoacetate can be further processed to two equivalents of Acetyl-CoA (AcCoA) through the AcCoA synthetase (AcsA) and the AcCoA acetyltransferase (BktB), both of which were upregulated between 1 and 5 days (AcsA was downregulated in the first hours of incubation). The intermediate product acetoacetyl-CoA could also be fed back toward PHB synthesis through the enzyme PhaB, which however was not differentially expressed (DE). Net-flux will therefore end up in the formation of AcCoA, to deliver a metabolically pluripotent compound, as well as to avoid a futile cycle of PHB synthesis and degradation. Secondly, acetoacetate can be converted to acetone via the acetoacetate decarboxylase, however this enzyme was not found in our data, nor is it mapped for C. metallidurans in the KEGG database. Decarboxylation could also theoretically happen through the reverse reaction catalyzed by the acetone carboxylase with concomitant ATP formation. Notably, the acetone carboxylase subunits AcxAB were found highly upregulated throughout most sample time points (downregulated only at 5 and 24 h), hinting at a potential involvement of the enzyme in this pathway. Subunit AcxC and the corresponding regulator AcxR were only identified at 5 and 24 h. Deletion of acxR (which resulted in the loss of acetone carboxylase production and growth on acetone or isopropanol ; ) resulted in a higher proportion of cells entering the VBNC state compared to the parental strain but did not affect the overall resuscitation behavior. It is therefore possible that besides AcCoA production, acetoacetate is used for the formation of ATP during increased energy requirements of cells facing Cu stress. However, literature is not clear about whether this reaction is favorable in physiological conditions, and acetoacetate decarboxylation was even reported to happen spontaneously in aqueous solution . Nonetheless, following these results, we can contemplate that through degradation of PHB granules, a pool of AcCoA is generated that might be used to reactivate the central carbon metabolism of dormant cells. This consideration prompted us to investigate whether intracellular PHB levels are different before and after resuscitation. To this end, the PHB content of cells was quantified via a Nile red assay . The median RFU (mRFU), which correlates with intracellular PHB levels and can thus be used for relative quantification , was 1170 for the starting condition. In addition, control cells had relatively constant fluorescence over all time points (mRFU between 758 and 878). Using Mood's median test, we could verify that mRFU values were significantly higher for Cu-treated cells at all time points (Table ). However, due to the very large number of recorded events, we resorted to the estimation of effect sizes in the Cu-treated condition using Cramér's V . A high effect size was detected by comparing the 5-h Cu sample to the starting condition (Fig. ), suggesting a relative change in PHB levels. This result was surprising as our proteomics data showed no considerable upregulation of PHB metabolizing enzymes in the first few hours of Cu treatment. Mostly, low effect sizes were detected when comparing Cu-treated samples at adjacent time points (Table , Cu T vs Cu T+1 ), indicating only small correlations between incubation time and changes in PHB levels. One single medium effect size between 24 and 48 h of Cu treatment could however corroborate the increased expression of PHB metabolizing enzymes starting at these time points (Fig. ). While the log-transformed RFU of cells at 5 h of Cu treatment seemed to follow a normal density function, transition to a bimodal distribution could be observed starting from 24 h, with skew toward lower RFU values (Fig. and ). Cu-treated cells clearly show a more heterogenous composition at 120 h than at 5 h, suggesting that a subpopulation with lower PHB content is present (Fig. ). This could indicate the emergence of a subpopulation that more actively metabolizes PHB, corroborating the strong upregulation of PHB degrading enzymes observed during resuscitation. Interestingly, a subpopulation with higher RFU was detected at 120 hours in the control condition, with an inverted proportion of cellular events compared to the Cu sample (Fig. ). Therefore, PHB synthesis in nontreated cells could be active to a similar extent as degradation in treated cells after several days of incubation in mineral water.
Cells in the resuscitation phase showed high upregulation of both isocitrate lyase homologues AceA and AceA2 (Fig. , particularly high AceA2 expression e.g. 6.53 Log 2 -FC at 4 days). Upregulation of this enzyme is an indicator for the glyoxylate cycle, which bypasses the two decarboxylation steps of the citric acid cycle to activate carbohydrate synthesis . Precursors of the glyoxylate cycle are oxaloacetate and AcCoA, the latter can be supplied from PHB degradation. Condensation of these two molecules catalyzed by the citrate synthase (GltA, non-DE) yields citrate (identical reaction to the citric acid cycle). Citrate can be converted to isocitrate by the aconitase Acn. AcnB was upregulated from day 2 onward (AcnA was non-DE). The highly upregulated isocitrate lyase cleaves isocitrate into succinate and glyoxylate. The second enzyme that is characteristic of the glyoxylate cycle, the malate synthase (AceB), uses glyoxylate and another equivalent of AcCoA to form malate. Even though AceB was non-DE, the bypass is driven forward since this reaction step is thought to be irreversible . The malate dehydrogenase Mdh (slightly upregulated at 4 and 24 h, else non-DE) closes the cycle by converting malate to oxaloacetate. The formed succinate is simultaneously converted to another equivalent of oxalacetate . Subsequently, it can be further processed to phosphoenolpyruvate through the phosphoenolpyruvate carboxykinase PckG, which was upregulated between days 2 and 5 (non-DE at time points before day 2). This marks the first step of gluconeogenesis. Catalyzing the rate-determining step of gluconeogenesis, the fructose-1,6-bisphosphatase Fbp2 was also upregulated from day 2 to 5. In addition, many other gluconeogenetic enzymes (e.g. GpmB, Pgk, CbbG, CbbA; ) followed the same expression profile (upregulation during resuscitation, non-DE during VBNC induction). Meanwhile, the competing isocitrate dehydrogenase (Icd), feeding into the citric acid cycle, was mostly non-DE. Interestingly, the corresponding kinase AceK was upregulated at days 4 and 5 (not detected or no significant differential expression before that). These results suggest that the glyoxylate shunt and subsequent gluconeogenesis are active during resuscitation.
Many pyruvate-metabolizing enzymes were significantly downregulated in VBNC and resuscitating cells (Fig. ). The pyruvate kinase PykA2, converting phosphoenolpyruvate to pyruvate, was distinctly downregulated until 5 h of Cu treatment (however, PykA1 and Pps were non-DE). Notably, we also observed decreased expression of enzymes involved in the interconversion of pyruvate and AcCoA: AceE (downregulated from 24 h onward) and LpdA2 (downregulated at most time points). Downregulation of these enzymes could be an important mechanism to avoid interference with the glyoxylate cycle. In addition, Ldh and Dld, enzymes involved in pyruvate-lactate conversions, as well as the malate dehydrogenase MaeB were downregulated at several time points (MaeB1 was non-DE). The aspartate aminotransferase AatA, catalyzing alanine-pyruvate conversion, was downregulated during the first few hours of incubation. Conversely, several enzymes of the Ilv and Leu families involved in the synthesis of branched amino acids were upregulated throughout Cu treatment, suggesting that adaptations in amino acid availability might be required at different stages of the Cu-induced VBNC state.
In our proteomic analysis, proteins involved in thiamine biosynthesis were found upregulated especially during the resuscitation phase (Fig. ). Most notably, ThiC and ThiO were upregulated between 1 and 5 days of Cu exposure (ThiO also between 1 and 4 h), and ThiE was slightly upregulated at these time points. The thiazole synthase ThiG was upregulated at 5 days but was not identified or non-DE in samples before that. ThiD was downregulated in the VBNC induction phase (between 1 and 4 h) and slightly upregulated afterward at 1 day of incubation (non-DE during the later phase). Other enzymes in the thiamine biosynthetic pathway (Dxs, IscS) were slightly upregulated only at 5 days (Dxs was also downregulated during the VBNC induction phase). The pathway leads to the formation of thiamine monophosphate, which can be further processed to thiamine pyrophosphate by the thiamine monophosphate kinase ThiL. ThiL was upregulated between 2 and 4 days, while the competing phosphatases PhoA and RsgA were non-DE or slightly downregulated. Interestingly, PhoA was upregulated at several time points until 1 day. In addition, many enzymes of the biotin synthesis pathway (BioABCDF, FabGI) were upregulated between 2 and 5 days but non-DE or downregulated at earlier time points, hinting at a potential role of this vitamin in resuscitation. Thiamine had previously been tested as a compound with potential resuscitation-promoting properties . Interestingly, supplementing mineral water with 5.6 mM thiamine at the start of incubation impeded loss of culturability in CH34 cells upon Cu treatment . This result suggests that abundant thiamine might prevent CH34 from entering the VBNC state. It is possible that this effect is caused by thiamine-mediated sequestration of Cu ions. However, when fully grown CH34 was challenged with a lethal dose of 5 mM CuSO 4 for 2 h, thiamine addition (equimolar) did not lower the toxic effect .
Water-borne cells of C. metallidurans CH34 were previously reported to enter the VBNC state upon Cu exposure . To further characterize this phenomenon, we extended the sample time points and confirmed reproducibility in a different water source. Shortly after the start of the Cu treatment, approximately 99.99% of cells lose their culturability. Coincidently, only approximately 10% of the cells lose membrane integrity during the first 5 h of incubation, indicating that the majority of nonculturable cells are still intact, i.e. VBNC. Therefore, VBNC cells cannot be distinguished from culturable cells by such a membrane permeability assay. The increase of the nonviable fraction at 24 h most likely stems from cells that are unable to resuscitate from the VBNC state. A shift from VBNC to the resuscitation phase could potentially be determined already after 5 h of Cu exposure. This time point shows a distinctly low number of up- and down-regulated proteins, which might reflect a reset of the cellular proteome mediating resuscitation. Accordingly, proteomic adaptations were most discernible either between 1 and 4 h or after 24 h of Cu treatment. For example, the COG classes “Cell cycle control, cell division, chromosome partitioning,” “Translation, ribosomal structure, and biogenesis,” and “Cell motility” contained particularly high proportions of upregulated proteins (around 50% of categorized proteins were upregulated), especially during resuscitation. In general, downregulated proteins were less abundant in these classes. Strong oxidative stress, e.g. mediated by redox-active transition metals like Cu, can cause heavy damage to ribosomes . We noticed that many RPs, represented in the COG class “Translation, ribosomal structure, and biogenesis,” were upregulated at most time points. Upregulation of RPs during the early incubation phase (1–4 h) could possibly counteract oxidative damage to secure unimpaired translation, which in turn is required for a proper adaptive response to the stressor. Interestingly, during the resuscitation phase (2–5 days), at least 80% of identified RPs were upregulated. A similar observation has been reported for E. coli , where the cellular ribosome content was linked to the resuscitation rate . Notably, enrichment of the ribosome KEGG-pathway has been monitored for Acidovorax citrulli cells resuscitating from a Cu-induced VBNC state . It can thus be argued that upregulation of RPs is needed to counteract acute oxidative stress and to help with enhanced translational requirements during resuscitation. One of such requirements could, for example, be the expression of proteins from the “Cell cycle control, cell division, chromosome partitioning” COG class. In fact, several proteins linked to cell division were upregulated during resuscitation. This could imply that resuscitating cells are establishing a set of proteins that allow them to initiate regrowth once favorable conditions are reached. In this regard, the strong upregulation of pMOL30-encoded proteins involved in the plasmid's partitioning and replication could hint at the need for appropriate maintenance and stability of pMOL30 as it carries metal determinants that play an important role during resuscitation. Cu resistance determinants encoded on pMOL30 include the periplasmic detoxification system Cop1, the inner membrane P IB1 -type ATPase CopF and the RND (resistance-nodulation-cell division) metal efflux system SilABCD, for all of which respective chromosomally encoded homologues exist (Cop2, CupA, Cus) . Differential expression of these systems (except Cus—not detected) was monitored throughout the Cu-induced VBNC state. Firstly, Cop1 proteins were generally upregulated in resuscitating cells (2–5 days, CopD1 was not detected) but not during the early stage of Cu treatment (1–24 h, mostly non-DE). This result stands in stark contrast to previous reports describing acute induction of the cop system in CH34 facing Cu stress . However, in these studies, Cu treatment was performed in growth medium, while in our experiment, water-borne cells of CH34 were exposed to Cu 2+ ions without addition of nutrients. Lack of energy resources could therefore be the reason for the delayed expression of proteins that are potentially vital for culturability. High upregulation of Cop1 at the later time points suggests that periplasmic detoxification is an important mechanism during resuscitation. This result is corroborated by the upregulation of the chromosomally encoded Cop2 proteins at 5 days (mostly not detected at other time points). Periplasmic detoxification could be aided by the putative cupredoxin-like copper-binding protein CopI, as well as CopF, an ATPase capable of transporting Cu ions to the periplasm. Both of these proteins were upregulated during the resuscitation phase but downregulated at early sample time points, hinting at impaired Cu ion export during the VBNC phase. Strong downregulation was also monitored for the chromosomally encoded cation-transporting ATPase CtpF, while CupA was mostly non-DE. Moreover, early phase downregulation of the Sil system with slight upregulation starting at 2 days of incubation supports the assumption that Cu ion export might be stalled in VBNC cells and activated during resuscitation. In addition, the plasmidic Cop system also contains several chaperone-like accessory proteins capable of binding Cu ions, however only two of them were identified in our data. CopK and CopH both showed drastic upregulation during resuscitation but were not found at earlier time points. CopK is a small periplasmic protein capable of binding Cu + and Cu 2+ at separate sites and is thought to act as a scavenger for periplasmic Cu-ions, delivering them to outer membrane proteins for efflux . CopH is a periplasmic, dimer-forming protein capable of binding 2 Cu ions per dimer and was reported to show significant homology to CzcE . Notably, the pMOL30-encoded protein CzcE showed the third highest Log 2 -FC (7.29) among all identified proteins (after CopH and PilJ). This protein had first been characterized to be induced by zinc and linked to a regulatory function in cobalt, zinc, and cadmium resistance in CH34 . It was later determined to be a periplasmic Cu-binding protein, putatively involved in Zn-Cu cross regulation . These proteins could potentially have a supportive function in periplasmic detoxification by sequestering accumulating Cu ions, thus preventing further cellular damage, and conveying them toward export systems. Moreover, a potential cross-regulatory function of CzcE with the Cop system is conceivable since no remarkable upregulation of other Czc proteins was monitored. Intriguingly, the Cop regulator CopR1 was only sporadically upregulated (1 and 4 h, 5 days), supporting the hypothesis that other regulatory systems are involved in the induction of Cop proteins. The HmzRS regulatory system, which was upregulated throughout Cu exposure, was reportedly linked to metal resistance due to its similarities to the Czc metal efflux system . A putative regulatory involvement of CzcE and HmzRS can thus not be ruled out. The present data suggests that despite CH34’s high level of metal resistance, it is not able to remain in a culturable state when Cu stress is combined with nutrient depletion. Although we observed short-term upregulation of metal efflux pumps (e.g. HmvB, NimB), this could be a futile effort since no sequestration of Cu-ions takes place. The cells might thus opt to accumulate Cu ions and transition into the VBNC state to sustain viability. Once certain amounts of Cu-binding proteins are expressed and located in the periplasm, cells might be able to sustainably deal with environmental Cu stress. Subsequently, Cu export systems could be activated, allowing resuscitation to happen. Elevated Cu concentrations can have diverse toxic effects on different cellular components, e.g. oxidative stress or mismetalation of cuproenzymes . It can therefore be expected that the Cu-induced VBNC state is paralleled by a broader cellular stress response than solely the activation of metal detoxifying systems. Our data shows that also other stress response systems are DE during the different VBNC phases. Firstly, we observed upregulation of terpenoid biosynthetic enzymes including the squalene synthase. It could be possible that CH34 cells produce squalene to counteract elevated levels of oxidative stress during the first few hours of Cu treatment. Several studies attest the antioxidant properties of this biomolecule . Squalene has also recently been found accumulated in VBNC cells of Campylobacter jejuni . Moreover, upregulation of superoxide dismutases SodB and SodC, their regulator SoxR, several universal stress proteins (UspA family) as well as different protein-modulating systems like chaperonins or the Clp protease, suggests that oxidative stress remediation might be activated, especially during early stage Cu treatment. In the proteomic response of Micrococcus luteus cells in a nutrient depletion-induced VBNC state, UspA and superoxide dismutase were detected, among others . Interestingly, in a transcriptional study investigating the Cu-induced VBNC state in Xanthomonas campestris , overexpression of Clp-related genes was linked to VBNC induction . Our results support the theory that distantly-related bacteria might share certain conserved proteins associated with dormancy. Such conserved proteins might not even depend on the kind of stressor. For example, the Clp protease as well as chaperonins GroES and GroEL were DE in CH34 cells in a VBNC state induced by desiccation and starvation . The same study reports differential expression of the LolA and GlmU proteins, both of which were also DE in our data. Besides LolA and GlmU, several other DE proteins were linked to the ‘Cell wall/membrane/envelope biogenesis’ COG class, which seemed to be activated during the VBNC induction phase. Another interesting observation was increased motility in Cu-treated cells. Several proteins of the Che and Pil families, as well as flagellin FliC2 were upregulated throughout Cu exposure. Higher abundance of such motility proteins could lead to increased formation of pili and/or flagella, potentially mediating the enhanced swarming potential of resuscitated cells. This is an interesting aspect as a shift toward higher motility would not be promptly expected for “dormant” cells. In natural habitats, CH34 might activate chemotaxis when facing Cu stress to facilitate migration toward less harmful environments. Enhanced cell motility could thus be important to reach favorable conditions where resuscitation can happen and might be tightly coordinated with Cu homeostasis. In a proteomic study of Vibrio cholerae VBNC cells, proteins linked to bacterial chemotaxis were reported to be accumulated . Chemotaxis proteins were also enriched in resuscitating Acidovorax citrulli cells following a Cu-induced dormant state . Our observations further strengthen the link between chemotaxis and the VBNC state. During the VBNC state and resuscitation, CH34 showed significant alterations in its central carbon and energy metabolism. Firstly, cells transitioning to VBNC particularly showed downregulation of pyruvate metabolizing enzymes. This result corresponds with the frequently reported link between the VBNC state and slowed-down carbon metabolism . We suggest that reactivation takes place via the accumulation of AcCoA produced via degradation of intracellular PHB granules. Most enzymes part of this metabolic pathway were found upregulated in cells after 1 day of Cu treatment until full resuscitation at day 5. PHB degradation in Cu-induced VBNC cells had already been observed for the plant pathogen Ralstonia solanacearum . In the closely related Cupriavidus necator , PHB accumulation was reported to prevent entry and/or to help recover from the VBNC state induced by low temperature . Obruca et al . showed that the monomer of PHB, 3-hydroxybutyrate (3HB), has protective effects on enzymes in conditions such as Cu-induced oxidative stress . The authors also suggest that intracellular accumulation of this metabolite might increase stress resistance by acting as a chemical chaperone. Based on our proteomics data, we need to consider that further processing of 3HB to AcCoA and subsequent feed into the glyoxylate cycle is probably favored in CH34. What remains unclear is whether PHB itself is formed before or during the transition into VBNC. When stationary-phase cells were stained with Nile Red, the mRFU (correlating with intracellular PHB levels) stayed relatively constant during 5 days of incubation in mineral water. In the Cu condition, the value increased approximately 20-fold after only 5 h of incubation, suggesting a drastic accumulation of PHB during VBNC formation. However, it could be possible that existing PHB granules are simply made more accessible to the dye as a consequence of Cu ion stress (e.g. by membrane permeabilization). This artifact seems even more likely considering that no remarkable upregulation of PHB metabolizing enzymes could be detected during the first few hours of incubation. However, upregulation of such enzymes was indeed observed during the resuscitation phase, and it is likely that a PHB-degrading subpopulation is present. We can thus assume an association between PHB degradation and restoration of culturability in CH34, even though effect sizes were low for most time points. Breakdown of intracellular PHB leads to a growing pool of AcCoA that could be used for a shift toward anabolic metabolism. This is done via activation of the glyoxylate cycle. The key enzyme for this pathway, isocitrate lyase AceA, was strongly upregulated in resuscitating cells. The glyoxylate cycle might be a mechanism to trigger resuscitation, bypassing the catabolic steps of the citrate cycle to activate gluconeogenesis. Accordingly, we monitored upregulation of the phosphoenolpyruvate carboxykinase PckG (which is linking the glyoxylate cycle to gluconeogenesis) and the rate-limiting gluconeogenic enzyme Fbp2 in resuscitating cells. The glyoxylate cycle has already been linked to the VBNC state in M. luteus and was hypothesized to help nutrient-deprived VBNC cells survive . A functional glyoxylate cycle has recently been described as a crucial metabolic pathway for viability in Pseudomonas aeruginosa VBNC cells . In addition, we found that enzymes involved in the biosynthesis of biotin and thiamine were upregulated, cofactors that are important for various reactions of carbohydrate metabolism . Thiamine supplementation to mineral water largely prevented VBNC formation and its biosynthesis was activated during resuscitation. This suggests that thiamine abundance could be an important factor for cell culturability. While stimulation of carbohydrate metabolism might be an explanation for this effect, it is also possible that VBNC-inducing Cu ions are simply sequestrated by thiamine, for instance through chelation by its thiazole moiety. However, no evidence for a thiamine-mediated decrease in bioavailable Cu levels could be obtained so far.
This study provides an in-depth, time-resolved look into the proteomic state of water-borne C. metallidurans CH34 transitioning to and resuscitating from a Cu-induced VBNC state. We found wide-ranging adaptations in the proteomes inherent to these unique cell states, including but not limited to the activation of Cu detoxification systems. While those played a crucial role during the resuscitation phase, a more general response to oxidative stress, comprising the enhanced production of RPs and chaperones, as well as cell wall modulations, were observed in the VBNC entry phase. Notably, induction of the VBNC state is correlated with downregulation of pyruvate metabolism. Our data suggests that central carbon and energy metabolism were gradually reactivated through degradation of intracellular PHB, yielding AcCoA, which can subsequently be used for gluconeogenesis via the glyoxylate shunt. Newly formed cellular resources could then possibly be used to deliver the energy needed for increased production of Cu-detoxifying proteins (e.g. Cop proteins), permitting resuscitation from the VBNC state. Furthermore, we also detected an increased expression of chemotaxis-related proteins in Cu-treated cells that was verified phenotypically through motility assays. To further investigate mechanisms that are controlling the VBNC state, we propose integrating metabolomic and proteomic data, which would improve our fundamental understanding of bacterial dormancy. This could help to identify potential weak-points in a unique bacterial survival strategy.
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Comprehensive use of cardiopulmonary exercise testing in pediatrics | fed26e63-caff-415b-8bca-d54cb127c365 | 10227478 | Pediatrics[mh] | The cardiopulmonary exercise test (CPET) allows the possibility to appoint the pathophysiological limitations of exercise and also the significance of functional impairment. It began to be use as a “gold standard” to evaluate the result of surgical, medical and rehabilitative treatment on cardiopulmonary function and to investigate the integrated physiological reactions to exercise in paediatric medicine. It is widely use in paediatric patients and adults and significantly improved the understanding of cardiopulmonary development in children and adolescents . A large number of research tools are used in clinical practice to assess physical fitness. These tools have a lot of advantages and disadvantages . Cardiopulmonary exercise test has become an chief clinical non-invasive tool to assess and predict the capacity of exercise in patients with heart failure and in different cardiac conditions. It supplies estimation of the exercise responses, affecting the cardiovascular, pulmonary, skeletal muscle, metabolism and the cellular system, which are not well reflected in individual organ systems by measuring function . Cardiopulmonary exercise test carry physiological parameters at rest and during progressive exercise. It determines the ability to produce energy at metabolically relevant time points as anaerobic threshold and the body’s cardiorespiratory fitness . Resting pulmonary and cardiac function cannot reliably estimate physical performance and functional capacity . Energetic human capacity is the most significant factor that sets the limits of physical capacity . The cardiopulmonary exercise test allows to assess body’s response during sub and maximal exercise. Mainly measurements include gas exchange parameters such as: oxygen consumption, carbon dioxide production, minute ventilation, ECG monitoring, blood pressure and pulse oximetry . In the latest years of CPET exploitation, the test has been appreciably identified by medical interest and as a physiological bases of different variables, which were before unknown and by accentuation proof for a multivariable approach. Most of problems with ventilation and its control were taken into consideration . An obstacle in performing the CPET test was mostly described as the financial barrier. Hospitals and institutions trying to initiate the action mentioned lack of funding as the most common reason for not being able to test .
We performed a literature search at Google Scholar, PubMed, Science Direct, available literature from the book from 2006 to 2019 and internet sources. The bibliography search was reviewed and performed using selected keywords. This study is based on analysis of literature about cardiopulmonary exercise test and cardiorespiratory fitness.
Physical effort requires coordinated actions of physiological mechanisms related to the functioning of the nervous system, circulatory system, respiratory system and internal function to cover the escalated energy demand of working muscles . Features which condition physical performance are: efficiency of aerobic muscle supply and activation of biochemical processes determining the use of oxygen energy sources, removal of catabolite, efficiency of thermoregulation and size and efficiency of energy substrates use. Considering on the subject about efficiency, we can not forget about the tolerance of fatigue changes during CPET test, which it can affect: aversion to effort or fear of effort. It can also occur pain, dyspnoea, palpitation, or excessive sweating . In the study of children and adolescents, we must also consider the race of subjects. Studies conducted on Caucasian race in the United Kingdom have shown that, English children have higher cardiovascular fitness than Indian children . Social, religious, linguistic and cultural traditions exclude involvement in physical activity, therefore their ability and approach to sport or recreation may be different from other children . The surveys on male and female have shown that in swimmers of age from 9 to 20 years from West Bengal, had importantly lower than norm value of VO 2max , than international athletes, which practiced endurance type of sport, however they had significantly higher VO 2max parameter than sedentary girls of West Bengal . Cardiorespiratory fitness, is a solid parameter to estimate the capacity of the cardiovascular system to overcome extended physical work. It has been depicted to be the most dominant predictor of death rate and morbidity, besides of classical cardiovascular disease, risk factors such as cholesterol, smoking cigarettes, hypertension, and diabetes mellitus type 1 (T1DM) and diabetes mellitus type 2 (T2DM) . In recent years, studies have documented the health benefits of regular physical activity. Nowadays it is highly appreciated that higher cardiorespiratory fitness and physical activity standards are beneficial for the diseases prophylaxis and prevention . Physical activity is essential for human health in every period of life, and it gains special value during the time of the fastest and most intense development, i.e. childhood . It has a positive effect on dealing with civilization diseases such as diabetes type 2 (T2DM), improves bone health, reduces the incidence of cancer, reduces signs of disability and extends life . Cardiopulmonary exercise test variables The aim of spirometry is the continuous survey of respiration (spirography) and respiratory gas metabolism . The tests are performed on a treadmill, cycle-ergometer or outdoor. Portable Ergospirometers are very often used to study physiological ventilation variables in field tests . There are 2 basic types of ergospirometers. The first one is an ergospirometer with a mixing chamber. The principle of its operation, is that during breathing, samples of exhaled gas are taken and collected in a reservoir (chamber), where they are mixed. The size of a single sample is proportional to the current tidal volume (VT). In every constant period of time, measurements are made of the gas composition in the chamber, which is a mixture of taken samples. Measurements of average O 2 and CO 2 concentrations are obtained, e.g. another set of measurement data every 10 seconds. The action of the second one is based on continuous sampling of breathing air with a constant gas sample volume. In this method, measurements of O 2 and CO 2 concentrations require the use of fast gas analysers, usually with a response time of less than 120 ms. In addition, synchronization of the flow, O 2 and CO 2 concentrations is required due to delays in the sample drain and in the gas sensor itself. Measurements of temporary O 2 and CO 2 concentrations, are obtained after each breath. The advantage of the “breath-by-breath” type ergospirometers compared to devices with a mixing chamber is the high accuracy of the measurement regardless of changing environmental conditions, because the concentration of O 2 and CO 2 is measured both during the inspiration and exhalation phases . The major results are schedule in the following order: maximal oxygen uptake (VO2max/peak), carbon dioxide emission (VCO 2 ), ventilatory threshold (VT), minute ventilation (VE), ventilatory equivalents for oxygen (VE/VO 2 ) and carbon dioxide (VE/VCO 2 ), respiratory exchange ratio (RER/R, VCO 2 /VO 2 ), heart rate (HR), saturation (SatO 2 ), ECG, blood pressure (BP) The most important parameter examined in the assessment of physical fitness is VO 2max , which we describe as the maximum integrated capacity of the pulmonary system, cardiovascular system and muscular system to uptake, transport and utilize O 2 . Through the value of the oxygen uptake kinetic reaction its survey is complex by the large “inter-breath” change in oxygen uptake in children during the test. It cuts the reliance in which kinetic variables can be asses and necessitates the measurement of variety identic transitions . VO2peak is highest speed attained at the end of the test . Ventilatory threshold is described as a the level at which, sudden growth in blood lactate is noticed. Output of lactic acid in the muscle rise curvilinear with increasing work load . We also pay attention to the carbon dioxide that it is the sum of exhaled CO 2 by a examined patient is an act of the substrate and metabolic rate utilized in oxidative metabolism. The sum of exhaled CO 2 by a examined patient is an act of the substrate and metabolic rate utilized in oxidative metabolism. The amount of carbo-dioxide exhaled in oxidative metabolism for each litre of oxygen consumed is named (RER/R) the respiratory exchange ratio . This parameter nearing to 0.7 if the dominant fuel is fat to 1.0 if the prevalent fuel is carbohydrate. During dynamic exercise, the heart rate (HR) increases in order to respond to higher oxygen demand. It is accompanied by an increase in the stroke volume of the heart, which reaches its maximum value already at 30–50% VO 2max . Enhanced work of the heart causes an augmentation in blood flow mainly in working skeletal muscles, heart and skin at the expense of a decrease in flow through the kidneys, liver and visceral organs. During physical effort, the body increases its oxygen demand, so the lung ventilation process potentiates. After beginning of training, there is an increase in VE (minute ventilation), the breathing cycle speeds up and gets deeper. The rapid increase in VE lasts a few seconds after initiation of activity, then this trend slows down until it reaches a level of stabilization. The transition phase occurs when you stop exercising operations. In the case of intense effort, the VE value enhance constantly, the steady state phase does not occur. During low intensity exercise, VE increases proportionally to VO 2 until it reaches 50–75% VO 2max . Parameters related to cardiopulmonary exercise test were divided into this, which characterize circulatory system, lung ventilation, metabolic changes and those which are enters into gas exchange in the lungs . Contraindications and savouireship Each patient should receive instructions and basic information on how the laboratory equipment works and what the test procedure consists of. The patient should avoid eating meals, smoking cigarettes and drinking alcohol at least 2 hours before the test. Patient should wear comfortable clothing and footwear. It should also be also follow the history of medications and perform resting supine ECG to identify individual for whom the test could be contraindicated or should be performed with special safety features . The basis that we can modify is the protocol with increasing linear load. It is able to choose Ramp or stepwise protocol. During the measurement process, the child should achieve a constant speed of 60 to 80 rpm. The load is gradually increased, depending on the chosen linear protocol. It is set to 1 W/1 kg of body weight as the basic load and increase the resistance every 10 seconds by 1 W. The load is heightened by increasing the resistance of the cycle-ergometer pedals. After reaching the desired parameters or when indicators to stop the examination appear, the doctor or paramedic decides to finish the survey. The test can also be interrupted at any time at the patient’s request or when disturbing symptoms appear. After the effort, a rest phase follows, then the patient is disconnected from the device and the electrodes are peeled off and discarded. The duration of the test lasts from 30 to 60 minutes . We increase the effort load to: Obtain the maximum rhythm frequency (220-age), occurrence of symptoms indicating need to end the test (maximum stress test limited by symptoms), achieving 85% of the maximum frequency rhythm (submaximal exercise test) . Absolute contraindications and exclusion criteria for children and adolescents are described in detail by American Heart Association (AHA). We can include among them: disagreement of person being examined/guardian, severe respiratory failure, congestive heart failure, active rheumatic fever with carditis, significant aortic stenosis, significant mitral valve stenosis, uncontrolled cardiac arrhythmias causing clinical symptoms or disadvantaging hemodynamics, severe arterial hypertension (systolic pressure & gt: 200 mm Hg and/or diastolic pressure & gt: 120 mm Hg), hypertrophic cardiomyopathy with former cases of collapse, diabetic children hypoglycaemia, hypoglycaemia above 250 mg/dl, severe disorders of other organs which may impact on the course of the effort or increase under their influence (e.g. infection, kidney failure, thyrotoxicosis), lower extremity phlebitis, physical disability which may prevent to perform safe and adequate test, mental disability preventing cooperation . However, some children, adolescents and adults noticed discomfort with the mouthpiece, facemask, or with nose clip. Consequently, all these inconvenience, should be reported before starting the CPET test. They serve to show the need for versatile initial patient assessment, and precise monitoring during the survey . Cardiopulmonary exercise test should be interpreted and controlled by a consultant with an experience in conducting the cardiopulmonary exercise testing. Furthermore, the individual performing the CPET test should be experienced in working on cardiopulmonary tests like also interpreting the outcomes . However, despite their precision and reproducibility, cardiopulmonary exercise testing physicians (cardiologists, pulmonologists, and physiologists) must be well trained to avoid misinterpretation pitfalls and above all, highly experienced in clinical practice and pathological conditions .
The aim of spirometry is the continuous survey of respiration (spirography) and respiratory gas metabolism . The tests are performed on a treadmill, cycle-ergometer or outdoor. Portable Ergospirometers are very often used to study physiological ventilation variables in field tests . There are 2 basic types of ergospirometers. The first one is an ergospirometer with a mixing chamber. The principle of its operation, is that during breathing, samples of exhaled gas are taken and collected in a reservoir (chamber), where they are mixed. The size of a single sample is proportional to the current tidal volume (VT). In every constant period of time, measurements are made of the gas composition in the chamber, which is a mixture of taken samples. Measurements of average O 2 and CO 2 concentrations are obtained, e.g. another set of measurement data every 10 seconds. The action of the second one is based on continuous sampling of breathing air with a constant gas sample volume. In this method, measurements of O 2 and CO 2 concentrations require the use of fast gas analysers, usually with a response time of less than 120 ms. In addition, synchronization of the flow, O 2 and CO 2 concentrations is required due to delays in the sample drain and in the gas sensor itself. Measurements of temporary O 2 and CO 2 concentrations, are obtained after each breath. The advantage of the “breath-by-breath” type ergospirometers compared to devices with a mixing chamber is the high accuracy of the measurement regardless of changing environmental conditions, because the concentration of O 2 and CO 2 is measured both during the inspiration and exhalation phases . The major results are schedule in the following order: maximal oxygen uptake (VO2max/peak), carbon dioxide emission (VCO 2 ), ventilatory threshold (VT), minute ventilation (VE), ventilatory equivalents for oxygen (VE/VO 2 ) and carbon dioxide (VE/VCO 2 ), respiratory exchange ratio (RER/R, VCO 2 /VO 2 ), heart rate (HR), saturation (SatO 2 ), ECG, blood pressure (BP) The most important parameter examined in the assessment of physical fitness is VO 2max , which we describe as the maximum integrated capacity of the pulmonary system, cardiovascular system and muscular system to uptake, transport and utilize O 2 . Through the value of the oxygen uptake kinetic reaction its survey is complex by the large “inter-breath” change in oxygen uptake in children during the test. It cuts the reliance in which kinetic variables can be asses and necessitates the measurement of variety identic transitions . VO2peak is highest speed attained at the end of the test . Ventilatory threshold is described as a the level at which, sudden growth in blood lactate is noticed. Output of lactic acid in the muscle rise curvilinear with increasing work load . We also pay attention to the carbon dioxide that it is the sum of exhaled CO 2 by a examined patient is an act of the substrate and metabolic rate utilized in oxidative metabolism. The sum of exhaled CO 2 by a examined patient is an act of the substrate and metabolic rate utilized in oxidative metabolism. The amount of carbo-dioxide exhaled in oxidative metabolism for each litre of oxygen consumed is named (RER/R) the respiratory exchange ratio . This parameter nearing to 0.7 if the dominant fuel is fat to 1.0 if the prevalent fuel is carbohydrate. During dynamic exercise, the heart rate (HR) increases in order to respond to higher oxygen demand. It is accompanied by an increase in the stroke volume of the heart, which reaches its maximum value already at 30–50% VO 2max . Enhanced work of the heart causes an augmentation in blood flow mainly in working skeletal muscles, heart and skin at the expense of a decrease in flow through the kidneys, liver and visceral organs. During physical effort, the body increases its oxygen demand, so the lung ventilation process potentiates. After beginning of training, there is an increase in VE (minute ventilation), the breathing cycle speeds up and gets deeper. The rapid increase in VE lasts a few seconds after initiation of activity, then this trend slows down until it reaches a level of stabilization. The transition phase occurs when you stop exercising operations. In the case of intense effort, the VE value enhance constantly, the steady state phase does not occur. During low intensity exercise, VE increases proportionally to VO 2 until it reaches 50–75% VO 2max . Parameters related to cardiopulmonary exercise test were divided into this, which characterize circulatory system, lung ventilation, metabolic changes and those which are enters into gas exchange in the lungs .
Each patient should receive instructions and basic information on how the laboratory equipment works and what the test procedure consists of. The patient should avoid eating meals, smoking cigarettes and drinking alcohol at least 2 hours before the test. Patient should wear comfortable clothing and footwear. It should also be also follow the history of medications and perform resting supine ECG to identify individual for whom the test could be contraindicated or should be performed with special safety features . The basis that we can modify is the protocol with increasing linear load. It is able to choose Ramp or stepwise protocol. During the measurement process, the child should achieve a constant speed of 60 to 80 rpm. The load is gradually increased, depending on the chosen linear protocol. It is set to 1 W/1 kg of body weight as the basic load and increase the resistance every 10 seconds by 1 W. The load is heightened by increasing the resistance of the cycle-ergometer pedals. After reaching the desired parameters or when indicators to stop the examination appear, the doctor or paramedic decides to finish the survey. The test can also be interrupted at any time at the patient’s request or when disturbing symptoms appear. After the effort, a rest phase follows, then the patient is disconnected from the device and the electrodes are peeled off and discarded. The duration of the test lasts from 30 to 60 minutes . We increase the effort load to: Obtain the maximum rhythm frequency (220-age), occurrence of symptoms indicating need to end the test (maximum stress test limited by symptoms), achieving 85% of the maximum frequency rhythm (submaximal exercise test) . Absolute contraindications and exclusion criteria for children and adolescents are described in detail by American Heart Association (AHA). We can include among them: disagreement of person being examined/guardian, severe respiratory failure, congestive heart failure, active rheumatic fever with carditis, significant aortic stenosis, significant mitral valve stenosis, uncontrolled cardiac arrhythmias causing clinical symptoms or disadvantaging hemodynamics, severe arterial hypertension (systolic pressure & gt: 200 mm Hg and/or diastolic pressure & gt: 120 mm Hg), hypertrophic cardiomyopathy with former cases of collapse, diabetic children hypoglycaemia, hypoglycaemia above 250 mg/dl, severe disorders of other organs which may impact on the course of the effort or increase under their influence (e.g. infection, kidney failure, thyrotoxicosis), lower extremity phlebitis, physical disability which may prevent to perform safe and adequate test, mental disability preventing cooperation . However, some children, adolescents and adults noticed discomfort with the mouthpiece, facemask, or with nose clip. Consequently, all these inconvenience, should be reported before starting the CPET test. They serve to show the need for versatile initial patient assessment, and precise monitoring during the survey . Cardiopulmonary exercise test should be interpreted and controlled by a consultant with an experience in conducting the cardiopulmonary exercise testing. Furthermore, the individual performing the CPET test should be experienced in working on cardiopulmonary tests like also interpreting the outcomes . However, despite their precision and reproducibility, cardiopulmonary exercise testing physicians (cardiologists, pulmonologists, and physiologists) must be well trained to avoid misinterpretation pitfalls and above all, highly experienced in clinical practice and pathological conditions .
Cardiopulmonary exercise test in clinical praxis is very useful and has potential indication for use in assessing the functional capacity of young people with moderate to severe valvular defects to evaluate for possible surgical intervention and to determine whether early fatigue is due to defect or deconditioning . Cardiopulmonary exercise test contains estimation of tolerance and intolerance during exercise, rating of patients with cardiovascular like: (heart failure, transplantation, cardiac rehabilitation, and exercise individualization) and respiratory diseases as: (chronic obstructive pulmonary disease (COPD), cystic fibrosis, interstitial lung diseases, pulmonary vascular disease and exercise-induced bronchospasm) and different clinical applicabilities like exercise rehabilitation, preoperative risk evaluation and exercise prescription to overall health improvement . The cardiopulmonary exercise test with survey of metabolic parameters, such as peak myocardial oxygen consumption and also exercise ventilation, may help in the clinical assessment of hypertrophic cardiomyopathy (HCM) patients in their functional capacity . Measurements of gas exchange are taking place more and more often in sports medicine. . It is a useful tool for assessing limitations during daily activities, that have a physiological basis on individual with chronic organ failure Cardiopulmonary exercise test is one of the most important diagnostic methods used in cardiology and sports medicine. Measurements, including gas exchange parameters during exercise, are characterized by a high prognostic value in patients not only with heart failure, but also with respiratory diseases . It would seems that it is impossible to perform a test on people with mucoviscoidosis. With the right approach and load dosing, Urquhart and Vendrusculo conducted a study on a group of 4 children from the age of 14 to 15. The measurement of performance and efficiency in cooperation with the musculoskeletal system and the cardiovascular system provided by CPET test adds more information to individualize exercise programmes for patients with highest risk suffering on cystic fibrosis . Also in patients with chronic obstructive pulmonary disease (COPD), VO2max/peak is the best indicator of aerobic fitness, as long as patients are able to exercise more than their limits . In studies conducted by Hunt et al . cardiorespiratory fitness on children was measured by FitnessGram assessment protocol. This is a good comparative method to the cardiopulmo-nary exercise test, because of the cost and the possibility of conducting it in the field. FitnesGram is usually use to estimate cardiorespiratory fitness and improve health and physical activity in children and adolescents .
Measurement of expiratory gas exchange during the test, physical activity is a repeatable and objective method, which enables accurate measurement of functional capacity. In this way, it is possible to detect the causes of reduced tolerance of effort, to notice the severity of many diseases, to monitor the effects of treatment and rehabilitation, but also to confirm the complete health and ability to exercise intensively.
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Assessing donor kidney function: the role of CIRBP in predicting delayed graft function post-transplant | 1ce104b7-6560-4c57-a9b8-b54a5a998064 | 11782215 | Surgical Procedures, Operative[mh] | Introduction Research has shown that delayed graft function (DGF) significantly reduces the overall survival rate of transplanted kidneys, increases the risk of rejection, prolongs hospital stay, and leads to higher healthcare costs . The adoption of extended-criteria donor (ECD) kidneys has expanded in recent years to address donor kidney shortages. However, this expansion has caused increased DGF incidence and graft loss . Thus, more accurate methods for assessing graft function and predicting DGF are essential to optimize the safe use of ECD donor kidneys. Current biomarkers for assessing kidney function are difficult to implement in clinical settings, and existing scoring systems lack sufficient validation . Serum creatinine is a kidney-specific metabolite most widely used as a marker for evaluating renal function . Its levels at various time points are also significant in predicting early outcomes in transplanted kidneys . However, serum creatinine levels are influenced by variables like age, sex, and muscle mass, and it does not accurately reflect tubular damage, limiting its clinical utility . Biomarkers such as exosomal miRNAs , transcriptomics UBD mRNA , and Klotho are associated with DGF; however, they have been studied in small sample sizes, and their detection methods are complex, hindering the establishment of a unified clinical diagnostic standard. The kidney donor profile index (KDPI) is a widely used indicator for predicting the long-term survival of transplanted kidneys across various kidney transplant centers. However, the KDPI lacks comprehensive patient data, limiting its accuracy and clinical applications . Several existing scoring systems exhibit limited predictive ability for transplant kidney outcomes . Although incorporating pathological findings of the transplanted kidney into these scoring systems improves the accuracy of DGF prediction, it remains time-consuming and does not support timely decision-making . Cold-inducible RNA-binding protein (CIRBP), has been classified as a damage-associated molecular pattern (DAMP), belongs to the heterologous ribonucleoprotein subgroup within the RNA-binding protein family . Also known as heterologous ribonucleoprotein A18, CIRBP responds to various stress conditions by modulating mRNA stability . Under normal conditions, CIRBP is expressed at low levels in tissues, resulting in low serum concentrations. However, when tissues experience damage from factors such as low temperature, oxidative stress, or ultraviolet irradiation, CIRBP expression increases significantly in the cytoplasm and is released extracellularly via the lysosomal and exosomal pathways . Serum CIRBP levels can increase within 6 h of tissue injury . During acute kidney injury (AKI), epithelial cells express and release large amounts of CIRBP, leading to cellular dysfunction by promoting the production of reactive oxygen species (ROS) and the cleavage of mitochondrial double-stranded DNA , inducing the release of inflammatory factors and apoptosis and accelerating AKI progression . Extracellular CIRBP worsens renal injury by binding to TREM-1 during ischemia-reperfusion injury (IRI) . Clinical studies have demonstrated that postoperative cardiac surgery patients with elevated serum CIRBP levels are more likely to develop AKI, with those exhibiting above-average concentrations experiencing more severe injury . Therefore, donor serum CIRBP levels reflect kidney injury before transplantation and can serve as a possible indicator for predicting post-transplant graft outcomes. Additionally, CIRBP is more clinically applicable than other biomarkers due to the ease of obtaining donor specimens and the convenience of the assay method. The purpose of this study was to investigate the relationship between serum CIRBP levels and kidney graft function following transplantation, with the hypothesis that elevated CIRBP levels are indicative of severe donor kidney injury and reduced post-transplant graft function.
Materials and methods 2.1 Study design This retrospective study involved deceased donors and recipients who had undergone successful kidney transplantation in our transplant center between 2016 and 2019. No organs were sourced from executed prisoners. Donors were excluded if they fulfilled any of the following criteria: (a) lacked a blood specimen, (b) had contaminated blood specimens affecting CIRBP levels, (c) were recipients who underwent combined multi-organ transplants, (d) were participating in other clinical trials, (e) had a history of organ transplantation, or (f) had other conditions deemed unsuitable for enrollment by the investigator. Blood specimens were collected from donors by Organ Procurement Organizations and used in various studies with the donors’ consent. All recipients were followed-up for a long period post-transplantation. This study was conducted in accordance with the ethical standards outlined in the Declaration of Helsinki and approved by the Medical Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University. Organs were allocated in a fair and transparent manner using the Chinese Organ Transplant Response System . The donor’s family voluntarily determined the type of donation, and the recipients underwent follow-up after transplantation. All participants provided informed consent before being included in the study, and their confidentiality was ensured throughout the research process. Data were anonymized to protect personal identities, and no patient-identifiable information was used in any part of the study. Donor data were obtained from the Chinese Computerized System for Human Organ Distribution and Sharing, completed by members of organ transplant organizations. Donor information was obtained from clinical electronic medical records. Recipient data were derived from electronic clinical records alone. Recipients were categorized into the DGF or immediate graft function (IGF) group according to their early renal function status. Donors were similarly classified into DGF and IGF groups, depending on the early renal function recovery of their paired recipients. DGF was defined as a reduction in blood creatinine concentration of less than 10% for three consecutive days within 1 week postoperatively, or if serum creatinine (SCr) did not decrease to 400 μmol/L within 1 week post-surgery . IGF was defined as the fast postoperative recovery of renal function with satisfactory diuresis and no further need for dialysis. Graft loss was defined as end stage renal disease requiring dialysis or retransplantation. ECD donors were defined as those over 60 years-old, or between 50 and 59 years old who met at least two of the following criteria: final serum creatinine exceeding 133 μmol/L, cause of death being cerebrovascular accident, or a history of hypertension . We converted human leukocyte antigen (HLA) mismatches into hierarchical profiles to facilitate data analysis: 0 to 1 mismatch as Level 1, 2 to 4 mismatches as Level 2, and 5 to 6 mismatches as Level 3. 2.2 Sample collection Prior to kidney procurement, 2 mL of fresh blood was drawn from the donor in the operating room using a vacuum blood collection needle and placed in a sterile test tube. The blood was left at room temperature for 1 hour and then centrifuged at 4°C, 2,500 rpm for 10 min. The resulting supernatant was separated, while the sediment was discarded. The supernatant was subsequently divided into aliquots, frozen, and stored at -80°C in the hospital. Before testing, the serum samples were thawed on crushed ice and centrifuged again at 1,000 g for 15 min at 4°C. 2.3 Measurement of cold inducible RNA binding protein level Serum CIRBP concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit (CSB-EL005440HU). Firstly, the protein standard was diluted per the instructions, and 100 µL of different concentrations of standard and samples (1:10 dilution) were added to a 96-well plate in duplicates, and the plates were incubated at 37°C for 2 h in the dark. Following incubation, the liquid from the wells was discarded, and 100 µL of pre-diluted anti-CIRBP biotin-labeled antibody solution was introduced into each well. After incubating at 37°C for 1 h, each well was washed at least three times with a washing solution before 100 μL of diluted horseradish peroxidase-labeled antibody was added to each well, following the same procedure as that in the previous step. After another 1-hour incubation at 37°C, we washed each well at least five times, to prevent false positive results. Thereafter, we added 90 µL of substrate solution to each well, and add 50 µL of termination solution was added to stop the reaction after a 15-min color development at 37°C. The optical density (OD) at 450 nm was measured within 5 minutes using an enzyme marker. A standard curve model was then generated from the OD values of the standard wells, and the CIRBP concentration of each sample was determined using this model. 2.4 Statistical analysis Age is presented as the mean ± standard deviation (SD). Student’s t-test was applied to compare the ages between the DGF and IGF groups. Body mass index (BMI), donor terminal creatinine, donor serum CIRBP, kidney donor risk index, KDPI, dialysis duration, and post-transplantation renal function values are presented as median [interquartile range], and these variables were compared between the DGF and IGF groups using the Mann-Whitney U test. Frequencies and percentages were used to present categorical variables, such as sex, cause of death, and dialysis modality. Comparisons between two groups were performed using the Chi-square tests when the sample size was ≥40 and Fisher’s exact tests when the sample size was <40. KDPI was calculated using standard methods . Factors associated with DGF and graft loss were confirmed using the Spearman correlation analysis. Each factor potentially associated with the occurrence of DGF after renal transplantation was initially analyzed using univariate logistic regression to statistically determine its influence on DGF. Independence of these factors was further evaluated through stepwise backward multivariate logistic regression, with entry criteria set at 0.05 and removal criteria at 0.1. This approach ensured that only meaningful parameters were retained in the final model. The predictive ability of CIRBP for DGF and graft loss was evaluated through receiver operating characteristics (ROC) curve analyses, with optimal cutoff points derived from Youden’s index, J, which is determined as J=sensitivity + specificity – Eqn. 1 . In addition, we applied forward stepwise multivariate linear regression analyses to identify the factors influencing recipient SCr concentrations at 6 months post-kidney transplantation. The Kaplan-Meier analysis was used to evaluate the impact of DGF occurrence and elevated donor plasma CIRBP levels on allograft survival time following kidney transplantation. All statistical tests were performed using SPSS version 27.0 (SPSS Inc., Chicago, IL, USA), and significance was determined by a P-value of less than 0.05.
Study design This retrospective study involved deceased donors and recipients who had undergone successful kidney transplantation in our transplant center between 2016 and 2019. No organs were sourced from executed prisoners. Donors were excluded if they fulfilled any of the following criteria: (a) lacked a blood specimen, (b) had contaminated blood specimens affecting CIRBP levels, (c) were recipients who underwent combined multi-organ transplants, (d) were participating in other clinical trials, (e) had a history of organ transplantation, or (f) had other conditions deemed unsuitable for enrollment by the investigator. Blood specimens were collected from donors by Organ Procurement Organizations and used in various studies with the donors’ consent. All recipients were followed-up for a long period post-transplantation. This study was conducted in accordance with the ethical standards outlined in the Declaration of Helsinki and approved by the Medical Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University. Organs were allocated in a fair and transparent manner using the Chinese Organ Transplant Response System . The donor’s family voluntarily determined the type of donation, and the recipients underwent follow-up after transplantation. All participants provided informed consent before being included in the study, and their confidentiality was ensured throughout the research process. Data were anonymized to protect personal identities, and no patient-identifiable information was used in any part of the study. Donor data were obtained from the Chinese Computerized System for Human Organ Distribution and Sharing, completed by members of organ transplant organizations. Donor information was obtained from clinical electronic medical records. Recipient data were derived from electronic clinical records alone. Recipients were categorized into the DGF or immediate graft function (IGF) group according to their early renal function status. Donors were similarly classified into DGF and IGF groups, depending on the early renal function recovery of their paired recipients. DGF was defined as a reduction in blood creatinine concentration of less than 10% for three consecutive days within 1 week postoperatively, or if serum creatinine (SCr) did not decrease to 400 μmol/L within 1 week post-surgery . IGF was defined as the fast postoperative recovery of renal function with satisfactory diuresis and no further need for dialysis. Graft loss was defined as end stage renal disease requiring dialysis or retransplantation. ECD donors were defined as those over 60 years-old, or between 50 and 59 years old who met at least two of the following criteria: final serum creatinine exceeding 133 μmol/L, cause of death being cerebrovascular accident, or a history of hypertension . We converted human leukocyte antigen (HLA) mismatches into hierarchical profiles to facilitate data analysis: 0 to 1 mismatch as Level 1, 2 to 4 mismatches as Level 2, and 5 to 6 mismatches as Level 3.
Sample collection Prior to kidney procurement, 2 mL of fresh blood was drawn from the donor in the operating room using a vacuum blood collection needle and placed in a sterile test tube. The blood was left at room temperature for 1 hour and then centrifuged at 4°C, 2,500 rpm for 10 min. The resulting supernatant was separated, while the sediment was discarded. The supernatant was subsequently divided into aliquots, frozen, and stored at -80°C in the hospital. Before testing, the serum samples were thawed on crushed ice and centrifuged again at 1,000 g for 15 min at 4°C.
Measurement of cold inducible RNA binding protein level Serum CIRBP concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit (CSB-EL005440HU). Firstly, the protein standard was diluted per the instructions, and 100 µL of different concentrations of standard and samples (1:10 dilution) were added to a 96-well plate in duplicates, and the plates were incubated at 37°C for 2 h in the dark. Following incubation, the liquid from the wells was discarded, and 100 µL of pre-diluted anti-CIRBP biotin-labeled antibody solution was introduced into each well. After incubating at 37°C for 1 h, each well was washed at least three times with a washing solution before 100 μL of diluted horseradish peroxidase-labeled antibody was added to each well, following the same procedure as that in the previous step. After another 1-hour incubation at 37°C, we washed each well at least five times, to prevent false positive results. Thereafter, we added 90 µL of substrate solution to each well, and add 50 µL of termination solution was added to stop the reaction after a 15-min color development at 37°C. The optical density (OD) at 450 nm was measured within 5 minutes using an enzyme marker. A standard curve model was then generated from the OD values of the standard wells, and the CIRBP concentration of each sample was determined using this model.
Statistical analysis Age is presented as the mean ± standard deviation (SD). Student’s t-test was applied to compare the ages between the DGF and IGF groups. Body mass index (BMI), donor terminal creatinine, donor serum CIRBP, kidney donor risk index, KDPI, dialysis duration, and post-transplantation renal function values are presented as median [interquartile range], and these variables were compared between the DGF and IGF groups using the Mann-Whitney U test. Frequencies and percentages were used to present categorical variables, such as sex, cause of death, and dialysis modality. Comparisons between two groups were performed using the Chi-square tests when the sample size was ≥40 and Fisher’s exact tests when the sample size was <40. KDPI was calculated using standard methods . Factors associated with DGF and graft loss were confirmed using the Spearman correlation analysis. Each factor potentially associated with the occurrence of DGF after renal transplantation was initially analyzed using univariate logistic regression to statistically determine its influence on DGF. Independence of these factors was further evaluated through stepwise backward multivariate logistic regression, with entry criteria set at 0.05 and removal criteria at 0.1. This approach ensured that only meaningful parameters were retained in the final model. The predictive ability of CIRBP for DGF and graft loss was evaluated through receiver operating characteristics (ROC) curve analyses, with optimal cutoff points derived from Youden’s index, J, which is determined as J=sensitivity + specificity – Eqn. 1 . In addition, we applied forward stepwise multivariate linear regression analyses to identify the factors influencing recipient SCr concentrations at 6 months post-kidney transplantation. The Kaplan-Meier analysis was used to evaluate the impact of DGF occurrence and elevated donor plasma CIRBP levels on allograft survival time following kidney transplantation. All statistical tests were performed using SPSS version 27.0 (SPSS Inc., Chicago, IL, USA), and significance was determined by a P-value of less than 0.05.
Results 3.1 Baseline characteristics illustrates the process of assembling the cohort, while and present the baseline characteristics of the 207 donor-recipient pairs engaged in the study. The cohort was stratified according to allograft function; 51 (25%) recipients developed DGF after kidney transplantation. The DGF and IGF groups were then compared. The mean ± SD donor age was 42.31 ± 14.25 years, and the median [interquartile range] BMI was 23.00 [21.40–25.40] kg/m 2 . Among the donors, 86% were male, 20% had hypertension, 46% died of cerebral hemorrhage, and 17% were extended-criteria donors; however, no factor significantly differed between the groups. As expected, the median plasma CIRBP level in the DGF group was 6.82 ng/mL, significantly higher than that in the IGF group (3.44 ng/mL; P<0.001). Similar results were observed for donor terminal SCr levels (158.00 vs. 109.00; P<0.001). However, we failed to observe the variation in donor KDPI values amidst the DGF and IGF groups (59% vs. 46%; P=0.215), consistent with our prediction that KDPI does not reliably predict early post-transplant renal function. The mean ± SD recipient age was 42.56 ± 10.80 years, with a median [interquartile range] BMI of 21.26 [19.84–22.86] kg/m 2 . Comparing the DGF and IGF groups, we found that male recipients (88% vs. 66%; P=0.002) and those with a higher BMI (22.64 vs. 20.96; P<0.001) had a greater likelihood of developing DGF. Additionally, recipients with DGF had distinctly higher SCr levels at 6 months post-transplant compared to those in the IGF group (171.00 vs. 121.00; P<0.001). No differences were found between the groups in terms of dialysis type, dialysis duration, HLA mismatch level, induction therapy, or immunosuppressant dosage. 3.2 Donor plasma CIRBP as a risk factor for DGF after kidney transplantation DGF occurred in recipients paired with 51 (25%) donors following kidney transplantation. Testing of donor plasma samples revealed that the median CIRBP concentration in the DGF group was 6.82 ng/mL, which was 1.98 times higher than that in the IGF group (6.82 vs. 3.44; P<0.001). Recipients with CIRBP concentrations exceeding 5.484 ng/mL were four times more likely to develop DGF than those with concentrations below 5.484 ng/mL (53% vs. 12%; P<0.001). When CIRBP levels exceeded 7.92 ng/mL, the probability of developing DGF was 100%, whereas levels below 2 ng/mL corresponded to 0% probability of developing DGF . Although the median terminal SCr concentration was notably higher in the DGF group than that in the IGF group, 19% of recipients developed DGF postoperatively even when the SCr concentration was < 100 μmol/L . Additionally, the scatter distribution of CIRBP resembled that of SCr , and Spearman’s correlation demonstrated a significant relationship between the two factors (r=0.140, P=0.045). Furthermore, the proportion of female recipients was notably lower in the DGF group than that in the IGF group (12% vs. 34%; P=0.002) , while no significant variation in KDPI was found between the two groups . Graft loss occurred in 10 recipients in the DGF group, showing a higher rate, compared to the IGF group (19.6% vs. 5.8%; P=0.003). To further clarify the role of CIRBP in DGF, a logistic regression analysis was conducted. Univariate analysis revealed that for every 1 μmol/L increase in donor terminal SCr, the risk of developing DGF increased by 0.7% (95% confidence interval [CI]: 1.004–1.010; P<0.001), for every 1 ng/mL rise in plasma CIRBP concentration, the risk increased by 73.3% (95% CI: 1.446–2.076; P<0.001), and female recipients had a 0.259-fold lower probability of developing DGF compared to males (95% CI: 0.104–0.646; P=0.004). Donor plasma CIRBP (OR=1.660; 95% CI: 1.376–2.004; P<0.001) and donor terminal SCr (OR=1.005; 95% CI: 1.001–1.009; P=0.025) were identified by multivariate analysis as independent risk factors for DGF development, while a protective factor was female sex of the recipient (OR=0.302; 95% CI: 0.103–0.885; P=0.029) . 3.3 Donor plasma CIRBP effectively predicts DGF The area under the ROC curve (ROC-AUC) of CIRBP was 0.801 (95% CI: 0.728–0.874; P<0.001), and the optimal cut-off value was 5.548 ng/mL, calculated using the Youden index, with a sensitivity of 65% and 83% specificity . This performance was markedly superior to that of donor-terminal creatinine (ROC-AUC=0.654; 95% CI: 0.560–0.749; P=0.001) and KDPI (ROC-AUC=0.558; 95% CI: 0.471–0.645; P=0.215) . Enhancing the predictive ability of CIRBP, ROC curve analysis was performed via logistic regression, separately fitting donor plasma CIRBP to donor terminal SCr levels and recipient sex. The best predictive model was represented by the formula: y=1.660×donor plasma CIRBP+3.312×male+1.005×donor terminal SCr-5.365 (ROC-AUC=0.835; 95% CI: 0.771–0.898; P<0.001). The optimal cutoff value was 0.308, having a sensitivity of 63% and 88% specificity and was superior to both the fitting model of plasma CIRBP and donor terminal SCr (ROC-AUC=0.808; 95% CI: 0.733–0.884; P<0.001) and the fitting model of plasma CIRBP and recipient sex (ROC-AUC 0.821; 95% CI: 0.753–0.888; P<0.001) . 3.4 Correlation between donor plasma CIRBP and recipient creatinine at 6 months post-transplantation At 6 months post-transplantation, recipient SCr concentration increased with increasing plasma CIRBP levels in donors . Meanwhile, the SCr concentration at 6 months post-transplantation increased with the KDPI value , consistent with previous findings, suggesting that the KDPI may help predict long-term donor kidney survival. However, no significant trend was noted for donor terminal SCr levels . Univariate linear regression analysis revealed significant linear correlations between SCr concentration at 6 months and donor plasma CIRBP (R²=0.08, P<0.001), KDPI (R²=0.07, P<0.001), female recipients (R²=0.06, P=0.002), donor age (R²=0.054, P=0.003), as well as donor cause of death being cerebral hemorrhage (R²=0.035, P=0.016) . Ultimately, only donor plasma CIRBP (R²=0.08, P=0.003), KDPI (R²=0.137, P=0.002), and female recipient status (R²=0.174, P=0.008) were retained in the multiple linear regression analyses . Conversely, donor terminal SCr levels were not found to be associated with recipient creatinine at 6 months post-transplantation. 3.5 Donor plasma CIRBP and its association with transplanted kidney survival time Recipients were categorized into high plasma CIRBP (≥5.484 ng/mL, n=60) and low plasma CIRBP (<5.484 ng/mL, n=147) groups based on the optimal cutoff value . We hypothesized that donor kidneys with higher plasma CIRBP concentrations would exhibit significantly shorter survival times compared to those with lower concentrations. Although our survival analysis showed a trend of reduced survival time for donor kidneys with plasma CIRBP concentrations >5.484 ng/mL, further statistical analysis did not support this hypothesis (P=0.167; ). However, when comparing graft survival times between the DGF and IGF groups, the Kaplan-Meier curves revealed that recipients who developed DGF had shorter long-term graft survival (P=0.002; ), consistent with findings from related studies. Univariate and multivariate analyses were conducted to identify the risk factors for kidney transplant loss. The results indicated that donor plasma CIRBP and donor terminal SCr were not significantly correlated with transplant loss (P=0.057 and P=0.231, respectively). Recipient female sex was the only significant factor correlated with reduced transplant loss risk (OR=0.125; 95% CI: 0.016–0.955; P=0.045) . Subsequently, ROC curve analyses were conducted to evaluate the association between transplanted kidney survival and donor-terminal SCr (ROC-AUC=0.420; 95% CI: 0.283–0.557; P=0.250), plasma CIRBP (ROC-AUC=0.601; 95% CI: 0.469–0.732; P=0.149), and KDPI (ROC-AUC=0.593; 95% CI: 0.457–0.728; P=0.183) . However, these analyses did not reveal any statistically significant differences.
Baseline characteristics illustrates the process of assembling the cohort, while and present the baseline characteristics of the 207 donor-recipient pairs engaged in the study. The cohort was stratified according to allograft function; 51 (25%) recipients developed DGF after kidney transplantation. The DGF and IGF groups were then compared. The mean ± SD donor age was 42.31 ± 14.25 years, and the median [interquartile range] BMI was 23.00 [21.40–25.40] kg/m 2 . Among the donors, 86% were male, 20% had hypertension, 46% died of cerebral hemorrhage, and 17% were extended-criteria donors; however, no factor significantly differed between the groups. As expected, the median plasma CIRBP level in the DGF group was 6.82 ng/mL, significantly higher than that in the IGF group (3.44 ng/mL; P<0.001). Similar results were observed for donor terminal SCr levels (158.00 vs. 109.00; P<0.001). However, we failed to observe the variation in donor KDPI values amidst the DGF and IGF groups (59% vs. 46%; P=0.215), consistent with our prediction that KDPI does not reliably predict early post-transplant renal function. The mean ± SD recipient age was 42.56 ± 10.80 years, with a median [interquartile range] BMI of 21.26 [19.84–22.86] kg/m 2 . Comparing the DGF and IGF groups, we found that male recipients (88% vs. 66%; P=0.002) and those with a higher BMI (22.64 vs. 20.96; P<0.001) had a greater likelihood of developing DGF. Additionally, recipients with DGF had distinctly higher SCr levels at 6 months post-transplant compared to those in the IGF group (171.00 vs. 121.00; P<0.001). No differences were found between the groups in terms of dialysis type, dialysis duration, HLA mismatch level, induction therapy, or immunosuppressant dosage.
Donor plasma CIRBP as a risk factor for DGF after kidney transplantation DGF occurred in recipients paired with 51 (25%) donors following kidney transplantation. Testing of donor plasma samples revealed that the median CIRBP concentration in the DGF group was 6.82 ng/mL, which was 1.98 times higher than that in the IGF group (6.82 vs. 3.44; P<0.001). Recipients with CIRBP concentrations exceeding 5.484 ng/mL were four times more likely to develop DGF than those with concentrations below 5.484 ng/mL (53% vs. 12%; P<0.001). When CIRBP levels exceeded 7.92 ng/mL, the probability of developing DGF was 100%, whereas levels below 2 ng/mL corresponded to 0% probability of developing DGF . Although the median terminal SCr concentration was notably higher in the DGF group than that in the IGF group, 19% of recipients developed DGF postoperatively even when the SCr concentration was < 100 μmol/L . Additionally, the scatter distribution of CIRBP resembled that of SCr , and Spearman’s correlation demonstrated a significant relationship between the two factors (r=0.140, P=0.045). Furthermore, the proportion of female recipients was notably lower in the DGF group than that in the IGF group (12% vs. 34%; P=0.002) , while no significant variation in KDPI was found between the two groups . Graft loss occurred in 10 recipients in the DGF group, showing a higher rate, compared to the IGF group (19.6% vs. 5.8%; P=0.003). To further clarify the role of CIRBP in DGF, a logistic regression analysis was conducted. Univariate analysis revealed that for every 1 μmol/L increase in donor terminal SCr, the risk of developing DGF increased by 0.7% (95% confidence interval [CI]: 1.004–1.010; P<0.001), for every 1 ng/mL rise in plasma CIRBP concentration, the risk increased by 73.3% (95% CI: 1.446–2.076; P<0.001), and female recipients had a 0.259-fold lower probability of developing DGF compared to males (95% CI: 0.104–0.646; P=0.004). Donor plasma CIRBP (OR=1.660; 95% CI: 1.376–2.004; P<0.001) and donor terminal SCr (OR=1.005; 95% CI: 1.001–1.009; P=0.025) were identified by multivariate analysis as independent risk factors for DGF development, while a protective factor was female sex of the recipient (OR=0.302; 95% CI: 0.103–0.885; P=0.029) .
Donor plasma CIRBP effectively predicts DGF The area under the ROC curve (ROC-AUC) of CIRBP was 0.801 (95% CI: 0.728–0.874; P<0.001), and the optimal cut-off value was 5.548 ng/mL, calculated using the Youden index, with a sensitivity of 65% and 83% specificity . This performance was markedly superior to that of donor-terminal creatinine (ROC-AUC=0.654; 95% CI: 0.560–0.749; P=0.001) and KDPI (ROC-AUC=0.558; 95% CI: 0.471–0.645; P=0.215) . Enhancing the predictive ability of CIRBP, ROC curve analysis was performed via logistic regression, separately fitting donor plasma CIRBP to donor terminal SCr levels and recipient sex. The best predictive model was represented by the formula: y=1.660×donor plasma CIRBP+3.312×male+1.005×donor terminal SCr-5.365 (ROC-AUC=0.835; 95% CI: 0.771–0.898; P<0.001). The optimal cutoff value was 0.308, having a sensitivity of 63% and 88% specificity and was superior to both the fitting model of plasma CIRBP and donor terminal SCr (ROC-AUC=0.808; 95% CI: 0.733–0.884; P<0.001) and the fitting model of plasma CIRBP and recipient sex (ROC-AUC 0.821; 95% CI: 0.753–0.888; P<0.001) .
Correlation between donor plasma CIRBP and recipient creatinine at 6 months post-transplantation At 6 months post-transplantation, recipient SCr concentration increased with increasing plasma CIRBP levels in donors . Meanwhile, the SCr concentration at 6 months post-transplantation increased with the KDPI value , consistent with previous findings, suggesting that the KDPI may help predict long-term donor kidney survival. However, no significant trend was noted for donor terminal SCr levels . Univariate linear regression analysis revealed significant linear correlations between SCr concentration at 6 months and donor plasma CIRBP (R²=0.08, P<0.001), KDPI (R²=0.07, P<0.001), female recipients (R²=0.06, P=0.002), donor age (R²=0.054, P=0.003), as well as donor cause of death being cerebral hemorrhage (R²=0.035, P=0.016) . Ultimately, only donor plasma CIRBP (R²=0.08, P=0.003), KDPI (R²=0.137, P=0.002), and female recipient status (R²=0.174, P=0.008) were retained in the multiple linear regression analyses . Conversely, donor terminal SCr levels were not found to be associated with recipient creatinine at 6 months post-transplantation.
Donor plasma CIRBP and its association with transplanted kidney survival time Recipients were categorized into high plasma CIRBP (≥5.484 ng/mL, n=60) and low plasma CIRBP (<5.484 ng/mL, n=147) groups based on the optimal cutoff value . We hypothesized that donor kidneys with higher plasma CIRBP concentrations would exhibit significantly shorter survival times compared to those with lower concentrations. Although our survival analysis showed a trend of reduced survival time for donor kidneys with plasma CIRBP concentrations >5.484 ng/mL, further statistical analysis did not support this hypothesis (P=0.167; ). However, when comparing graft survival times between the DGF and IGF groups, the Kaplan-Meier curves revealed that recipients who developed DGF had shorter long-term graft survival (P=0.002; ), consistent with findings from related studies. Univariate and multivariate analyses were conducted to identify the risk factors for kidney transplant loss. The results indicated that donor plasma CIRBP and donor terminal SCr were not significantly correlated with transplant loss (P=0.057 and P=0.231, respectively). Recipient female sex was the only significant factor correlated with reduced transplant loss risk (OR=0.125; 95% CI: 0.016–0.955; P=0.045) . Subsequently, ROC curve analyses were conducted to evaluate the association between transplanted kidney survival and donor-terminal SCr (ROC-AUC=0.420; 95% CI: 0.283–0.557; P=0.250), plasma CIRBP (ROC-AUC=0.601; 95% CI: 0.469–0.732; P=0.149), and KDPI (ROC-AUC=0.593; 95% CI: 0.457–0.728; P=0.183) . However, these analyses did not reveal any statistically significant differences.
Discussion To date, no study has established a correlation between CIRBP levels and kidney function after transplantation. In this study, CIRBP was identified for the first time as a risk factor for DGF development following kidney transplantation, demonstrating its potential to accurately predict DGF occurrence. Additionally, elevated donor plasma CIRBP concentrations were significantly associated with higher recipient SCr levels at 6 months post-transplant. IRI is a major cause of AKI . During IRI, CIRBP is expressed intracellularly and released into the bloodstream via lysosomal and exosomal pathways, subsequently binding to TREM-1 receptors and exacerbating inflammation and kidney injury. Relative clinical studies demonstrated that elevated CIRBP concentrations correlated with an increased risk of AKI following cardiac macrovascular surgery . Additionally, animal experiments by Cen et al. showed that IRI-induced kidney injury was significantly attenuated following CIRBP knockdown in mice . Therefore, we can hypothesize that elevated CIRBP concentrations in donor blood are associated with increased graft kidney injury severity and a higher DGF probability following kidney transplantation. In this study, ELISA was used to analyze donor plasma specimens. demonstrates a significant correlation between plasma CIRBP and SCr concentrations. Higher plasma CIRBP concentrations were associated with elevated SCr levels, and the CIRBP concentration in donors from the DGF group was twice that of donors from the IGF group. Following further statistical analysis, we confirmed that CIRBP is an independent risk factor and an effective predictor of DGF after renal transplantation. Related animal experiments have shown that intravenous CIRBP injection induces AKI in mice, whereas the administration of a TREM-1 blocking agent significantly reduces IRI . In the future, blocking agents targeting CIRBP can be used in donors with elevated plasma CIRBP concentrations prior to renal transplantation, potentially reducing graft damage and the incidence of postoperative DGF. In recent years, the exploration of predictors for DGF has continued; however, novel biomarkers generally face challenges, including complex detection methods, lack of uniform measurement standards, and the need for further cohort studies for validation , limiting their clinical application. Notably, researchers have also sought to combine and model DGF prediction using a variety of biomarkers that are easily acquired and detected but have limited predictive ability when used individually, achieving improved results. For instance, Rima et al. (2024) combined kidney injury molecule-1, interleukin-18, and clinical factors, reaching an ROC-AUC of 0.863 for DGF prediction . In our study, the ROC-AUC for DGF prediction using CIRBP alone was 0.801, and it shares the advantages of easy access to clinical specimens and convenient testing. Combining CIRBP with existing clinical models may enhance both the accuracy and clinical utility of DGF diagnosis. Additionally, we observed that female recipients were significantly less likely to develop DGF than male recipients, suggesting a protective effect against DGF. Compared with men, women have higher levels of estrogen, which can increase autoantibody expression, inhibit Th1 cell-mediated responses, decrease the production of adhesion molecules as well as ROS in the vasculature, and elevate nitric oxide synthase expression, which are factors that play various roles in different diseases . Hence, we hypothesize that the inflammatory response in the vascular endothelium is attenuated by estrogen in women compared with men, leading to slower progression of chronic renal fibrosis in women, resulting in a reduced likelihood of DGF and extended survival of transplanted kidneys in female recipients. However, specific mechanisms underlying these effects in renal transplantation require further investigation. Our findings revealed that elevated donor plasma CIRBP concentrations were associated with higher recipient SCr levels at 6 months post-kidney transplantation. Chronic graft dysfunction is a pressing issue in renal transplantation, primarily caused by chronic fibrosis and rejection following kidney injury . Bolourani et al. demonstrated that extracellular CIRBP triggers an inflammatory phenotype in pulmonary fibroblasts via a mechanism dependent on TLR4 . Therefore, it is reasonable to hypothesize that elevated plasma CIRBP concentrations are associated with more severe graft injury, leading to increased chronic renal fibrosis post-transplantation and resulting in higher recipient SCr levels at 6 months. However, this hypothesis requires further validation and targeting CIRBP may be a potential therapeutic strategy for chronic graft renal failure in future research. The survival time in the donor kidneys with elevated plasma CIRBP concentrations was not significantly different from that in the donor kidneys with low plasma CIRBP concentrations, although a decreasing trend was observed. During IRI, damaged cells release DAMPs that promote inflammation and activate and recruit T cells to the transplanted kidney via dendritic cells, exacerbating the injury and inducing rejection . CIRBP, a newly recognized DAMP molecule, shares similar properties, and its increased concentration should correlate with more severe kidney injury and a higher likelihood of transplant failure in the postoperative period. Possibly owing to the limited number of studies on recipients and the short follow-up period, female sex of recipients was the only observed protective factor that correlated with kidney graft loss in this study. However, in our study, recipients who developed DGF after renal transplantation had longer graft survival compared to those without DGF, consistent with findings from related studies . DGF development significantly increases the incidence of posttransplant adverse events. In a 2021 study by Phillips et al. , of 4,714 kidney transplant recipients, 1,847 developed DGF, which gave rise to a 1.7-fold enhance in the risk of graft loss and a 1.8-fold increase in recipient death for those with DGF lasting more than 14 d. In a 2019 study, Kim et al. reported a 2.4-fold increase in the 30-d readmission rate among 269 recipients who developed DGF. Similarly, Yousif et al. , in a 2022 study, reported a 1.6-fold increase in acute rejection among 578 renal transplant recipients who developed DGF. Therefore, prediction and prevention of DGF are crucial. Unlike the extracellular CIRBP discussed herein, intracellular CIRBP plays a critical role in maintaining cellular stability by stabilizing RNA, preventing RNA degradation, and regulating RNA transcription . Related studies have shown that intracellular CIRBP in kidney cells can regulate the expression of hypoxia-inducible factor-1α, thereby reducing oxidative stress damage caused by IRI . Furthermore, a recent study demonstrated that CIRBP effectively attenuates hypothermic damage and inhibits ferroptosis in cardiac myocytes during heart transplantation . These findings suggest that, in the future, upregulating CIRBP expression in ECD donor kidneys while blocking the pathway of extracellular free CIRBP may reduce the risk of transplantation failure. This approach could potentially facilitate the re-expansion of the criteria for ECD donor kidneys and help address the critical shortage of donor kidneys. Our study had certain limitations. It was a relatively small, retrospective, single-center study involving 207 cases, which restricted the range of factors available for analysis. Additionally, we were unable to obtain and analyze tissue specimens from zero-time biopsies of the transplanted kidneys. Plasma CIRBP levels in donors can be influenced by inflammation or trauma to the central nervous system , and as a result, are expected to vary with the duration of brain death in brain-dead donors. However, our study did not account for the important factor of donor brain death duration. In the future, it will be essential to collect additional zero puncture specimens from donor kidneys for pathological testing, record the duration of brain death and related confounders for analysis and conduct a multicenter, large-sample study to further validate our findings. In conclusion, our preliminary study identified a correlation between CIRBP and transplant renal function, establishing donor plasma CIRBP as an independent risk factor for DGF after renal transplantation. Therefore, donor plasma CIRBP levels are expected to become a new index for evaluating transplanted kidney function and antagonizing CIRBP may represent a novel therapeutic approach to reducing the risk of kidney transplant failure, particularly in ECD donor kidney transplantation.
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Triolein emulsion enhances temozolomide brain delivery: an experimental study in rats | 07b36fa6-fae1-43bf-a658-7aba7b77b38a | 8583762 | Pharmacology[mh] | The blood–brain barrier (BBB) prevents noxious substances from penetrating the brain parenchyma covering the vessel and is a major obstacle for drug delivery to the central nervous system. The prognosis of patients with primary brain tumors remains poor because the BBB acts as a physiological barrier that limits approximately 98% and 100% of small- and large-molecule drugs, respectively, from reaching the parenchymal tissue (Pardridge, ). Although considerable progress has been made in the development of therapeutic techniques, such as surgery, radiotherapy, photodynamic therapy, and chemotherapy, the clinical outcome of patients with gliomas remains poor with a 5-year survival rate of <3% for those with glioblastoma (Catuogno et al., ). The heterogeneous integrity of the BBB poses a challenge for drug delivery across the BBB (Abbott, ). Generally, glioma progresses when the permeability of the BBB is enhanced compared to that of normal brain tissue (Cruceru et al., ). However, the BBB in peripheral glioma remains intact because tumor cells that have escaped migrate to the surrounding brain parenchyma, resulting in a highly refractory nature of the malignant glioma within a 2–3 cm margin of the surgical resection cavity (Veringa et al., ; Zhan & Lu, ). Injecting a triolein emulsion into the carotid artery immediately opens the BBB temporarily and reversibly via tight junctions, resulting in increased vascular permeability (Kim et al., ; Ryu et al., ; Sol et al., ). 8-Carbamoyl-3-methyl-imidazo-[5,1-d]-1,2,3,5-tetrazin-4-(3H) (temozolomide, TMZ; SCH 52365), an alkylating agent and imidazotetrazine derivative that exhibits broad-spectrum antitumor activity against murine tumors, is used in the treatment of malignant gliomas (Ruggiero et al., ; Stevens et al., ). TMZ is rapidly degraded to its active metabolite, 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), which then rapidly destroys the inactive derivatives, n-5-aminoimidazole-4-carboxamide and methyldiazonium cation (Denny et al., ). For the estimation of TMZ, chromatographic and spectrophotometric methods, such as high-performance liquid chromatography (HPLC) with ultraviolet detection and LC coupled with tandem mass spectrometry (MS/MS) are the most frequently used techniques. TMZ has been extracted from biological samples using liquid–liquid extraction or solid-phase extraction (Estlin et al., ; Meany et al., ). Desorption electrospray ionization (DESI)-MS imaging of biological tissues is an efficient and highly sensitive MS ionization technique for imaging lipids and metabolites from biological tissues (Eberlin et al., ). DESI is clinically applicable because it enables the analysis of biomolecules in the x and y directions via spraying of charged droplets and provides chemical information that can be displayed as two-dimensional (2 D) images (Agar et al., ). Increasing the vascular permeability of TMZ using triolein emulsion injection would make it the most effective chemotherapeutic agent currently available for the treatment of malignant glioma. To the best of our knowledge, no study of the enhancement of the permeability of TMZ in the brain using a triolein emulsion has been reported using HPLC analysis or DESI-MS imaging. Thus, the aim of the present study was to evaluate the difference in the concentration of TMZ in the rat brain with and without the administration of triolein emulsion into the carotid artery using MS techniques.
Drugs, reagents, and surgical material TMZ, triolein, and trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). Silk and ketamine hydrochloride were purchased from Ailee (Busan, Korea) and Huons (Gyeonggi-do, Korea), respectively, whereas Rompun (xylazine) was purchased from Bayer Korea (Seoul, Korea). Dimethyl sulfoxide (DMSO) was purchased from Daejung (Siheung-si, Korea). Intravenous (IV) catheters and 2-mL syringes were purchased from Dukwoo Medical Co., Ltd. (Gyeonggi-do, Korea) and Korea Vaccine (Seoul, Korea), respectively. The three-way stopcock was purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Animal model establishment The Institutional Animal Review Board of the Biomedical Research Institute approved all experimental protocols (approval no: 2020-009-A1C0). The experiments were performed using 10-week-old male Sprague-Dawley rats (SAMTACO, Osan, Korea) weighing approximately 300 g after 2–3 days of acclimation. All animals were kept in a semi-specific pathogen-free environment maintained at 18–22 °C under a 12-h light/dark cycle and were allowed access to water and food ad libitum. Animals were anesthetized using an intramuscular injection of ketamine hydrochloride (2.5 mg/kg) and xylazine (0.125 mg/kg) and were allowed to breathe ambient air spontaneously during the procedure. To determine whether triolein enhanced the permeability of TMZ, three groups of rats were used. Group 1 (negative control) was untreated, group 2 and 4 (experimental groups) were treated with triolein and TMZ, and group 3 and 5 (positive control groups) were treated with TMZ alone. The TMZ injection was administered at 20 mg/kg to groups 2 ( n = 8) and 3 ( n = 8), and a parallel set of groups 4 ( n = 8) and 5 ( n = 8) were administered 10 mg/kg. Experimental protocol Experimental protocol used in the present study was described as a schema in the . First of all, TE was infused into the right common carotid artery. Just after that, TMZ injection was followed. Before euthanization of the rat, trypan blue was injected intravenously at the tail vein. Euthanization of the rats was performed 1 hour after TE infusion using carbon dioxide gas. Brain tissue was obtained and two different studies were performed. One was the experiment for quantitative analysis of TMZ concentration using the HPLC method, and another for qualitative analysis of TMZ concentration using the DESI-MS imaging method. Triolein emulsion infusion The hair on the right cervical area of each rat was removed using an electric razor and a longitudinal incision was made. The right common carotid artery was isolated, and the right external carotid artery was ligated with a silk thread under an operating microscope (Olympus SZX7-STU2, Tokyo, Japan) to ensure that the target drug only passed through the right internal carotid artery. A 24-gauge IV catheter (Angiocath; Becton Dickinson) was inserted into the right common carotid artery. A 1-mL syringe containing 50 μL neutral triglyceride triolein (molecular weight [MW] = 885.5 g/mol) and a 10-mL syringe containing 10 mL normal saline were connected to the three-way stopcock. Triolein emulsion (50 μL/10 mL, 0.05%) was prepared by mixing triolein and normal saline using a vigorous to-and-fro movement of the syringes for 3 min. Triolein emulsion (2 mL) was injected into the right common carotid artery of each rat over a period of 30 s. TMZ preparation TMZ was administered at doses of 20 mg/kg and 10 mg/kg to the respective groups. The TMZ formula was administered intravenously to patients at doses of 50–200 mg/m 2 as a solution in 3% DMSO in the initial phase I trial. The animals in the present study received 20 and 10 mg/kg TMZ, and a 10 mg/kg rat dose was equivalent to a 150 mg/m 2 adult human dose (Patel et al., ). TMZ was dissolved in DMSO at concentrations of 36 and 18 mg/mL and then further diluted with normal saline. Specifically, 0.167 mL TMZ-DMSO stock was mixed with 1.833 mL normal saline, and a 2-mL solution was prepared immediately before the injection to avoid any possible degradation of TMZ. TMZ injection and brain tissue harvesting The rats were administered 2 mL TMZ via an intracarotid arterial injection (36 and 18 mg/kg) immediately after triolein emulsion infusion. For the gross evaluation of the opening of the BBB, trypan blue (2 mL) was intravenously injected before euthanization. All the rats were euthanized 1 hour after TE infusion using carbon dioxide gas. Trypan blue-stained brain tissue samples were harvested and placed in 10 mL 0.5 M HCl buffer solution for HPLC analysis. Quantitative HPLC and statistical analyses The brain tissue samples were chopped, sonicated for 10 min at 20–25 °C, centrifuged at 3000 × g at 20–25 °C, and then, the supernatants were collected, followed by subsequent centrifugation at 17,000 × g for 30 min and the supernatant was collected for HPLC analysis (Shimadzu, Kyoto, Japan) to determine the concentration of TMZ. The HPLC system was fitted out with an SPD-20A Prominence UV/Vis detector, an LC-20AT liquid chromatograph, a CT-20A Prominence column oven, a DGU-20ASR degassing unit and an SIL-20 Prominence autosampler. A VDSpher ® PUR 100 C18-E column (5 µm, 150 × 4.6 mm, VDS Optilab, Berlin, Germany) was used. The internal standard equation was predetermined and the TMZ concentrations were analyzed in a mobile phase consisting of 8:2 volumes of 0.5% (v/v) acetic acid:methanol at a flow rate of 1.1 mL/min, oven temperature of 40 °C, and wavelength of 330 nm. The internal standard equation for TMZ based on the TMZ concentration and area under the curve (AUC) was calculated using a linear regression method. TMZ quantification was validated using an internal standard equation. The detection range was 0.064–40 µg/mL TMZ and an AUC of 2764.9–1514221.3. The equation of TMZ concentration and AUC was “y = 37815x + 2554.7” and R 2 = 1. The method was fully validated for precision, accuracy, selectivity, and linearity (Gilant et al., ). The AUC values of the TMZ peak at a retention time of 2.480 min were calculated as the concentration of TMZ in the brain. TMZ concentrations were compared between groups, and the TMZ concentration ratios of the right to left hemisphere of each group were calculated. All data analyses were conducted using a two-tailed unpaired t-test using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA), and the statistical significance was set at p < .05. Qualitative analysis using DESI-MS imaging DESI-MS imaging experiments were performed using a Waters XEVO G2-XS quadrupole time-of-flight (Q-ToF) mass spectrometer (Waters, Milford, MA, USA). The DESI ion source (Waters) was mounted on a mass spectrometer and controlled using Omni Spray software (Prosolia, Indianapolis, IN, USA). The DESI source was initially set up using a rock spray solvent of 0.1% formic acid in acetonitrile:water (95:5, v/v) and 0.2 ng/μL leucine enkephalin (m/z 556.2771 in the ESI + mode) was added as the internal standard and lock mass compound. The flow rate was 2 μL/min in the positive ion mode. All MS parameters were recorded under the following condition: capillary voltage, 5 keV; sample cone voltage, 40 V; source and desolvation temperatures, 150 °C and 250 °C, respectively; desolvation and cone gas flow rates, 600 L/h and 50 L/h, respectively; gas pressure, 4.5 bar; spray voltage, 4.5 kV; spray, sprayer incidence, and collection angles, 60°, 75°, and 10°, respectively; sprayer-to-inlet and sprayer-to-sample distances, 3 mm and 1 mm, respectively; source temperature, 150 °C; and source offset, 80 V. Prior to image acquisition, the detected ion intensity of the red Sharpie marker pen (rhodamine) [M + H] + at m/z 443.23 for positive ion mode was verified to conduct the experiment. The emitter capillary protrusion was initially optimized by observing the removal of material from an ink patch on a glass slide. The mass spectra were acquired in the range of m/z 50–700 for all MS analyses. Furthermore, 1000 of the most intense peaks were observed. All m/z values were extracted at a mass window of 0.02 Da. The total scan time was determined based on the pixel size and scan speed. The square pixel size was 100 μm for the imaging MS, whereas the scan speeds were 100–110 μm/s. The time required for imaging the brain samples was 230–240 min. All imaging data were acquired and analyzed directly using high-definition imaging (HDI) version 1.4 in combination with MassLynx version 4.1 (Waters, Milford, MA, USA). The HDI imaging software enabled the acquisition and processing of data from the DESI-MS imaging experiments.
TMZ, triolein, and trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). Silk and ketamine hydrochloride were purchased from Ailee (Busan, Korea) and Huons (Gyeonggi-do, Korea), respectively, whereas Rompun (xylazine) was purchased from Bayer Korea (Seoul, Korea). Dimethyl sulfoxide (DMSO) was purchased from Daejung (Siheung-si, Korea). Intravenous (IV) catheters and 2-mL syringes were purchased from Dukwoo Medical Co., Ltd. (Gyeonggi-do, Korea) and Korea Vaccine (Seoul, Korea), respectively. The three-way stopcock was purchased from Becton Dickinson (Franklin Lakes, NJ, USA).
The Institutional Animal Review Board of the Biomedical Research Institute approved all experimental protocols (approval no: 2020-009-A1C0). The experiments were performed using 10-week-old male Sprague-Dawley rats (SAMTACO, Osan, Korea) weighing approximately 300 g after 2–3 days of acclimation. All animals were kept in a semi-specific pathogen-free environment maintained at 18–22 °C under a 12-h light/dark cycle and were allowed access to water and food ad libitum. Animals were anesthetized using an intramuscular injection of ketamine hydrochloride (2.5 mg/kg) and xylazine (0.125 mg/kg) and were allowed to breathe ambient air spontaneously during the procedure. To determine whether triolein enhanced the permeability of TMZ, three groups of rats were used. Group 1 (negative control) was untreated, group 2 and 4 (experimental groups) were treated with triolein and TMZ, and group 3 and 5 (positive control groups) were treated with TMZ alone. The TMZ injection was administered at 20 mg/kg to groups 2 ( n = 8) and 3 ( n = 8), and a parallel set of groups 4 ( n = 8) and 5 ( n = 8) were administered 10 mg/kg.
Experimental protocol used in the present study was described as a schema in the . First of all, TE was infused into the right common carotid artery. Just after that, TMZ injection was followed. Before euthanization of the rat, trypan blue was injected intravenously at the tail vein. Euthanization of the rats was performed 1 hour after TE infusion using carbon dioxide gas. Brain tissue was obtained and two different studies were performed. One was the experiment for quantitative analysis of TMZ concentration using the HPLC method, and another for qualitative analysis of TMZ concentration using the DESI-MS imaging method.
The hair on the right cervical area of each rat was removed using an electric razor and a longitudinal incision was made. The right common carotid artery was isolated, and the right external carotid artery was ligated with a silk thread under an operating microscope (Olympus SZX7-STU2, Tokyo, Japan) to ensure that the target drug only passed through the right internal carotid artery. A 24-gauge IV catheter (Angiocath; Becton Dickinson) was inserted into the right common carotid artery. A 1-mL syringe containing 50 μL neutral triglyceride triolein (molecular weight [MW] = 885.5 g/mol) and a 10-mL syringe containing 10 mL normal saline were connected to the three-way stopcock. Triolein emulsion (50 μL/10 mL, 0.05%) was prepared by mixing triolein and normal saline using a vigorous to-and-fro movement of the syringes for 3 min. Triolein emulsion (2 mL) was injected into the right common carotid artery of each rat over a period of 30 s.
TMZ was administered at doses of 20 mg/kg and 10 mg/kg to the respective groups. The TMZ formula was administered intravenously to patients at doses of 50–200 mg/m 2 as a solution in 3% DMSO in the initial phase I trial. The animals in the present study received 20 and 10 mg/kg TMZ, and a 10 mg/kg rat dose was equivalent to a 150 mg/m 2 adult human dose (Patel et al., ). TMZ was dissolved in DMSO at concentrations of 36 and 18 mg/mL and then further diluted with normal saline. Specifically, 0.167 mL TMZ-DMSO stock was mixed with 1.833 mL normal saline, and a 2-mL solution was prepared immediately before the injection to avoid any possible degradation of TMZ.
The rats were administered 2 mL TMZ via an intracarotid arterial injection (36 and 18 mg/kg) immediately after triolein emulsion infusion. For the gross evaluation of the opening of the BBB, trypan blue (2 mL) was intravenously injected before euthanization. All the rats were euthanized 1 hour after TE infusion using carbon dioxide gas. Trypan blue-stained brain tissue samples were harvested and placed in 10 mL 0.5 M HCl buffer solution for HPLC analysis.
The brain tissue samples were chopped, sonicated for 10 min at 20–25 °C, centrifuged at 3000 × g at 20–25 °C, and then, the supernatants were collected, followed by subsequent centrifugation at 17,000 × g for 30 min and the supernatant was collected for HPLC analysis (Shimadzu, Kyoto, Japan) to determine the concentration of TMZ. The HPLC system was fitted out with an SPD-20A Prominence UV/Vis detector, an LC-20AT liquid chromatograph, a CT-20A Prominence column oven, a DGU-20ASR degassing unit and an SIL-20 Prominence autosampler. A VDSpher ® PUR 100 C18-E column (5 µm, 150 × 4.6 mm, VDS Optilab, Berlin, Germany) was used. The internal standard equation was predetermined and the TMZ concentrations were analyzed in a mobile phase consisting of 8:2 volumes of 0.5% (v/v) acetic acid:methanol at a flow rate of 1.1 mL/min, oven temperature of 40 °C, and wavelength of 330 nm. The internal standard equation for TMZ based on the TMZ concentration and area under the curve (AUC) was calculated using a linear regression method. TMZ quantification was validated using an internal standard equation. The detection range was 0.064–40 µg/mL TMZ and an AUC of 2764.9–1514221.3. The equation of TMZ concentration and AUC was “y = 37815x + 2554.7” and R 2 = 1. The method was fully validated for precision, accuracy, selectivity, and linearity (Gilant et al., ). The AUC values of the TMZ peak at a retention time of 2.480 min were calculated as the concentration of TMZ in the brain. TMZ concentrations were compared between groups, and the TMZ concentration ratios of the right to left hemisphere of each group were calculated. All data analyses were conducted using a two-tailed unpaired t-test using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA), and the statistical significance was set at p < .05.
DESI-MS imaging experiments were performed using a Waters XEVO G2-XS quadrupole time-of-flight (Q-ToF) mass spectrometer (Waters, Milford, MA, USA). The DESI ion source (Waters) was mounted on a mass spectrometer and controlled using Omni Spray software (Prosolia, Indianapolis, IN, USA). The DESI source was initially set up using a rock spray solvent of 0.1% formic acid in acetonitrile:water (95:5, v/v) and 0.2 ng/μL leucine enkephalin (m/z 556.2771 in the ESI + mode) was added as the internal standard and lock mass compound. The flow rate was 2 μL/min in the positive ion mode. All MS parameters were recorded under the following condition: capillary voltage, 5 keV; sample cone voltage, 40 V; source and desolvation temperatures, 150 °C and 250 °C, respectively; desolvation and cone gas flow rates, 600 L/h and 50 L/h, respectively; gas pressure, 4.5 bar; spray voltage, 4.5 kV; spray, sprayer incidence, and collection angles, 60°, 75°, and 10°, respectively; sprayer-to-inlet and sprayer-to-sample distances, 3 mm and 1 mm, respectively; source temperature, 150 °C; and source offset, 80 V. Prior to image acquisition, the detected ion intensity of the red Sharpie marker pen (rhodamine) [M + H] + at m/z 443.23 for positive ion mode was verified to conduct the experiment. The emitter capillary protrusion was initially optimized by observing the removal of material from an ink patch on a glass slide. The mass spectra were acquired in the range of m/z 50–700 for all MS analyses. Furthermore, 1000 of the most intense peaks were observed. All m/z values were extracted at a mass window of 0.02 Da. The total scan time was determined based on the pixel size and scan speed. The square pixel size was 100 μm for the imaging MS, whereas the scan speeds were 100–110 μm/s. The time required for imaging the brain samples was 230–240 min. All imaging data were acquired and analyzed directly using high-definition imaging (HDI) version 1.4 in combination with MassLynx version 4.1 (Waters, Milford, MA, USA). The HDI imaging software enabled the acquisition and processing of data from the DESI-MS imaging experiments.
The whole brain and tissue sections of the treated hemispheres were stained blue with trypan blue in the experimental groups (group 2 and 4, ). Some rats showed blue staining of the contralateral hemisphere because of the bilateral supply of the cerebral artery. The rats of the group 1 (negative control group, n = 2) or the positive control groups (group 3 and 5) did not reveal staining with trypan blue. Quantitative HPLC and statistical analyses TMZ 20 mg/kg treatment TMZ was not detected in the bilateral hemispheres of group 1 rats (negative control group). The mean ± standard deviation (SD) of TMZ concentration was 12.85 ± 5.02 and 5.48 ± 2.56 µg/g in the ipsilateral and contralateral hemispheres of the group 2 rats (experimental group), respectively. The mean ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere in group 2 was 2.40 ± 0.36 . In group 3 (positive control group), the mean ± SD concentrations of TMZ were 3.72 ± 0.44 and 3.32 ± 0.40 µg/g in the ipsilateral and contralateral hemispheres, respectively. The mean ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere of group 3 was 1.13 ± 0.14 . Thus, triolein emulsion significantly (two-tailed unpaired t -test, p < .05) enhanced the delivery of TMZ, which was 2.12 (2.40/1.13) times higher than that of TMZ administered alone . TMZ 10 mg/kg treatment The mean ± SD of TMZ concentration was 3.77 ± 1.32 µg/g and 1.57 ± 0.76 µg/g in the treated and contralateral hemispheres of the group 4 rats (experimental group), respectively. Thus, the mean ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere in group 4 was 2.54 ± 0.47 . In group 5 (positive control group), the mean ± SD of TMZ concentration was 1.26 ± 0.26 µg/g and 1.14 ± 0.21 µg/g in the ipsilateral and contralateral hemispheres, respectively. The concentration ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere in group 5 was 1.11 ± 0.09 . Thus, in the presence of triolein emulsion, the delivery of TMZ was significantly (two-tailed unpaired t -test, p < .05) higher (2.28, 2.54/1.11 times) than that observed without the emulsion . Qualitative analysis (DESI-MS imaging for TMZ concentration) TMZ was identified at m/z 217 because of the sodium adduct ions [M + Na] + 194 → 217. The signal intensity of TMZ was not detected using DESI-MS imaging in group 1 (negative control group). In group 2 and 4 (experimental groups), the signal intensity of TMZ was remarkably high in the ipsilateral hemisphere . In group 3 and 5 (positive control groups), the ipsilateral hemisphere showed minimal or no TMZ signal . The signal intensities of the rats treated with TMZ 20 mg/kg (group 2) tended to be higher than those of the TMZ 10 mg/kg treated rats (group 4).
TMZ 20 mg/kg treatment TMZ was not detected in the bilateral hemispheres of group 1 rats (negative control group). The mean ± standard deviation (SD) of TMZ concentration was 12.85 ± 5.02 and 5.48 ± 2.56 µg/g in the ipsilateral and contralateral hemispheres of the group 2 rats (experimental group), respectively. The mean ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere in group 2 was 2.40 ± 0.36 . In group 3 (positive control group), the mean ± SD concentrations of TMZ were 3.72 ± 0.44 and 3.32 ± 0.40 µg/g in the ipsilateral and contralateral hemispheres, respectively. The mean ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere of group 3 was 1.13 ± 0.14 . Thus, triolein emulsion significantly (two-tailed unpaired t -test, p < .05) enhanced the delivery of TMZ, which was 2.12 (2.40/1.13) times higher than that of TMZ administered alone . TMZ 10 mg/kg treatment The mean ± SD of TMZ concentration was 3.77 ± 1.32 µg/g and 1.57 ± 0.76 µg/g in the treated and contralateral hemispheres of the group 4 rats (experimental group), respectively. Thus, the mean ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere in group 4 was 2.54 ± 0.47 . In group 5 (positive control group), the mean ± SD of TMZ concentration was 1.26 ± 0.26 µg/g and 1.14 ± 0.21 µg/g in the ipsilateral and contralateral hemispheres, respectively. The concentration ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere in group 5 was 1.11 ± 0.09 . Thus, in the presence of triolein emulsion, the delivery of TMZ was significantly (two-tailed unpaired t -test, p < .05) higher (2.28, 2.54/1.11 times) than that observed without the emulsion .
TMZ was not detected in the bilateral hemispheres of group 1 rats (negative control group). The mean ± standard deviation (SD) of TMZ concentration was 12.85 ± 5.02 and 5.48 ± 2.56 µg/g in the ipsilateral and contralateral hemispheres of the group 2 rats (experimental group), respectively. The mean ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere in group 2 was 2.40 ± 0.36 . In group 3 (positive control group), the mean ± SD concentrations of TMZ were 3.72 ± 0.44 and 3.32 ± 0.40 µg/g in the ipsilateral and contralateral hemispheres, respectively. The mean ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere of group 3 was 1.13 ± 0.14 . Thus, triolein emulsion significantly (two-tailed unpaired t -test, p < .05) enhanced the delivery of TMZ, which was 2.12 (2.40/1.13) times higher than that of TMZ administered alone .
The mean ± SD of TMZ concentration was 3.77 ± 1.32 µg/g and 1.57 ± 0.76 µg/g in the treated and contralateral hemispheres of the group 4 rats (experimental group), respectively. Thus, the mean ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere in group 4 was 2.54 ± 0.47 . In group 5 (positive control group), the mean ± SD of TMZ concentration was 1.26 ± 0.26 µg/g and 1.14 ± 0.21 µg/g in the ipsilateral and contralateral hemispheres, respectively. The concentration ratio ± SD of the ipsilateral hemisphere to the contralateral hemisphere in group 5 was 1.11 ± 0.09 . Thus, in the presence of triolein emulsion, the delivery of TMZ was significantly (two-tailed unpaired t -test, p < .05) higher (2.28, 2.54/1.11 times) than that observed without the emulsion .
TMZ was identified at m/z 217 because of the sodium adduct ions [M + Na] + 194 → 217. The signal intensity of TMZ was not detected using DESI-MS imaging in group 1 (negative control group). In group 2 and 4 (experimental groups), the signal intensity of TMZ was remarkably high in the ipsilateral hemisphere . In group 3 and 5 (positive control groups), the ipsilateral hemisphere showed minimal or no TMZ signal . The signal intensities of the rats treated with TMZ 20 mg/kg (group 2) tended to be higher than those of the TMZ 10 mg/kg treated rats (group 4).
In the present study, we focused on evaluating the enhancement of TMZ delivery in the rat brain by co-administration of triolein emulsion infusion through the carotid artery. The concentration of TMZ was approximately two times higher in the rat brain in the presence of the triolein emulsion than it was without the emulsion. The ratios of the TMZ concentration in the ipsilateral hemisphere to that in the contralateral hemisphere were 2.13 and 2.18 times higher following the injections of TMZ 20 mg/kg and 10 mg/kg, respectively, than they were before treatment and were significantly different from those of the control group. These results indicate that the vascular permeability of the brain was significantly increased after triolein emulsion was injected into the cerebral artery. Triolein, a major constituent of the bone marrow and the main cause of clinical fat embolism, induces vasogenic edema immediately after it is injected into the carotid artery (Kim et al., ). Cerebral infarction may occur when triolein is administered as a bolus injection, but not when it is injected as an emulsion (Kim et al., ). Thus, the development of cerebral fat embolisms depends on the morphology of the embolized fat and the size of the fat particles. The increased vasogenic edema induced by triolein emulsion is reversible and does not cause any significant histological changes, hemodynamic occlusive appearance, or metabolic differences in the brain (Kim et al., , ; Baik et al., ). The reversible and temporal increase in vasogenic edema is because of the increased vascular permeability of the cerebral artery and can be useful in the adjuvant treatment of intractable brain diseases, such as malignant tumors, encephalopathy, epilepsy, and neurodegenerative disorders. The molecular mechanism mediating the increase in vascular permeability induced by triolein emulsion is unknown, and the morphological pathway mainly involves tight junctions and minimal transcytosis in the cerebral endothelium (Sol et al., ). Recently, a study using triolein emulsion infusion into the hepatic arteries was reported to increase the doxorubicin concentration approximately twofold and suggests that a triolein infusion might be a useful adjuvant treatment of liver cancer (Kim et al., ). Currently, despite aggressive combination treatments, the prognosis of malignant glioma remains poor because of the unique structure of the brain. In particularly, the BBB, which consists of endothelial cells with tight junctions lining the microvasculature of the brain, impedes the penetration of drugs. TMZ, which is the only chemotherapeutic agent that improves the survival rate of patients with malignant gliomas, is lipophilic and penetrates the BBB (Brada et al., ). The penetration of TMZ administered orally or intravenously into the brain has been shown to be variable (20%–39%) based on the brain/plasma AUC ratio (Reyderman et al., ; Ostermann et al., ). TMZ exerts chemotherapeutic activity following its conversion to linear triazine MTIC, which is known to have an important chemotherapeutic effect mediated by a potent alkylating activity. TMZ inserts a methyl group into the purines and pyrimidines of the DNA, resulting in cell death (Zhang et al., ). The 2-year survival rate of patients with malignant gliomas was slightly higher at 24% with combined radiation and TMZ therapy than it was at 10% with radiation treatment alone (Stupp et al., ). Several new techniques have been investigated for promoting the delivery of TMZ through the BBB using MS for quantification in animal models (Zhang et al., ; Stupp et al., ). Focused ultrasound (FUS) has recently been used to overcome the challenges of penetrating the BBB and enhances the delivery of anticancer agents (Zhang et al., ). After oral administration of TMZ (50 mg/kg), FUS enhanced the penetration in mouse brains by 2.7 times the levels observed in the group administered TMZ alone (Liu et al., ). Regadenoson, which is used as a cardiac stress agent in patients who are unable to walk on a treadmill test, has also been used to transiently disrupt the BBB, which subsequently increases TMZ levels in the rat brain (Stupp et al., ). The brain TMZ concentration after oral administration of 50 mg/kg was 1.59 times higher in the presence of regadenoson than it was with TMZ alone (Jackson et al., ). These results are quite similar to those of the present study, although the administration route and amount of TMZ were different. HPLC and LC-MS/MS are the most common and validated techniques for the analysis of TMZ concentrations in animal models and patients (Patel et al., ; Brada et al., ; Ostermann et al., ; Kim et al., ; Diez et al., ). Furthermore, studies using these techniques have shown delayed TMZ peak concentration at 2.5 h and a mean half-life of 1.5 h after infusion through the cerebrospinal fluid (CSF). The delivery of TMZ from the plasma to the CSF was substantial (CSF:plasma AUC ratio, 0.33) (Patel et al., ). MTIC, the active form of TMZ, is produced by chemical degradation at physiological pH. MTIC forms methyl adducts at the N 7 -position of guanine, N 3 -position of adenine, and O 6 -position of guanine. Furthermore, O 6 -methylguanine appears to be a critical cytotoxic molecule (Gibson et al., ). MTIC is then degraded to the inactive derivatives 5-aminoimidazole-4-carboxamide and methyldiazonium cation (Marchesi et al., ). However, prolonged treatment results in resistance and low efficacy of subsequent therapy because of DNA damage repair enzymes, which leads to the recurrence of malignancy in 60%–75% of patients (Chamberlain, ; Beier et al., ). For successful treatment, high doses of TMZ may be needed, which results in increased toxicity and several complications. To overcome the shortcomings of TMZ therapy, a delivery carrier is required to increase its effects at the target site. Several carriers of TMZ have been reported, but they lack tumor-specific delivery (Tentori & Graziani, ). An efficient drug delivery system for glioma therapy should target the tumor and be able to cross the BBB. The qualitative analysis of TMZ concentration in the present study showed a remarkably higher signal intensity in the treated hemispheres than in the contralateral hemispheres and control groups. In addition, the signal intensity tended to be stronger after higher amounts of TMZ were injected. The signal intensity observed with DESI-MS imaging correlated with the TMZ concentrations determined using HPLC. Thus, DESI-MS imaging appeared to be suitable for determining TMZ concentration levels in tissues and organs. In the control groups, the signal intensity was not changed or was slightly increased in the treated hemispheres, depending on the acquisition scale. The mild signal intensity detected using DESI-MS imaging in the control group in the present study indicated that TMZ was delivered at normal levels to the brain parenchyma through the BBB. The concentration in the brain parenchyma was nearly 30% of the plasma concentration (Agarwala & Kirkwood, ). Presumably, this is the first study to report the use of HPLC analysis and DESI-MS imaging of TMZ. DESI-MS imaging is an emerging and powerful technique that has been recently developed for the imaging of lipids or metabolites in tissues (Takats et al., ; Wiseman et al., ). Clinically, this technique has been used to identify malignant tissues in brain, breast, or prostate cancers (Eberlin et al., ; Santagata et al., ; Kerian et al., ; Calligaris et al., ). The outstanding advantage of the DESI-MS technique is the time efficiency gained because no complex sample preparation or separation techniques, such as extraction and chromatography, is required. In addition, this technique has high discriminating power, sensitivity, specificity, and the throughput and ability to analyze specimens in a wide mass spectrum from simple amino acids to drug molecules, alkaloids, terpenoids, and steroids to peptides and proteins (Cooks et al., ; Lostun et al., ). The technique is also extremely efficient for surface analysis, mapping, and forensic applications, especially because of it has minimal destructiveness and enables possible in-situ measurements (Takats et al., ; Cooks et al., ). The present technique could be applicable in many clinical fields, such as malignant brain tumors of primary or metastatic cancers, retinal or testicular malignancies under conditions of having blood-tissue barriers, refractory hepatic or pancreatic malignancies, of the hypovascular conditions showing difficulty in chemotherapy. Enhancement of drug delivery by triolein emulsion is independent of the organs that the technique was used (Kim et al., , , , ; Lee et al., ). In conclusion, the results of the present study demonstrate that triolein emulsion enhances the penetrability of the BBB, thereby providing a potential new strategy to enhance the therapeutic effect of TMZ. Furthermore, HPLC and DESI-MS imaging are suitable for the quantitative and qualitative analyses, respectively, of TMZ concentrations in brain tissue.
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Oral gel loaded with penciclovir–lavender oil nanoemulsion to enhance bioavailability and alleviate pain associated with herpes labialis | d12061bf-29cf-4448-9c81-8244a1ca1de8 | 8175053 | Pharmacology[mh] | Introduction Oral health is considered a major constituent of overall health and involves much more than having healthy teeth. While the mouth harbors numerous harmless bacteria under control, improper oral care can consequently lead to uncontrolled growth of bacteria and subsequent tissue damage, resulting in tooth and gum impairment. In addition, some diseases, such as diabetes, HIV/AIDS, and osteoporosis, can adversely affect oral health, which could further lead to other issues for instance endocarditis, cardiovascular diseases, pregnancy and birth complications, and pneumonia (Li et al., ). Infections in the oral cavity can be caused by bacteria, fungi, or viruses (Bandara & Samaranayake, ). Among these, viral infections are less common but are manifested as ulcers or blisters that can be detrimental and hence lead to poor quality of life. Herpes labialis, or cold sore, is a viral infection caused by herpes simplex virus type 1 (HSV-1) and is usually characterized by painful skin or mucosal lesions (Santosh & Muddana, ). Several therapeutic strategies have been proposed against herpes labialis along with both short-term and long-term preventive treatments. Amongst the commonly used, both acyclovir and penciclovir (PV) creams are found to be effective against herpes labialis (Opstelten et al., ). Topical applications of zinc oxide or zinc sulfate, anesthetic, and antiviral creams can achieve good results upon prompt use, among which antiviral creams are considered the most effective therapeutic strategy. PV has been proven to be effective against both HSV-1 and HSV-2 in recurrent herpes labialis (Spruance et al., ). Compared to acyclovir and valacyclovir creams, PV cream has been reported to significantly reduce the time of lesion healing and decrease lesion area and pain. One major advantage of PV is that its active form shows a very long half-life in HSV-infected cells (Schmid-Wendtner & Korting, ). Nevertheless, since it has a poor oral bioavailability of 5–10%, topical formulations with liposomes, microemulsion, and microemulsion gels have been proposed (Yang & Wang, ; Zhu et al., , ). Further, these advanced delivery systems can also reduce the adverse effects of PV (Zhu et al., ). The formulation components of micro- and nanoemulsions contribute to the enhancement of drug bioavailability from these advanced nanostructured delivery systems (Hosny et al., ). Mainly, the oil phase for these formulations is selected based on its power to solubilize the drug and, less frequently, on the therapeutic purpose of the system. In this context, essential oils are ideal for providing the oil phase and additional antiviral activity, specifically against HSV-1 (Hosny et al., ; Alghaith et al., ). It is pertinent to mention that lavender oil (LO) from Lavandula plants (Lavender) shows significant antiviral activity (Alghaith et al., ), and LO creams have demonstrated successful treatment against herpes labialis lesions (Altaei & Ahmed, ). Moreover, the anesthetic activity of LO can be of immense use in alleviating pain of lesions in herpes labialis (Ghelardini et al., ). Noteworthy, the chemical constituents of lavender vary depending on its variety (Białoń et al., ) and primarily include linalool, linalyl acetate, lavandulol acetate, and β-caryophyllene oxide (Dong et al., ). Compounds, such as α-terpineol, 4-terpinenol, and linalool, have shown specific antiviral activity (Król et al., ), while the local anesthetic activity of linalool and linalyl acetate from LO has been established (Koulivand et al., ). Additionally, 4-terpinenol, thymol, and carvacrol are known to exert anti-inflammatory actions, providing additional benefits against herpes labialis (Król et al., ). Self-nanoemulsifying drug delivery systems (SNEDDS) are widely used to enhance the solubility and bioavailability of drugs with poor aqueous solubility. On dilution with a physiological medium, SNEDDS produce nano-sized drug-loaded oil droplets that are primarily responsible for the improved absorption and bioavailability (Cherniakov et al., ), which is least affected by the presence or absence of food (Charman et al., ). Furthermore, SNEDDS offer high physical and chemical stabilities and can be converted into other dosage forms, such as tablets and capsules (Shahba et al., ). Therefore, the use of LO as an oil phase to formulate PV-loaded SNEDDS would be a promising strategy to explore the combined effects of these components. Considering that micro- and nanoemulsion-based systems for oral application suffer from poor residence in the oral cavity, adequate retention of the PV-loaded system is essential for effective therapy of herpes labialis. Fortunately, oral gels can satisfactorily solve this issue by providing sustained or controlled release of therapeutic agents for an enhanced effect (Miyazaki et al., ; Aslani et al., ). Since prolonged exposure of herpes labialis lesions to PV can improve therapeutic efficacy, incorporating SNEDDS in a gel base would greatly offer an additional advantage. The formulation of SNEDDS and oral gels is multifactorial process, involving the drug, excipients, and methods. However, the process of selecting the optimal formula using conventional experiments can be highly complex and time-consuming. Comparatively, the design of experiments approach which involves simultaneous and systematic evaluation of the formulation or process variables with the minimum number of experiment trials, offers distinct advantages and reaching the optimum formula rapidly (N Politis et al., ). Therefore, this study aimed to formulate PV-loaded LO (PV-LO) in the form of SNEDDS (PV-LO-SNEDDS) by employing a distance quadratic mixture design and its subsequent incorporation in an oral gel. It was anticipated that the optimized PV-LO-SNEDDS-loaded oral gel (O3) would enhance the bioavailability of PV, whereas the use of LO as an oil phase would contribute to the anesthetic activity and may also alleviate pain associated with herpes labialis. Most importantly, the incorporation of the optimized PV-LO-SNEDDS within the oral gel base would ensure intimate contact with oral mucosa, and support the prolonged release of PV to relieve herpes labials.
Materials and methods 2.1. Materials PV was obtained as a gift sample from Qingdao Sigma Chemical Co., Ltd. Shandong, China. Basil oil, thyme oil, peppermint oil, rosemary oil, LO, citronella oil, verbena oil, and camphor oil were procured from Jiangxi Origin Aromatics Co., Ltd. Jiangxi, China. Tween80, Span80, Steareth-2, Steareth-21, Labrasol, Labrafil 1944, Brij 30, Lauroglycol-FCC, Lutrol-E400, propylene glycol, and Transcutol were obtained as gift samples from Gattefosse (Saint-Priest, France). 2.2. Estimation of PV solubility in various SNEDDS components In this study, all previously-mentioned essential oils were studied for the solubility of PV. Meanwhile, Tween80:Span80 in the ratio 5.33:4.67, Steareth-2: Steareth-21 in the ratio 4.9:5.1, Labrasol:Labrafil 1944 in the ratio 6:4, and Brij 30 were evaluated for the solubility of PV to use as the surfactants in the proposed SNEDDS. As the reported HLB for LO is 10 so the different pairs of surfactant in each surfactant mixture were mixed in ratios which allow the HLB for the mixture equal 10. In addition, Lauroglycol-FCC, Lutrol-E400, propylene glycol, and Transcutol were studied as co-surfactants. The solubility of PV was assessed by shaking an excess of PV with 2 mL of the tested samples at 25 ± 0.5 °C for 48 h. The samples were then centrifuged (1200 rpm, 15 min), diluted with methanol, and quantified by UV–Vis spectrophotometry at 260 nm. 2.3. Pseudoternary phase diagram in various solvent systems A pseudoternary phase diagram for locating the nanoemulsion region with LO (oil phase), Labrasol:Labrafil 1944 in the ratio 6:4 (surfactant mixture), and Lauroglycol-FCC (co-surfactant) was prepared. The total composition of the three components was maintained at 100% with 100 mg PV. The definite compositions were prepared for the identification of the nanoemulsion region in the diagram. 2.4. Preparation and optimization of PV-LO-loaded SNEDDS In order to optimize compositions of the selected oil, surfactant, and co-surfactant phases, a distance quadratic mixture design was used. Three independent variables were studied for this purpose: Factor A was LO in the range of 11–25%; Factor B was the surfactant mixture (Labrasol:Labrafil 1944) in the range of 39–53%; and Factor C was Lauroglycol-FCC co-surfactant in the range of 36–50%. The globule size and stability index were taken as the responses for the evaluation and optimization of the PV-LO loaded SNEDDS formulation. A total of 15 runs were executed randomly and contained 100 mg PV in each formulation . About 1 g of each mixture was prepared by simply mixing the three components (oil, surfactant, and co-surfactant). 2.4.1. Preparation of PV-LO-loaded SNEDDS The PV-LO-SNEDDS formulations were prepared by mixing and vortexing the specified proportions of the LO (oil), Labrasol:Labrafil 1944 in the ratio 6:4 (surfactant mixture), and Lauroglycol-FCC (co-surfactant) for 5 min. Finally, the mixture was allowed to achieve equilibrium for 12 h in a shaker water bath set at 100 rpm and 37 °C. 2.4.2. Evaluation of the PV-LO-loaded SNEDDS 2.4.2.1. Determination of globule size of the PV-LO SNEDDS The aqueous dispersions of the PV-LO SNEDDS obtained after each trial were subjected to 10 times dilution with distilled water, then the diluted samples were evaluated for globule size (Zetatrac, Microtrac, Montgomeryville, PA). 2.4.2.2. Determination of stability index The stability index was estimated by exposing the PV-LO-SNEDDS formulations to consecutive freeze-thaw cycles thrice at a freezing temperature of −25 °C for 12 h and thaw temperature of 25 °C for 12 h. The stability index was determined using the initial and final globule sizes using (Sindi et al., ): (1) Stability index of SNEDDS = Original globule size – Change in globule size Original globule size × 100 2.4.3. Optimization of PV-LO-loaded SNEDDS The optimization of the formulation was performed by selecting the optimal percentages of LO (Factor A), surfactant mixture (Factor B), and Lauroglycol-FCC (Factor C). The minimum value for globule size and maximum value for stability index were analyzed as the constraints on response variables. 2.5. Development of the chitosan oral gel loaded with PV-LO-SNEDDs The chitosan-based oral gel was prepared by loading of PV-LO-SNEDDs into a chitosan hydrogel. The hydrogel was first prepared by dispersing 200 mg chitosan in 10 mL of 1.5% dilute aqueous acetic acid, then stirring at 500 rpm and 25 ± 1 °C. Afterward, the required quantity of optimized PV-LO-SNEDDS was dispersed into the chitosan hydrogel with a final PV concentration of 1%. Other hydrogel samples were also prepared in a similar manner and stored overnight under refrigeration until further use (Sindi et al., ). 2.6. Rheological characterization of the chitosan oral gel loaded with PV-LO-SNEDDs The rheology of chitosan oral gel loaded with PV-LO-SNEDDs (O3) was compared with plain chitosan hydrogel (O1). Samples (1 g each) were evaluated at 25 ± 1 °C (Brookfield viscometer, spindle 52) at shear rates of 2, 10, 20, 30, 40, 50, and 60 s −1 to obtain the flow curves. The viscosities at a maximum (η max ) and minimum (η min ) rate of shear were determined according to a previous study (Sindi et al., ), and Farrow’s constant (n) was calculated using : (2) Log G = n Log F – Log ɳ where G is the shear rate; ɳ is viscosity; F is the shear stress; and n is Farrow’s constant. 2.7. In vitro release of the PV-LO-SNEDDS-loaded chitosan oral hydrogel The release of PV from the O2, O3, marketed PV cream (1%), and 1% PV aqueous suspension was studied using a modified Type I USP apparatus in accordance with a reported procedure (Sindi et al., ). In this work, the previously activated membrane (100-µm pore size) was tied to the lower part of a cylindrical tube (length of 10 cm and diameter of 2.7 cm), instead of a basket. Accurately weighed samples containing 10 mg PV were placed inside the tubes, then the apparatus was set at 50 rpm and 37 ± 0.5 °C with phosphate-buffered saline (pH 6.8, 250 mL) as the medium. The dissolution samples were withdrawn at predetermined time intervals; 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h with replenishment of lost medium volume after each sampling with the fresh medium. The filtered (0.45 µm) samples were analyzed using high-performance liquid chromatography. Methanol mixed with 0.1 M ammonium acetate buffer at pH 6.0 in a volume ratio of 1:10 was used as the mobile phase. The flow rate of the mobile phase through the 5-μm ODS2 column at 35 °C was 1 mL/min and detected at 260 nm. 2.8. Ex vivo mucosal permeation studies A reported procedure was followed to study the ex vivo mucosal permeation (Sindi et al., ). Fresh sheep buccal mucosa of dimension 2 × 2 cm with a diffusion area of 1.75 cm 2 was used as the membrane barrier in the Franz diffusion cell containing 8 mL of phosphate-buffered saline at pH 6.8 and 37 ± 1 °C as the receptor medium. The mucosal permeation of PV from O2, O3, marketed PV cream (1%), and 1% PV aqueous dispersion was determined. Samples were collected and the PV content was determined by HPLC. The filtered (0.45 µm) samples were analyzed using high-performance liquid chromatography. Methanol mixed with 0.1 M ammonium acetate buffer at pH 6.0 in a volume ratio of 1:10 was used as the mobile phase. The flow rate of the mobile phase through the 5-μm ODS2 column at 35 °C was 1 mL/min and detected at 260 nm. The cumulative PV permeation, steady-state flux (Jss), permeability coefficient (PC), enhancement factor (EF), relative permeation rate (RPR), and diffusion coefficient (D) were determined for the samples. 2.9. In vivo evaluation of the optimized PV-LO SNEDDs loaded chitosan oral hydrogel The in vivo study was performed on three groups of male Wistar rats (200–250 g), which was approved by the local Institutional Review Board for Preclinical & Clinical Research (Approval No. 22-04-21). Group 1 orally ingested the O2 formulation at a PV dose of 10 mg/kg body weight. In Group 2, the animals were orally administered the optimized PV-LO SNEDDs-loaded chitosan oral hydrogel (O3) at a PV dose of 10 mg/kg. Group 3 animals were administered transdermal application of marketed PV 1% cream at a PV dose of 10 mg/kg. Finally, the pharmacokinetic parameters of PV including maximum plasma level (C max ), time to reach maximum plasma level (t max ), area under plasma concentration–time curve (AUC), and elimination rate constant (K), were determined (Kinetica version 4, Thermo Electron Corporation, Waltham, MA) and compared.
Materials PV was obtained as a gift sample from Qingdao Sigma Chemical Co., Ltd. Shandong, China. Basil oil, thyme oil, peppermint oil, rosemary oil, LO, citronella oil, verbena oil, and camphor oil were procured from Jiangxi Origin Aromatics Co., Ltd. Jiangxi, China. Tween80, Span80, Steareth-2, Steareth-21, Labrasol, Labrafil 1944, Brij 30, Lauroglycol-FCC, Lutrol-E400, propylene glycol, and Transcutol were obtained as gift samples from Gattefosse (Saint-Priest, France).
Estimation of PV solubility in various SNEDDS components In this study, all previously-mentioned essential oils were studied for the solubility of PV. Meanwhile, Tween80:Span80 in the ratio 5.33:4.67, Steareth-2: Steareth-21 in the ratio 4.9:5.1, Labrasol:Labrafil 1944 in the ratio 6:4, and Brij 30 were evaluated for the solubility of PV to use as the surfactants in the proposed SNEDDS. As the reported HLB for LO is 10 so the different pairs of surfactant in each surfactant mixture were mixed in ratios which allow the HLB for the mixture equal 10. In addition, Lauroglycol-FCC, Lutrol-E400, propylene glycol, and Transcutol were studied as co-surfactants. The solubility of PV was assessed by shaking an excess of PV with 2 mL of the tested samples at 25 ± 0.5 °C for 48 h. The samples were then centrifuged (1200 rpm, 15 min), diluted with methanol, and quantified by UV–Vis spectrophotometry at 260 nm.
Pseudoternary phase diagram in various solvent systems A pseudoternary phase diagram for locating the nanoemulsion region with LO (oil phase), Labrasol:Labrafil 1944 in the ratio 6:4 (surfactant mixture), and Lauroglycol-FCC (co-surfactant) was prepared. The total composition of the three components was maintained at 100% with 100 mg PV. The definite compositions were prepared for the identification of the nanoemulsion region in the diagram.
Preparation and optimization of PV-LO-loaded SNEDDS In order to optimize compositions of the selected oil, surfactant, and co-surfactant phases, a distance quadratic mixture design was used. Three independent variables were studied for this purpose: Factor A was LO in the range of 11–25%; Factor B was the surfactant mixture (Labrasol:Labrafil 1944) in the range of 39–53%; and Factor C was Lauroglycol-FCC co-surfactant in the range of 36–50%. The globule size and stability index were taken as the responses for the evaluation and optimization of the PV-LO loaded SNEDDS formulation. A total of 15 runs were executed randomly and contained 100 mg PV in each formulation . About 1 g of each mixture was prepared by simply mixing the three components (oil, surfactant, and co-surfactant). 2.4.1. Preparation of PV-LO-loaded SNEDDS The PV-LO-SNEDDS formulations were prepared by mixing and vortexing the specified proportions of the LO (oil), Labrasol:Labrafil 1944 in the ratio 6:4 (surfactant mixture), and Lauroglycol-FCC (co-surfactant) for 5 min. Finally, the mixture was allowed to achieve equilibrium for 12 h in a shaker water bath set at 100 rpm and 37 °C. 2.4.2. Evaluation of the PV-LO-loaded SNEDDS 2.4.2.1. Determination of globule size of the PV-LO SNEDDS The aqueous dispersions of the PV-LO SNEDDS obtained after each trial were subjected to 10 times dilution with distilled water, then the diluted samples were evaluated for globule size (Zetatrac, Microtrac, Montgomeryville, PA). 2.4.2.2. Determination of stability index The stability index was estimated by exposing the PV-LO-SNEDDS formulations to consecutive freeze-thaw cycles thrice at a freezing temperature of −25 °C for 12 h and thaw temperature of 25 °C for 12 h. The stability index was determined using the initial and final globule sizes using (Sindi et al., ): (1) Stability index of SNEDDS = Original globule size – Change in globule size Original globule size × 100 2.4.3. Optimization of PV-LO-loaded SNEDDS The optimization of the formulation was performed by selecting the optimal percentages of LO (Factor A), surfactant mixture (Factor B), and Lauroglycol-FCC (Factor C). The minimum value for globule size and maximum value for stability index were analyzed as the constraints on response variables.
Preparation of PV-LO-loaded SNEDDS The PV-LO-SNEDDS formulations were prepared by mixing and vortexing the specified proportions of the LO (oil), Labrasol:Labrafil 1944 in the ratio 6:4 (surfactant mixture), and Lauroglycol-FCC (co-surfactant) for 5 min. Finally, the mixture was allowed to achieve equilibrium for 12 h in a shaker water bath set at 100 rpm and 37 °C.
Evaluation of the PV-LO-loaded SNEDDS 2.4.2.1. Determination of globule size of the PV-LO SNEDDS The aqueous dispersions of the PV-LO SNEDDS obtained after each trial were subjected to 10 times dilution with distilled water, then the diluted samples were evaluated for globule size (Zetatrac, Microtrac, Montgomeryville, PA). 2.4.2.2. Determination of stability index The stability index was estimated by exposing the PV-LO-SNEDDS formulations to consecutive freeze-thaw cycles thrice at a freezing temperature of −25 °C for 12 h and thaw temperature of 25 °C for 12 h. The stability index was determined using the initial and final globule sizes using (Sindi et al., ): (1) Stability index of SNEDDS = Original globule size – Change in globule size Original globule size × 100
Determination of globule size of the PV-LO SNEDDS The aqueous dispersions of the PV-LO SNEDDS obtained after each trial were subjected to 10 times dilution with distilled water, then the diluted samples were evaluated for globule size (Zetatrac, Microtrac, Montgomeryville, PA).
Determination of stability index The stability index was estimated by exposing the PV-LO-SNEDDS formulations to consecutive freeze-thaw cycles thrice at a freezing temperature of −25 °C for 12 h and thaw temperature of 25 °C for 12 h. The stability index was determined using the initial and final globule sizes using (Sindi et al., ): (1) Stability index of SNEDDS = Original globule size – Change in globule size Original globule size × 100
Optimization of PV-LO-loaded SNEDDS The optimization of the formulation was performed by selecting the optimal percentages of LO (Factor A), surfactant mixture (Factor B), and Lauroglycol-FCC (Factor C). The minimum value for globule size and maximum value for stability index were analyzed as the constraints on response variables.
Development of the chitosan oral gel loaded with PV-LO-SNEDDs The chitosan-based oral gel was prepared by loading of PV-LO-SNEDDs into a chitosan hydrogel. The hydrogel was first prepared by dispersing 200 mg chitosan in 10 mL of 1.5% dilute aqueous acetic acid, then stirring at 500 rpm and 25 ± 1 °C. Afterward, the required quantity of optimized PV-LO-SNEDDS was dispersed into the chitosan hydrogel with a final PV concentration of 1%. Other hydrogel samples were also prepared in a similar manner and stored overnight under refrigeration until further use (Sindi et al., ).
Rheological characterization of the chitosan oral gel loaded with PV-LO-SNEDDs The rheology of chitosan oral gel loaded with PV-LO-SNEDDs (O3) was compared with plain chitosan hydrogel (O1). Samples (1 g each) were evaluated at 25 ± 1 °C (Brookfield viscometer, spindle 52) at shear rates of 2, 10, 20, 30, 40, 50, and 60 s −1 to obtain the flow curves. The viscosities at a maximum (η max ) and minimum (η min ) rate of shear were determined according to a previous study (Sindi et al., ), and Farrow’s constant (n) was calculated using : (2) Log G = n Log F – Log ɳ where G is the shear rate; ɳ is viscosity; F is the shear stress; and n is Farrow’s constant.
In vitro release of the PV-LO-SNEDDS-loaded chitosan oral hydrogel The release of PV from the O2, O3, marketed PV cream (1%), and 1% PV aqueous suspension was studied using a modified Type I USP apparatus in accordance with a reported procedure (Sindi et al., ). In this work, the previously activated membrane (100-µm pore size) was tied to the lower part of a cylindrical tube (length of 10 cm and diameter of 2.7 cm), instead of a basket. Accurately weighed samples containing 10 mg PV were placed inside the tubes, then the apparatus was set at 50 rpm and 37 ± 0.5 °C with phosphate-buffered saline (pH 6.8, 250 mL) as the medium. The dissolution samples were withdrawn at predetermined time intervals; 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h with replenishment of lost medium volume after each sampling with the fresh medium. The filtered (0.45 µm) samples were analyzed using high-performance liquid chromatography. Methanol mixed with 0.1 M ammonium acetate buffer at pH 6.0 in a volume ratio of 1:10 was used as the mobile phase. The flow rate of the mobile phase through the 5-μm ODS2 column at 35 °C was 1 mL/min and detected at 260 nm.
Ex vivo mucosal permeation studies A reported procedure was followed to study the ex vivo mucosal permeation (Sindi et al., ). Fresh sheep buccal mucosa of dimension 2 × 2 cm with a diffusion area of 1.75 cm 2 was used as the membrane barrier in the Franz diffusion cell containing 8 mL of phosphate-buffered saline at pH 6.8 and 37 ± 1 °C as the receptor medium. The mucosal permeation of PV from O2, O3, marketed PV cream (1%), and 1% PV aqueous dispersion was determined. Samples were collected and the PV content was determined by HPLC. The filtered (0.45 µm) samples were analyzed using high-performance liquid chromatography. Methanol mixed with 0.1 M ammonium acetate buffer at pH 6.0 in a volume ratio of 1:10 was used as the mobile phase. The flow rate of the mobile phase through the 5-μm ODS2 column at 35 °C was 1 mL/min and detected at 260 nm. The cumulative PV permeation, steady-state flux (Jss), permeability coefficient (PC), enhancement factor (EF), relative permeation rate (RPR), and diffusion coefficient (D) were determined for the samples.
In vivo evaluation of the optimized PV-LO SNEDDs loaded chitosan oral hydrogel The in vivo study was performed on three groups of male Wistar rats (200–250 g), which was approved by the local Institutional Review Board for Preclinical & Clinical Research (Approval No. 22-04-21). Group 1 orally ingested the O2 formulation at a PV dose of 10 mg/kg body weight. In Group 2, the animals were orally administered the optimized PV-LO SNEDDs-loaded chitosan oral hydrogel (O3) at a PV dose of 10 mg/kg. Group 3 animals were administered transdermal application of marketed PV 1% cream at a PV dose of 10 mg/kg. Finally, the pharmacokinetic parameters of PV including maximum plasma level (C max ), time to reach maximum plasma level (t max ), area under plasma concentration–time curve (AUC), and elimination rate constant (K), were determined (Kinetica version 4, Thermo Electron Corporation, Waltham, MA) and compared.
Results and discussion 3.1. Estimation of PV solubility in various SNEDDS components Various essential oils, including basil, thyme, peppermint, rosemary, lavender, citronella, verbena, and camphor, were studied on the basis of their reported anesthetic activity, under the presumption that they exert a beneficial effect against pain associated with herpes labials. Such an advantage of essential oils has been proven in nanostructured carriers (Lai et al., ). The PV solubility in these oils was measured in an attempt to determine the most appropriate oil phase to be used. indicates that all the essential oils exhibited good solubility of PV; this is in agreement with a report denoting that essential oils enhance the solubility of poorly water-soluble drugs (Hosny et al., ). Among the studied oils, PV was found to have a significantly higher solubility in LO with a value of 445 ± 17 mg/mL compared to the other oils ( p value <.05). Therefore, LO was selected as the oil phase in the proposed PV-loaded SNEDDS. Given the fact that the required HLB for LO is 10, surfactant mixtures that provide an HLB value of 10 were studied. Amongst the surfactants studied, PV was found to have significantly higher solubility of 105 ± 9 mg/mL in Labrasol:Labrafil 1944 (6:4) ( p value <.05), as shown in . Comparing all co-surfactants, further indicates that Lauroglycol-FCC demonstrated a significantly higher solubility (79 ± 4 mg/mL; p value <.05). Therefore, LO, Labrasol:Labrafil 1944 (6:4), and Lauroglycol-FCC were selected as the oil phase, surfactant mixture, and co-surfactant, respectively, for the formulation of PV-loaded SNEDDS. In a similar study, a system containing Labrafil 1944, Labrasol, and Lauroglycol-FCC was reported to enhance the bioavailability of Coenzyme Q10 in a SNEDDS formulation (Balakrishnan et al., ). Therefore, the components of the proposed PV-LO-SNEDDS appeared promising for further screening and optimization. 3.2. Pseudoternary-phase diagram Identification of the suitable levels for the formulation of PV-loaded SNEDDS was carried out by constructing a pseudoternary-phase diagram . The nanoemulsion region was observed when the LO concentration ranged between 11% and 27%, Labrasol:Labrafil 1944 (6:4) surfactant mixture level between 39% and 62%, and Lauroglycol-FCC level between 36% and 61%. 3.3. Preparation and optimization of PV-LO loaded SNEDDS provides the obtained globule size and stability index for each run suggested by the software. 3.3.1. Globule size A special quadratic model was suggested for the globule size response with a model F value of 6409.04, and the lack of fit was not significant. Furthermore, adequate precision was acceptable with a value of 254.829, which is sufficiently large to use the design space. The analysis of variance (ANOVA) data for the globule size of PV-LO-SNEDDS is provided in . Globule size was calculated using based on the L-pseudo components. The trace plot was obtained to determine the effect of the individual design components on the response and, therefore, serves as the perturbation plot of the non-mixture design. The response trace plot in reveals that LO (Factor A) has the greatest influence and surfactant mixture (Factor B) has the least influence on the globule size of PV-LO-SNEDDS. The 2D simplex contour and 3D plots also confirm the significant effects of LO (Factor A) and Lauroglycol-FCC (Factor C) on globule size. The interaction effects displayed in these plots show that minimum values for globule size were observed at low values of Factor A, intermediate values of Factor B, and high values of Factor C. It has been established that higher oil content leads to larger globule size, and hence, the present observation regarding Factor A is acceptable (Sakeena et al., ). In addition, co-surfactants have been reported to reduce the interfacial tension of globules to values below that produced by a surfactant alone (Gurram et al., ). Thus, the observed effect of Lauroglycol-FCC as a co-surfactant in the PV-LO-SNEDDS is justified. However, the negligible effect of Labrasol:Labrafil 1944 (ratio of 6:4) as the surfactant mixture was rather unexpected. While high HLB surfactants generally decrease the globule size of o/w type nanoemulsions, surfactants with low HLB values have been proven to increase the globule size (Niamprem et al., ). Importantly, low HLB-value surfactants provide poor spreading and emulsification properties compared to high HLB-value surfactants (Eid et al., ). The very low Labrafil 1944 HLB of 3 (Jakab et al., ) and Labrafil 1944 oil-like properties (Balakrishnan et al., ), might be the reasons that contribute to the diminished effect of the surfactant mixture on globule size. (3) Globule size = + 312.49 A + 250.95 B + 294.69 C − 250.63 AB − 321.54 AC + 187.93 BC + 7228 . 98 A 2 BC − 3204 . 43 AB 2 C − 11323 . 45 ABC 2 3.3.2. Stability index A special quadratic model was suggested for the globule size response with a model F value of 383.97, for which the lack of fit was not significant, and the adequate precision value was 52.6782. The analysis of variance (ANOVA) data for the stability index of PV-LO-SNEDDS are shown in . The equation for the stability index derived in terms of the L-pseudo components is given in . The response trace plot for the stability index further reveals that the effect of the factors follows the order: Lauroglycol-FCC (Factor C) > LO (Factor A) > surfactant mixture (Factor B). The 2D simplex contour and 3D plots show that the stability index drastically increased when Factor C was increased. Considering that co-surfactants have been reported to increase the interfacial fluidity and disordering of a surfactant film (Isailović et al., ), a significant effect of Lauroglycol-FCC as the co-surfactant in PV-LO-SNEDDS on the stability index was anticipated. Moreover, the stability index was found to decrease when Factor A was increased, of note, the lowest stability index was obtained at the highest level of Factor A. Considering that higher oil content leads to larger globule size and subsequent reduction in droplet stability, such a decrease in stability index can be expected (Sakeena et al., ). The effect of surfactant mixture on stability index can be justified by the same explanations provided for its effect on the globule size, whereby the combination of both low and high HLB value surfactants exhibited an intermediate effect on the stability index. (4) Stability index = + 71.53 A + 76.52 B + 92.84 C + 3.88 AB + 21.37 AC + 8.57 BC + 304 . 97 A 2 BC + 226 . 71 AB 2 C − 374 . 99 ABC 2 3.3.3. Optimization of PV-LO-loaded SNEDDS The optimum formula in was found to contain 14% Factor A, 40.5% Factor B, 45.5% Factor C, and 100 mg PV. Then, the prepared PV-LO-SNEDDS using this optimized formula exhibited a globule size of 157 ± 9 nm and stability index of more than 88 ± 4%; values which are close to the predicted values. 3.4. Rheological evaluation of the PV-LO SNEDDs-loaded chitosan oral hydrogel The rheological parameters of the chitosan hydrogel loaded with the optimized PV-LO-SNEDDS (O3) were determined in comparison to a plain chitosan hydrogel (O1) . The rheological behavior of the formulations depends on both the material properties and process attributes (Ghica et al., ). The values of Farrow’s constant were more than 1 for both O1 and O3 samples, indicating a pseudoplastic behavior (El-Leithy et al., ). In this study, the chitosan hydrogel was prepared with 200 mg chitosan in 10 mL dilute aqueous acetic acid, corresponding to a concentration of 0.2 g/L. It has been reported that chitosan concentrations above 0.50 g/dL show a pseudoplastic or shear-thinning behavior (Kienzle-Sterzer et al., ). Thus, the pseudoplastic or shear-thinning properties of O1 and O3 gels can be justified. This was further confirmed from the plots of viscosity versus the rate of shear , whereby decreased viscosity of both samples was observed at higher shear rates, which is a characteristic of shear-thinning systems. Nonetheless, the minimum and maximum viscosities were higher for the O3 formulation compared to the O1 hydrogel. Previously, a published study has demonstrated the enhanced hydrogel viscosity after the inclusion of lipid-based nanostructures (Padamwar & Pokharkar, ), thereby, this may explain the higher viscosity of O3 with embedded SNEDDS compared to O1. Noteworthy, in addition to the advantage of the O3 formulation for sustaining PV release, also, the addition of lipid nanostructures will not prevent the gelation of chitosan but rather increases the rheological moduli by a significant proportion (Billard et al., ). The thixotropy of the O1 and O3 formulations can be elucidated from the rheograms of the shear rate versus shear stress as shown in . Specifically, the area of the formed loops represents the thixotropic behavior of the formulation (Ghica et al., ). Thus, the hysteresis loops of the O3 formulation exhibit a larger area than those of the plain chitosan hydrogel, thus it can be suggested that O3 has a slightly higher thixotropic behavior. This further implies that the O3 formulation with greater thixotropy can provide a better-sustained release of PV, easy application at the buccal mucosal surface, as well as an enhanced retention of the gel in the buccal mucosal surface (Lee et al., ). The plots for both shear rate and shear stress on logarithmic scales in provide a better delineation of the individual flow curves for further comparison (Jeong, ). Specifically, Farrow’s constant for the samples was determined from the slope of these plots. Thereafter, the rheological behaviors of the samples were evaluated based on the reciprocal of Farrow’s constant, or the slope of log shear stress versus log shear rate, whereby values less than one for the slope is indicative of a shear-thinning system (Wilkes, ). The results denote that O3 demonstrates a greater shear-thinning behavior compared to O1. Similarly, some evidence has demonstrated a combined pseudoplastic and thixotropic behavior for oral gel in a previous report (Hosny et al., ). 3.5. In vitro release of PV from PV-LO-SNEDDS loaded chitosan oral hydrogel (O3) A further comparative in vitro release study of the PV profile from O2, O3, commercial PV cream (1%), and 1% PV aqueous dispersion was carried out, and the results are shown in . A significant increase, in addition to a sustained PV release was observed from the O3 compared to other samples. These observations can be described according to two aspects. First, the formulation of SNEDDS contributes to an enhanced drug dissolution and release of poorly soluble drugs (Kazi et al., ). Second, the polymer gel acts as a barrier for drug diffusion from the formulation into the medium, thus resulting in its release in a sustained manner (Xu et al., ). This effect is evident from the lower drug release from O2 compared to the 1% aqueous suspension of PV and the combined effect of enhanced and sustained release from the O3 formulation. Such a pattern is highly beneficial in certain conditions such as herpes labialis, wherein both enhanced concentration and a sustained release of PV are desired criteria. Meanwhile, other samples also showed a sustained release pattern, which could probably be explained due to the low dissolution and slow release of PV from these systems. The significantly higher PV release from O3 compared to the marketed PV cream revealed the importance of the O3 formulation in the management of herpes labialis. 3.6. Ex vivo mucosal permeation studies Ex vivo permeability studies in sheep buccal mucosa confirmed that the O3 formulation significantly enhanced PV permeation ( p value <.05) compared to all the other formulations . The flux of permeation of PV followed the order: O3 > marketed PV (1%) cream > O2 > 1% PV aqueous suspension. Obviously, the PCs also showed the same order since the same dose of PV was loaded for all samples. The calculated RPR with respect to the marketed PV (1%) cream was also highest for O3 with a value of 2.069. Nevertheless, the O3 formulation increased PV permeation by 4.777 times compared to the 1% PV aqueous suspension, while the marketed PV (1%) cream could only enhance PV permeation by 2.308 times. Interestingly, chitosan can improve the paracellular permeation of drugs across the epithelium of mucosa (Thanou et al., ), which is facilitated by the reversible interaction of protonated chitosan with tight junction constituents that result in a widened paracellular path (Thanou et al., ). This effect can be held responsible for the higher permeation of PV from O2 compared to the 1% aqueous suspension. Thus, the flux of permeation, permeation coefficient, diffusion coefficient, and RPR values were higher for O2 compared to the 1% aqueous suspension. In addition, essential oils like LO have demonstrated permeation-enhancing effects (de Matos et al., ), wherein linalool as a major constituent of LO can significantly influence permeation (Kamatou & Viljoen, ). Herein, only the O3 formulation contains LO; thus, the enhanced permeation of PV can be contributed to the presence of LO as the oil phase of SNEDDS. In addition, the SNEDDS formulation in O3 is also highly responsible for improving the permeation and spreadability of PV across buccal mucosa (Khan et al., ; Salem et al., ). 3.7. In vivo evaluation of the optimized PV-LO-SNEDDs loaded chitosan oral hydrogel The pharmacokinetic data obtained from the in vivo studies are provided in , which demonstrates that the C max , AUC 0–t , and AUC 0–inf were significantly higher ( p value<.05) for O3 in comparison with the other formulations. Specifically, the increased C max and AUC values of O3 can be contributed to the enhanced absorption of chitosan via the increased paracellular permeation of drugs across the mucosa epithelium (Thanou et al., ). Due to the fact that the O2 formulation also contains chitosan, yet shows the least values for these pharmacokinetic parameters; this indicates that the increased C max and AUC values of O3 could be attributed to the presence of SNEDDS. This result is in agreement with previous reports demonstrating the similar enhancement of C max and AUC of poorly water-soluble drugs by the SNEDDS formulation (Yoo et al., ; Tripathi et al., ). Indeed, the enhanced and faster absorption results in the reduction of T max , which was observed for the O3 formulation with the lowest T max . When the drug is delivered as a sustained-release product, its elimination half-life increases and, subsequently, the k el value is lowered (Kim et al., ). Therefore, the sustained delivery of PV from O3 was also confirmed by the lowest value of 0.534 ± 0.05 h −1 for this formulation. On the other hand, the relative bioavailability of the optimized O3 formula compared to the O2 oral gel and marketed PV 1% cream was found to be 245% and 180%, respectively. Importantly, these high relative bioavailability values confirm the efficacy of the O3 formulation in enhancing the bioavailability of PV.
Estimation of PV solubility in various SNEDDS components Various essential oils, including basil, thyme, peppermint, rosemary, lavender, citronella, verbena, and camphor, were studied on the basis of their reported anesthetic activity, under the presumption that they exert a beneficial effect against pain associated with herpes labials. Such an advantage of essential oils has been proven in nanostructured carriers (Lai et al., ). The PV solubility in these oils was measured in an attempt to determine the most appropriate oil phase to be used. indicates that all the essential oils exhibited good solubility of PV; this is in agreement with a report denoting that essential oils enhance the solubility of poorly water-soluble drugs (Hosny et al., ). Among the studied oils, PV was found to have a significantly higher solubility in LO with a value of 445 ± 17 mg/mL compared to the other oils ( p value <.05). Therefore, LO was selected as the oil phase in the proposed PV-loaded SNEDDS. Given the fact that the required HLB for LO is 10, surfactant mixtures that provide an HLB value of 10 were studied. Amongst the surfactants studied, PV was found to have significantly higher solubility of 105 ± 9 mg/mL in Labrasol:Labrafil 1944 (6:4) ( p value <.05), as shown in . Comparing all co-surfactants, further indicates that Lauroglycol-FCC demonstrated a significantly higher solubility (79 ± 4 mg/mL; p value <.05). Therefore, LO, Labrasol:Labrafil 1944 (6:4), and Lauroglycol-FCC were selected as the oil phase, surfactant mixture, and co-surfactant, respectively, for the formulation of PV-loaded SNEDDS. In a similar study, a system containing Labrafil 1944, Labrasol, and Lauroglycol-FCC was reported to enhance the bioavailability of Coenzyme Q10 in a SNEDDS formulation (Balakrishnan et al., ). Therefore, the components of the proposed PV-LO-SNEDDS appeared promising for further screening and optimization.
Pseudoternary-phase diagram Identification of the suitable levels for the formulation of PV-loaded SNEDDS was carried out by constructing a pseudoternary-phase diagram . The nanoemulsion region was observed when the LO concentration ranged between 11% and 27%, Labrasol:Labrafil 1944 (6:4) surfactant mixture level between 39% and 62%, and Lauroglycol-FCC level between 36% and 61%.
Preparation and optimization of PV-LO loaded SNEDDS provides the obtained globule size and stability index for each run suggested by the software. 3.3.1. Globule size A special quadratic model was suggested for the globule size response with a model F value of 6409.04, and the lack of fit was not significant. Furthermore, adequate precision was acceptable with a value of 254.829, which is sufficiently large to use the design space. The analysis of variance (ANOVA) data for the globule size of PV-LO-SNEDDS is provided in . Globule size was calculated using based on the L-pseudo components. The trace plot was obtained to determine the effect of the individual design components on the response and, therefore, serves as the perturbation plot of the non-mixture design. The response trace plot in reveals that LO (Factor A) has the greatest influence and surfactant mixture (Factor B) has the least influence on the globule size of PV-LO-SNEDDS. The 2D simplex contour and 3D plots also confirm the significant effects of LO (Factor A) and Lauroglycol-FCC (Factor C) on globule size. The interaction effects displayed in these plots show that minimum values for globule size were observed at low values of Factor A, intermediate values of Factor B, and high values of Factor C. It has been established that higher oil content leads to larger globule size, and hence, the present observation regarding Factor A is acceptable (Sakeena et al., ). In addition, co-surfactants have been reported to reduce the interfacial tension of globules to values below that produced by a surfactant alone (Gurram et al., ). Thus, the observed effect of Lauroglycol-FCC as a co-surfactant in the PV-LO-SNEDDS is justified. However, the negligible effect of Labrasol:Labrafil 1944 (ratio of 6:4) as the surfactant mixture was rather unexpected. While high HLB surfactants generally decrease the globule size of o/w type nanoemulsions, surfactants with low HLB values have been proven to increase the globule size (Niamprem et al., ). Importantly, low HLB-value surfactants provide poor spreading and emulsification properties compared to high HLB-value surfactants (Eid et al., ). The very low Labrafil 1944 HLB of 3 (Jakab et al., ) and Labrafil 1944 oil-like properties (Balakrishnan et al., ), might be the reasons that contribute to the diminished effect of the surfactant mixture on globule size. (3) Globule size = + 312.49 A + 250.95 B + 294.69 C − 250.63 AB − 321.54 AC + 187.93 BC + 7228 . 98 A 2 BC − 3204 . 43 AB 2 C − 11323 . 45 ABC 2 3.3.2. Stability index A special quadratic model was suggested for the globule size response with a model F value of 383.97, for which the lack of fit was not significant, and the adequate precision value was 52.6782. The analysis of variance (ANOVA) data for the stability index of PV-LO-SNEDDS are shown in . The equation for the stability index derived in terms of the L-pseudo components is given in . The response trace plot for the stability index further reveals that the effect of the factors follows the order: Lauroglycol-FCC (Factor C) > LO (Factor A) > surfactant mixture (Factor B). The 2D simplex contour and 3D plots show that the stability index drastically increased when Factor C was increased. Considering that co-surfactants have been reported to increase the interfacial fluidity and disordering of a surfactant film (Isailović et al., ), a significant effect of Lauroglycol-FCC as the co-surfactant in PV-LO-SNEDDS on the stability index was anticipated. Moreover, the stability index was found to decrease when Factor A was increased, of note, the lowest stability index was obtained at the highest level of Factor A. Considering that higher oil content leads to larger globule size and subsequent reduction in droplet stability, such a decrease in stability index can be expected (Sakeena et al., ). The effect of surfactant mixture on stability index can be justified by the same explanations provided for its effect on the globule size, whereby the combination of both low and high HLB value surfactants exhibited an intermediate effect on the stability index. (4) Stability index = + 71.53 A + 76.52 B + 92.84 C + 3.88 AB + 21.37 AC + 8.57 BC + 304 . 97 A 2 BC + 226 . 71 AB 2 C − 374 . 99 ABC 2 3.3.3. Optimization of PV-LO-loaded SNEDDS The optimum formula in was found to contain 14% Factor A, 40.5% Factor B, 45.5% Factor C, and 100 mg PV. Then, the prepared PV-LO-SNEDDS using this optimized formula exhibited a globule size of 157 ± 9 nm and stability index of more than 88 ± 4%; values which are close to the predicted values.
Globule size A special quadratic model was suggested for the globule size response with a model F value of 6409.04, and the lack of fit was not significant. Furthermore, adequate precision was acceptable with a value of 254.829, which is sufficiently large to use the design space. The analysis of variance (ANOVA) data for the globule size of PV-LO-SNEDDS is provided in . Globule size was calculated using based on the L-pseudo components. The trace plot was obtained to determine the effect of the individual design components on the response and, therefore, serves as the perturbation plot of the non-mixture design. The response trace plot in reveals that LO (Factor A) has the greatest influence and surfactant mixture (Factor B) has the least influence on the globule size of PV-LO-SNEDDS. The 2D simplex contour and 3D plots also confirm the significant effects of LO (Factor A) and Lauroglycol-FCC (Factor C) on globule size. The interaction effects displayed in these plots show that minimum values for globule size were observed at low values of Factor A, intermediate values of Factor B, and high values of Factor C. It has been established that higher oil content leads to larger globule size, and hence, the present observation regarding Factor A is acceptable (Sakeena et al., ). In addition, co-surfactants have been reported to reduce the interfacial tension of globules to values below that produced by a surfactant alone (Gurram et al., ). Thus, the observed effect of Lauroglycol-FCC as a co-surfactant in the PV-LO-SNEDDS is justified. However, the negligible effect of Labrasol:Labrafil 1944 (ratio of 6:4) as the surfactant mixture was rather unexpected. While high HLB surfactants generally decrease the globule size of o/w type nanoemulsions, surfactants with low HLB values have been proven to increase the globule size (Niamprem et al., ). Importantly, low HLB-value surfactants provide poor spreading and emulsification properties compared to high HLB-value surfactants (Eid et al., ). The very low Labrafil 1944 HLB of 3 (Jakab et al., ) and Labrafil 1944 oil-like properties (Balakrishnan et al., ), might be the reasons that contribute to the diminished effect of the surfactant mixture on globule size. (3) Globule size = + 312.49 A + 250.95 B + 294.69 C − 250.63 AB − 321.54 AC + 187.93 BC + 7228 . 98 A 2 BC − 3204 . 43 AB 2 C − 11323 . 45 ABC 2
Stability index A special quadratic model was suggested for the globule size response with a model F value of 383.97, for which the lack of fit was not significant, and the adequate precision value was 52.6782. The analysis of variance (ANOVA) data for the stability index of PV-LO-SNEDDS are shown in . The equation for the stability index derived in terms of the L-pseudo components is given in . The response trace plot for the stability index further reveals that the effect of the factors follows the order: Lauroglycol-FCC (Factor C) > LO (Factor A) > surfactant mixture (Factor B). The 2D simplex contour and 3D plots show that the stability index drastically increased when Factor C was increased. Considering that co-surfactants have been reported to increase the interfacial fluidity and disordering of a surfactant film (Isailović et al., ), a significant effect of Lauroglycol-FCC as the co-surfactant in PV-LO-SNEDDS on the stability index was anticipated. Moreover, the stability index was found to decrease when Factor A was increased, of note, the lowest stability index was obtained at the highest level of Factor A. Considering that higher oil content leads to larger globule size and subsequent reduction in droplet stability, such a decrease in stability index can be expected (Sakeena et al., ). The effect of surfactant mixture on stability index can be justified by the same explanations provided for its effect on the globule size, whereby the combination of both low and high HLB value surfactants exhibited an intermediate effect on the stability index. (4) Stability index = + 71.53 A + 76.52 B + 92.84 C + 3.88 AB + 21.37 AC + 8.57 BC + 304 . 97 A 2 BC + 226 . 71 AB 2 C − 374 . 99 ABC 2
Optimization of PV-LO-loaded SNEDDS The optimum formula in was found to contain 14% Factor A, 40.5% Factor B, 45.5% Factor C, and 100 mg PV. Then, the prepared PV-LO-SNEDDS using this optimized formula exhibited a globule size of 157 ± 9 nm and stability index of more than 88 ± 4%; values which are close to the predicted values.
Rheological evaluation of the PV-LO SNEDDs-loaded chitosan oral hydrogel The rheological parameters of the chitosan hydrogel loaded with the optimized PV-LO-SNEDDS (O3) were determined in comparison to a plain chitosan hydrogel (O1) . The rheological behavior of the formulations depends on both the material properties and process attributes (Ghica et al., ). The values of Farrow’s constant were more than 1 for both O1 and O3 samples, indicating a pseudoplastic behavior (El-Leithy et al., ). In this study, the chitosan hydrogel was prepared with 200 mg chitosan in 10 mL dilute aqueous acetic acid, corresponding to a concentration of 0.2 g/L. It has been reported that chitosan concentrations above 0.50 g/dL show a pseudoplastic or shear-thinning behavior (Kienzle-Sterzer et al., ). Thus, the pseudoplastic or shear-thinning properties of O1 and O3 gels can be justified. This was further confirmed from the plots of viscosity versus the rate of shear , whereby decreased viscosity of both samples was observed at higher shear rates, which is a characteristic of shear-thinning systems. Nonetheless, the minimum and maximum viscosities were higher for the O3 formulation compared to the O1 hydrogel. Previously, a published study has demonstrated the enhanced hydrogel viscosity after the inclusion of lipid-based nanostructures (Padamwar & Pokharkar, ), thereby, this may explain the higher viscosity of O3 with embedded SNEDDS compared to O1. Noteworthy, in addition to the advantage of the O3 formulation for sustaining PV release, also, the addition of lipid nanostructures will not prevent the gelation of chitosan but rather increases the rheological moduli by a significant proportion (Billard et al., ). The thixotropy of the O1 and O3 formulations can be elucidated from the rheograms of the shear rate versus shear stress as shown in . Specifically, the area of the formed loops represents the thixotropic behavior of the formulation (Ghica et al., ). Thus, the hysteresis loops of the O3 formulation exhibit a larger area than those of the plain chitosan hydrogel, thus it can be suggested that O3 has a slightly higher thixotropic behavior. This further implies that the O3 formulation with greater thixotropy can provide a better-sustained release of PV, easy application at the buccal mucosal surface, as well as an enhanced retention of the gel in the buccal mucosal surface (Lee et al., ). The plots for both shear rate and shear stress on logarithmic scales in provide a better delineation of the individual flow curves for further comparison (Jeong, ). Specifically, Farrow’s constant for the samples was determined from the slope of these plots. Thereafter, the rheological behaviors of the samples were evaluated based on the reciprocal of Farrow’s constant, or the slope of log shear stress versus log shear rate, whereby values less than one for the slope is indicative of a shear-thinning system (Wilkes, ). The results denote that O3 demonstrates a greater shear-thinning behavior compared to O1. Similarly, some evidence has demonstrated a combined pseudoplastic and thixotropic behavior for oral gel in a previous report (Hosny et al., ).
In vitro release of PV from PV-LO-SNEDDS loaded chitosan oral hydrogel (O3) A further comparative in vitro release study of the PV profile from O2, O3, commercial PV cream (1%), and 1% PV aqueous dispersion was carried out, and the results are shown in . A significant increase, in addition to a sustained PV release was observed from the O3 compared to other samples. These observations can be described according to two aspects. First, the formulation of SNEDDS contributes to an enhanced drug dissolution and release of poorly soluble drugs (Kazi et al., ). Second, the polymer gel acts as a barrier for drug diffusion from the formulation into the medium, thus resulting in its release in a sustained manner (Xu et al., ). This effect is evident from the lower drug release from O2 compared to the 1% aqueous suspension of PV and the combined effect of enhanced and sustained release from the O3 formulation. Such a pattern is highly beneficial in certain conditions such as herpes labialis, wherein both enhanced concentration and a sustained release of PV are desired criteria. Meanwhile, other samples also showed a sustained release pattern, which could probably be explained due to the low dissolution and slow release of PV from these systems. The significantly higher PV release from O3 compared to the marketed PV cream revealed the importance of the O3 formulation in the management of herpes labialis.
Ex vivo mucosal permeation studies Ex vivo permeability studies in sheep buccal mucosa confirmed that the O3 formulation significantly enhanced PV permeation ( p value <.05) compared to all the other formulations . The flux of permeation of PV followed the order: O3 > marketed PV (1%) cream > O2 > 1% PV aqueous suspension. Obviously, the PCs also showed the same order since the same dose of PV was loaded for all samples. The calculated RPR with respect to the marketed PV (1%) cream was also highest for O3 with a value of 2.069. Nevertheless, the O3 formulation increased PV permeation by 4.777 times compared to the 1% PV aqueous suspension, while the marketed PV (1%) cream could only enhance PV permeation by 2.308 times. Interestingly, chitosan can improve the paracellular permeation of drugs across the epithelium of mucosa (Thanou et al., ), which is facilitated by the reversible interaction of protonated chitosan with tight junction constituents that result in a widened paracellular path (Thanou et al., ). This effect can be held responsible for the higher permeation of PV from O2 compared to the 1% aqueous suspension. Thus, the flux of permeation, permeation coefficient, diffusion coefficient, and RPR values were higher for O2 compared to the 1% aqueous suspension. In addition, essential oils like LO have demonstrated permeation-enhancing effects (de Matos et al., ), wherein linalool as a major constituent of LO can significantly influence permeation (Kamatou & Viljoen, ). Herein, only the O3 formulation contains LO; thus, the enhanced permeation of PV can be contributed to the presence of LO as the oil phase of SNEDDS. In addition, the SNEDDS formulation in O3 is also highly responsible for improving the permeation and spreadability of PV across buccal mucosa (Khan et al., ; Salem et al., ).
In vivo evaluation of the optimized PV-LO-SNEDDs loaded chitosan oral hydrogel The pharmacokinetic data obtained from the in vivo studies are provided in , which demonstrates that the C max , AUC 0–t , and AUC 0–inf were significantly higher ( p value<.05) for O3 in comparison with the other formulations. Specifically, the increased C max and AUC values of O3 can be contributed to the enhanced absorption of chitosan via the increased paracellular permeation of drugs across the mucosa epithelium (Thanou et al., ). Due to the fact that the O2 formulation also contains chitosan, yet shows the least values for these pharmacokinetic parameters; this indicates that the increased C max and AUC values of O3 could be attributed to the presence of SNEDDS. This result is in agreement with previous reports demonstrating the similar enhancement of C max and AUC of poorly water-soluble drugs by the SNEDDS formulation (Yoo et al., ; Tripathi et al., ). Indeed, the enhanced and faster absorption results in the reduction of T max , which was observed for the O3 formulation with the lowest T max . When the drug is delivered as a sustained-release product, its elimination half-life increases and, subsequently, the k el value is lowered (Kim et al., ). Therefore, the sustained delivery of PV from O3 was also confirmed by the lowest value of 0.534 ± 0.05 h −1 for this formulation. On the other hand, the relative bioavailability of the optimized O3 formula compared to the O2 oral gel and marketed PV 1% cream was found to be 245% and 180%, respectively. Importantly, these high relative bioavailability values confirm the efficacy of the O3 formulation in enhancing the bioavailability of PV.
Conclusions Based on the solubility studies, LO, Labrasol:Labrafil 1944 (6:4), and Lauroglycol-FCC were selected as the oil, surfactant mixture, and co-surfactant, respectively. LO had the highest influence on globule size, whereas the stability index was most influenced by Lauroglycol-FCC. The optimized formula contained 14% LO, 40.5% Labrasol:Labrafil 1944 in the ratio of 6:4, 45.5% Lauroglycol-FCC, and 100 mg PV. The rheological characterization study of the optimized gel (O3) revealed its combined pseudoplastic and thixotropic behavior. The permeation studies in sheep buccal mucosa confirmed that the O3 formulation can significantly improve PV permeation, while the pharmacokinetic data indicate that O3 can enhance the bioavailability of PV and provide a sustained release. Altogether, these findings validate the usefulness of the O3 formulation, containing the optimized PV-LO-SNEDDS formulation in chitosan hydrogel, as a promising therapeutic approach against herpes labialis.
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Oral Health Research in the WHO African Region between 2011 and 2022: A Scoping Review | d09fdeac-3f68-4799-9687-b639741e1236 | 11562290 | Dentistry[mh] | Oral diseases are among the most common noncommunicable diseases (NCDs), with impact at social, economic, and health system levels. An estimated 3.5 billion individuals have active oral disease, giving rise to pain, disfigurement, social seclusion, emotional anguish, and, in severe cases, even death . The burden of oral diseases significantly overshadows the collective prevalence of all 5 major NCDs—mental disorders, cardiovascular disease, diabetes mellitus, chronic respiratory diseases, and cancers—underscoring the critical importance of addressing oral health globally . The African region is one of the World Health Organization’s (WHO’s) 6 regions worldwide and comprises 47 Member States. Due to various socioeconomic, cultural, and environmental factors , oral health remains a significant public health concern in the region. Around 44% of the population was estimated to suffer from oral diseases in 2019, with the most significant increase in major oral disease cases in the previous 30 y among all WHO regions . The most prevalent and severe oral diseases include dental caries, periodontal diseases, oral and oropharyngeal cancer, orofacial trauma, oral manifestations of HIV infection, congenital disabilities, and noma. The burden of these oral diseases creates notable disparities, impacting marginalized communities throughout their life span . Despite efforts to enhance the region’s oral health status, the prevailing actions in recent decades have been characterized by fragmented and specialized approaches rather than implementing integrated and economically efficient strategies. The WHO Regional Oral Health Strategy 2016–2025, addressing oral diseases as part of NCDs, was published in 2016 . This publication puts forth strategies to aid Member States in prioritizing oral health by incorporating oral diseases into the prevention and management of NCDs within the framework of Universal Health Coverage (UHC) . One strategic objective of the Regional Oral Health strategy is “to improve integrated surveillance of oral diseases, monitoring, and evaluation of programmes and research” . Beyond the scope of regional strategies, the global political guidance for oral health also underscored the significance of oral health research, particularly emphasizing the requirement for contextually relevant and constantly updated research that centers on the public health side of oral health (Seventy-fifth World Health Assembly and World Health Organization 2022; ). Although the number of publications by countries in the WHO African region has recently increased , authorities still report a lack of evidence for decision-making in oral health strategies . The aim of this scoping review was to determine the oral health research scope and output in the WHO African region between January 2011 and December 2022.
The reporting of this scoping review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) checklist ( Appendix Table 1 ) . The protocol was registered on the Open Science Framework (Labarca et al. 2023), and the search strategy was registered in the CABI Digital Library . Eligibility Criteria We included primary and secondary studies published in English, French, or Portuguese between January 1, 2011, and December 31, 2022, addressing oral health–related research having individuals, groups, or populations as their unit of analysis. We excluded in vitro and in vivo studies focusing on cells, biomarkers, or animals. Eligible studies included clinical trials, cohort studies, case-control studies, cross-sectional studies, ecological studies, systematic reviews (with or without meta-analysis), policy briefs, and evidence-informed guidelines. These reports either addressed a topic relevant to the WHO African region assessed using the title and study objective or were conducted in a country in the region. Search Strategy and Information Sources We conducted a systematic search in the following electronic databases: PubMed, EMBASE, Epistemonikos (curating primary and secondary studies from the Cochrane Database of Systematic Reviews [CDSR], PubMed, EMBASE, CINAHL [Cumulative Index to Nursing and Allied Health Literature], PsycINFO, LILACS, Database of Abstracts of Reviews of Effects [DARE], the Campbell Collaboration online library, the JBI Database of Systematic Reviews and Implementation Reports, and the EPPI-Centre Evidence Library), Scopus, and African regional databases such as Regional African Index Medicus and the African Journal Online ( Appendix Table 2 ). We adapted the PubMed search strategy to the syntax and thesauri of other databases. In addition, to identify articles that might have been missed in the electronic searches, we conducted cross-citation searches in Google Scholar and Microsoft Academic, using each included study as the index reference. We reviewed the reference list of each included study and the abstracts of the International Association for Dental, Oral, and Craniofacial Research (IADR) General and Regional Sessions. We searched dissertations and theses in the ProQUEST database and for ongoing or recently completed systematic reviews in PROSPERO. The retrieved records were uploaded into Covidence to ensure an efficient workflow and remove overlapping records across databases. Selection of Studies We screened the retrieved records using title and abstract and, in the second stage, using full-text reports independently and in duplicate by 15 previously calibrated reviewers (κ = 0.91). We solved disagreements by consensus. If differences persisted, a third external reviewer acted as a referee. If information was missing to ascertain eligibility, we contacted the study’s corresponding author via e-mail. Data Extraction and Analysis Ten reviewers, independently and in duplicate, extracted data from the included studies using a piloted data extraction template. Reviewers were trained to ensure consistency in data extraction (κ = 0.81). Disagreements in the extracted data were solved by consensus or adjudication by an additional third reviewer when consensus was elusive. We extracted variables associated with the journal of publication (name, country, editorial focus on African-oriented research [determined by the journal description and editorial scope reported in the journal’s webpage, indices, and aggregators]), study authors (number of authors, country of the study, affiliations, collaborations within and outside the WHO African region, for African authors their position in the article and corresponding assignment), primary and secondary studies (title, year of publication, keywords, and number of citations in Google Scholar, funding source, declaration of conflict of interest). In addition, we classified the studies according to 4 taxonomies: 1. Research type as defined by WHO in their systematic approach for undertaking a research priority–setting exercise : a. Problem: research to measure the size of the health problem through epidemiology, estimating the disease burden, and other forms of data collection b. Cause: research to understand the causal agents, risk factors, and determinants of the health issue (this research may include, for instance, a study of infection cycles, vectors, the role of socioeconomic factors, environment, diet, and the interaction of multiple factors) c. Solution: research to develop new interventions, including therapeutics, devices, procedures, policy interventions, public health campaigns, and so on d. Implementation: research to translate new interventions into policy and practice and understand the barriers to delivering known interventions e. Evaluation: research to monitor and evaluate the effectiveness or health impact of an intervention or program 2. Research designs: clinical trials, cohort studies, case-control studies, cross-sectional studies, ecological studies, systematic reviews (with or without meta-analysis), policy briefs, and evidence-informed guidelines 3. Study disciplines as categorized by the American Dental Association (ADA): dental public health, general dentistry, pediatric dentistry, oral and maxillofacial surgery, oral and maxillofacial pathology, oral medicine, periodontics, orthodontics and dentofacial orthopedics, oral and maxillofacial radiology, endodontics, prosthodontics, basic science–related research (translational research), orofacial pain, and dental anesthesiology 4. Oral health topics and subcategories. In an iterative process, we extracted the study’s primary disease, condition, or specific research topic and subcategories that further described the topic by reviewing the title, keywords, and full-text article. We used descriptive statistics (frequencies and percentages) to summarize quantitative data. A country-level impact factor was determined by calculating the ratio of the total number of citations received by records from a particular country to the overall number of studies conducted within that country. Each record covering diverse topics was individually classified for each relevant theme. This approach was implemented to maintain transparency and methodological clarity in handling multifaceted records, allowing for a nuanced analysis of each record and topic. In addition, we conducted a collaboration mapping analysis. We created a database including all relevant study citation data (title, abstract, year of publication), authors’ names, academic or research institution (affiliation), city, and country of the institution. These data were obtained using publicly available Application Programming Interface (API) from the US National Library of Medicine and Elsevier. We retrieved records based on a query including eligible studies’ PubMed Identifier (PMID) and other database-specific identifiers using Python . The list of articles was exported using a .csv file. A new script was created using C# to read the .csv file and prepare the data set for analysis. We conducted an iterative deserialization process to transform the original output generated from the APIs into human-readable text and identify different source data formats. We implemented exception functions to standardize the data set and stored it in a Structure Query Language (SQL) relational database for transactional purposes. The data set was used to create an interactive data visualization tool in the form of a collaboration map, using Shiny, a web application framework for R developed by RStudio . Additional information regarding the collaboration map is available on GitHub .
We included primary and secondary studies published in English, French, or Portuguese between January 1, 2011, and December 31, 2022, addressing oral health–related research having individuals, groups, or populations as their unit of analysis. We excluded in vitro and in vivo studies focusing on cells, biomarkers, or animals. Eligible studies included clinical trials, cohort studies, case-control studies, cross-sectional studies, ecological studies, systematic reviews (with or without meta-analysis), policy briefs, and evidence-informed guidelines. These reports either addressed a topic relevant to the WHO African region assessed using the title and study objective or were conducted in a country in the region.
We conducted a systematic search in the following electronic databases: PubMed, EMBASE, Epistemonikos (curating primary and secondary studies from the Cochrane Database of Systematic Reviews [CDSR], PubMed, EMBASE, CINAHL [Cumulative Index to Nursing and Allied Health Literature], PsycINFO, LILACS, Database of Abstracts of Reviews of Effects [DARE], the Campbell Collaboration online library, the JBI Database of Systematic Reviews and Implementation Reports, and the EPPI-Centre Evidence Library), Scopus, and African regional databases such as Regional African Index Medicus and the African Journal Online ( Appendix Table 2 ). We adapted the PubMed search strategy to the syntax and thesauri of other databases. In addition, to identify articles that might have been missed in the electronic searches, we conducted cross-citation searches in Google Scholar and Microsoft Academic, using each included study as the index reference. We reviewed the reference list of each included study and the abstracts of the International Association for Dental, Oral, and Craniofacial Research (IADR) General and Regional Sessions. We searched dissertations and theses in the ProQUEST database and for ongoing or recently completed systematic reviews in PROSPERO. The retrieved records were uploaded into Covidence to ensure an efficient workflow and remove overlapping records across databases.
We screened the retrieved records using title and abstract and, in the second stage, using full-text reports independently and in duplicate by 15 previously calibrated reviewers (κ = 0.91). We solved disagreements by consensus. If differences persisted, a third external reviewer acted as a referee. If information was missing to ascertain eligibility, we contacted the study’s corresponding author via e-mail.
Ten reviewers, independently and in duplicate, extracted data from the included studies using a piloted data extraction template. Reviewers were trained to ensure consistency in data extraction (κ = 0.81). Disagreements in the extracted data were solved by consensus or adjudication by an additional third reviewer when consensus was elusive. We extracted variables associated with the journal of publication (name, country, editorial focus on African-oriented research [determined by the journal description and editorial scope reported in the journal’s webpage, indices, and aggregators]), study authors (number of authors, country of the study, affiliations, collaborations within and outside the WHO African region, for African authors their position in the article and corresponding assignment), primary and secondary studies (title, year of publication, keywords, and number of citations in Google Scholar, funding source, declaration of conflict of interest). In addition, we classified the studies according to 4 taxonomies: 1. Research type as defined by WHO in their systematic approach for undertaking a research priority–setting exercise : a. Problem: research to measure the size of the health problem through epidemiology, estimating the disease burden, and other forms of data collection b. Cause: research to understand the causal agents, risk factors, and determinants of the health issue (this research may include, for instance, a study of infection cycles, vectors, the role of socioeconomic factors, environment, diet, and the interaction of multiple factors) c. Solution: research to develop new interventions, including therapeutics, devices, procedures, policy interventions, public health campaigns, and so on d. Implementation: research to translate new interventions into policy and practice and understand the barriers to delivering known interventions e. Evaluation: research to monitor and evaluate the effectiveness or health impact of an intervention or program 2. Research designs: clinical trials, cohort studies, case-control studies, cross-sectional studies, ecological studies, systematic reviews (with or without meta-analysis), policy briefs, and evidence-informed guidelines 3. Study disciplines as categorized by the American Dental Association (ADA): dental public health, general dentistry, pediatric dentistry, oral and maxillofacial surgery, oral and maxillofacial pathology, oral medicine, periodontics, orthodontics and dentofacial orthopedics, oral and maxillofacial radiology, endodontics, prosthodontics, basic science–related research (translational research), orofacial pain, and dental anesthesiology 4. Oral health topics and subcategories. In an iterative process, we extracted the study’s primary disease, condition, or specific research topic and subcategories that further described the topic by reviewing the title, keywords, and full-text article. We used descriptive statistics (frequencies and percentages) to summarize quantitative data. A country-level impact factor was determined by calculating the ratio of the total number of citations received by records from a particular country to the overall number of studies conducted within that country. Each record covering diverse topics was individually classified for each relevant theme. This approach was implemented to maintain transparency and methodological clarity in handling multifaceted records, allowing for a nuanced analysis of each record and topic. In addition, we conducted a collaboration mapping analysis. We created a database including all relevant study citation data (title, abstract, year of publication), authors’ names, academic or research institution (affiliation), city, and country of the institution. These data were obtained using publicly available Application Programming Interface (API) from the US National Library of Medicine and Elsevier. We retrieved records based on a query including eligible studies’ PubMed Identifier (PMID) and other database-specific identifiers using Python . The list of articles was exported using a .csv file. A new script was created using C# to read the .csv file and prepare the data set for analysis. We conducted an iterative deserialization process to transform the original output generated from the APIs into human-readable text and identify different source data formats. We implemented exception functions to standardize the data set and stored it in a Structure Query Language (SQL) relational database for transactional purposes. The data set was used to create an interactive data visualization tool in the form of a collaboration map, using Shiny, a web application framework for R developed by RStudio . Additional information regarding the collaboration map is available on GitHub .
We identified 24,014 records across databases. After removing duplicates, 18,992 remained for title and abstract screening. Out of 1,720 records selected for full-text screening, 1,379 proved eligible ( Appendix Fig. 1 and Appendix Table 3 ). The average number of articles published per year between 2011 and 2022 was 115 ± 11.7 (SD), with a trend line that suggests no significant fluctuations during the past decade ( Appendix Fig. 2 ). Type of Research, Designs, and Study Discipline According to the WHO research type classification ( n = 1,933 [more than 1 research type category was addressed in a single article]), more than half of the entire oral health research output of the region ( n = 1,018, 52.7%) focused on measuring the magnitude of a health problem, estimating the burden of disease (problem). One-third ( n = 584, 30.2%) of the research output aimed to identify causal agents, risk factors, and determinants of health (cause). Research addressing solution, implementation, and evaluation categories combined did not account for more than 17.1% ( n = 331) of the total research output . When classifying articles according to their design, more than three-quarters were cross-sectional studies ( n = 1,053, 76.4%), while cohort, case-control, and clinical trials accounted for 21.1% ( n = 292) of the total research output. No more than 1.4% of all articles focused on evidence synthesis (systematic reviews with or without meta-analysis and policy briefs). Implementation tools like evidence-informed practice guidelines were infrequently published ( n = 3, 0.2%) . Across disciplines, more than half of the entire research output concerned dental public health ( n = 503, 21.5%), general dentistry ( n = 473, 20.2%), and pediatric dentistry ( n = 280, 12.0%) ( Appendix Fig. 3 ). Research Efforts (Number of Investigators) per Oral Health Topic The classification of articles per oral health topic across investigators ( n = 5,440) indicated that approximately half ( n = 2,768, 50.9%) of the research efforts focused on dental caries (939 investigators, 17.3%), oral health status assessment (629 investigators, 11.6%), periodontal disease (410 investigators, 7.5%), oral mucosa lesions (399 investigators, 7.3%), and oral health education (391 investigators, 7.2%). Noma was addressed in 6 studies in our sample ( and Appendix Table 4 ). In addition, we identified 294 unique articles (27.2%) that focused on public health–related topics (e.g., oral health status, oral health education, oral health systems, and oral health needs) conducted by 1,296 investigators. For this topic, most research efforts were dedicated to the following subcategories: assessment of oral health situation (epidemiological studies) (524 investigators, 40.4%) and health care professionals’ education (173 investigators, 13.3%). Relevant areas to inform policy and system-level interventions, including community outreach and promotion (84 investigators, 6.5%), oral health services (79 investigators, 6.1%), oral health care access (58 investigators, 4.5%), and workforce studies (26 investigators, 2.0%), did not individually reach beyond 6.5% of total research efforts ( Appendix Fig. 4 ). Journals Publishing African Oral Health Research Articles about oral health research in the WHO African region were published in African and non-African dental journals. One-third of the articles ( n = 497, 36.04%) were published in 75 journals with an editorial focus on Africa. Of the 20 journals with the most publications, 12 (60%) had an African editorial scope. The journals with the most publications were BMC Oral Health , the Nigerian Journal of Clinical Practice , and the Pan African Medical Journal ( Appendix Table 5 ). Research Efforts per Country and across Collaborations Nigeria and South Africa had the highest participation in oral health research studies (i.e., where the study was conducted) in the WHO African region ( n = 912, 55.3%). The individual research output contribution of the remaining countries ranged between 1.1% and 4.6%. The most prolific research partnerships included authors from multiple countries in the region ( n = 1,195, 57.7%), while national collaborations represented 18.2% ( n = 2,070) of the total partnerships. A total of 73 articles addressed research topics pertaining to the WHO African region and were neither published by researchers affiliated with any entity within the WHO African region nor involved collaboration with African researchers ( , Collaboration map [ https://tmdapp.shinyapps.io/dental-map-affiliations/ ]).
According to the WHO research type classification ( n = 1,933 [more than 1 research type category was addressed in a single article]), more than half of the entire oral health research output of the region ( n = 1,018, 52.7%) focused on measuring the magnitude of a health problem, estimating the burden of disease (problem). One-third ( n = 584, 30.2%) of the research output aimed to identify causal agents, risk factors, and determinants of health (cause). Research addressing solution, implementation, and evaluation categories combined did not account for more than 17.1% ( n = 331) of the total research output . When classifying articles according to their design, more than three-quarters were cross-sectional studies ( n = 1,053, 76.4%), while cohort, case-control, and clinical trials accounted for 21.1% ( n = 292) of the total research output. No more than 1.4% of all articles focused on evidence synthesis (systematic reviews with or without meta-analysis and policy briefs). Implementation tools like evidence-informed practice guidelines were infrequently published ( n = 3, 0.2%) . Across disciplines, more than half of the entire research output concerned dental public health ( n = 503, 21.5%), general dentistry ( n = 473, 20.2%), and pediatric dentistry ( n = 280, 12.0%) ( Appendix Fig. 3 ).
The classification of articles per oral health topic across investigators ( n = 5,440) indicated that approximately half ( n = 2,768, 50.9%) of the research efforts focused on dental caries (939 investigators, 17.3%), oral health status assessment (629 investigators, 11.6%), periodontal disease (410 investigators, 7.5%), oral mucosa lesions (399 investigators, 7.3%), and oral health education (391 investigators, 7.2%). Noma was addressed in 6 studies in our sample ( and Appendix Table 4 ). In addition, we identified 294 unique articles (27.2%) that focused on public health–related topics (e.g., oral health status, oral health education, oral health systems, and oral health needs) conducted by 1,296 investigators. For this topic, most research efforts were dedicated to the following subcategories: assessment of oral health situation (epidemiological studies) (524 investigators, 40.4%) and health care professionals’ education (173 investigators, 13.3%). Relevant areas to inform policy and system-level interventions, including community outreach and promotion (84 investigators, 6.5%), oral health services (79 investigators, 6.1%), oral health care access (58 investigators, 4.5%), and workforce studies (26 investigators, 2.0%), did not individually reach beyond 6.5% of total research efforts ( Appendix Fig. 4 ).
Articles about oral health research in the WHO African region were published in African and non-African dental journals. One-third of the articles ( n = 497, 36.04%) were published in 75 journals with an editorial focus on Africa. Of the 20 journals with the most publications, 12 (60%) had an African editorial scope. The journals with the most publications were BMC Oral Health , the Nigerian Journal of Clinical Practice , and the Pan African Medical Journal ( Appendix Table 5 ).
Nigeria and South Africa had the highest participation in oral health research studies (i.e., where the study was conducted) in the WHO African region ( n = 912, 55.3%). The individual research output contribution of the remaining countries ranged between 1.1% and 4.6%. The most prolific research partnerships included authors from multiple countries in the region ( n = 1,195, 57.7%), while national collaborations represented 18.2% ( n = 2,070) of the total partnerships. A total of 73 articles addressed research topics pertaining to the WHO African region and were neither published by researchers affiliated with any entity within the WHO African region nor involved collaboration with African researchers ( , Collaboration map [ https://tmdapp.shinyapps.io/dental-map-affiliations/ ]).
This study identified the oral health research scope and output in the WHO African region between 2011 and 2022. We estimated that nearly 50% of scientific production originated from Nigeria. Although having a very high rate of production of research publications, Nigeria also has, by far, the most dental schools of any country in the WHO African region. In addition, collaboration networks among authors and institutions within the region and globally were predominantly linked to Nigeria and South Africa, which could be explained, in part, by the number of dental schools in these countries. A significant portion of publications are found in journals without a specific focus on the WHO African region. Most studies were cross-sectional and focused primarily on dental public health, general dentistry, and pediatric dentistry. Most publications from the region centered on the most prevalent oral diseases, including dental caries, periodontal diseases, other oral cavity lesions, and dental and oral cavity anomalies. On the other hand, noma, a disease associated with high mortality and currently a public health priority in several countries in the region , was only addressed in 6 studies in our sample. The large disparity in dental research output among countries in the region was consistent with a previous study that indicated that among all African countries, South Africa, Egypt, and Nigeria accounted for 60% of the articles indexed in PubMed between 1996 and 2005 . A difference in our study compared to theirs is that we considered the regional definition by WHO, which excludes Egypt. A more recent overview evaluated oral health publications across different African subregions between 2005 and 2010 and concluded that of 935 publications, encompassing all types of articles except editorials and letters, around 68% originated from Nigeria and South Africa . The lack of scientific production and dissemination in low-income African countries is troubling, considering their high disease burdens, pronounced health inequities, and pressing need for scientific data . Research published in European or North American journals often excludes topics relevant to low-income African countries . When considering the research type according to WHO’s research prioritization criteria, approximately 53% were related to the “problem” category and 30% to the “cause” category. In our study, the number of publications addressing “solution,” “evaluation,” and “implementation” categories was scant, highlighting the need to redirect efforts toward generating evidence on the effectiveness, safety, and impact of oral health interventions. Our findings differ from a previous WHO report on general health research globally, which showed that out of 2,145 publications between 2002 and 2017, the highest proportion of the 5 categories (cause, problem, solution, evaluation, and implementation) corresponded to implementation (43%) . Previous studies have emphasized the need to incorporate aspects related to dissemination and implementation into research agendas in clinical, community, and policy environments in dentistry . In our review, approximately 76% of the included articles were cross-sectional, aligning with research aimed at describing a “problem,” measuring health issues extent through epidemiology, estimating disease burden, and other data collection forms . A bibliometric study that analyzed articles published in PubMed-indexed journals between 2008 and 2018 in the WHO African region also identified a prevalence of cross-sectional designs in half of the articles . Our results also showed that oral health research in the region primarily originates from university-based dental schools. This poses a challenge for a region where 11 countries lack dental schools . While these countries might have medical or public health schools partially addressing oral health issues, the absence of dental schools can still pose a substantial challenge in fostering dedicated dental research . A recent study suggested that none of the 3,300 most-cited articles in dentistry originated from countries in the WHO Africa region . The number of publications in African journals presents an opportunity to reach local audiences on regional interest topics. A factor contributing to the limited visibility of local publications is that these journals are not included in large databases like the US National Library of Medicine (MEDLINE) and the region lacks a country- or regional-based knowledge translation platform to share these local publications . Local and global evidence are integral to the knowledge base informing oral health policies . The limited visibility problem is not solely related to the source of scientific information and its dissemination but also the ability to access such information. While Internet users in Africa have increased, the region still has the lowest Internet penetration rates globally, with highly unequal distribution between the WHO African region and northern African countries . One strength of this review was the implementation of a comprehensive search strategy across major health databases, including African regional databases such as Regional African Index Medicus and the African Journal Online. This approach allowed us to identify scientific articles published in journals from the WHO Africa region, not only in English but also in French and Portuguese, the working languages of the WHO Regional Committee in the African region. Additionally, including multilingual articles enhances the representation of diverse research outputs. Another strength was identifying countries with the highest production of scientific publications, pinpointing collaborations with authors outside the WHO Africa region, and identifying prevalent research topics. Our review also has some limitations. Age groups other than those typically classified under pediatric dentistry or special subpopulations were not specifically categorized. This issue may limit the generalizability of the findings to specific age ranges within populations. In addition, we cannot rule out the possible exclusion of nonindexed predatory journals known to impact the scientific production of the region. Although we have included scientific articles written in the working languages of the WHO Regional Committee in the African region, it is possible that this choice may have inadvertently omitted research from countries and journals utilizing languages other than the aforementioned. Another limitation of our study is the absence of information regarding the specific expertise of the authors of the reviewed papers. Although not a standard in scoping reviews , our review did not include an assessment of the methodological quality of the included studies, which may limit the ability to make further inferences about investigators’ training and research design competence. The findings of this study highlight the necessity of strengthening oral health research in the WHO African region. It emphasizes the importance of establishing core evidence-producing hubs for decision-making originating from local universities and other regional institutions. Such an approach ensures that decisions remain rooted in local expertise, reducing reliance on external collaborations that may risk scientific colonization. Research hubs must be led and driven by African investigators. This is not a call for deprioritizing international partnerships but an opportunity for oral health research organizations in the WHO African region to define their destiny . The recent WHO Oral Health Action Plan includes, as one of its strategic objectives, the continuous development and updating of context-specific research focusing on public health aspects related to oral health . The lack of funding and infrastructure and insufficient collaboration among local researchers are possible explanations for our results . Opportunities exist in the region to enhance the capacity for oral health research. For example, “Research for Health: A Strategy for the African Region 2016–2025” exists, which supports Member States in fostering the development of a functional national health research system by establishing research for health governance, improving building and sustenance of human, physical, and institutional research capacities; strengthening production and use of research to enhance the performance of health systems; establishing sustainable research for health financing; and establishing mechanisms for tracking health research investments. In addition, a research network exists, such as the Consortium for Advanced Research Training in Africa (CARTA), a program designed to strengthen research infrastructure and capacity at African universities. CARTA aims to develop local research capacity to comprehend the determinants of population health and effectively intervene to enhance health outcomes . This places science in the service of public health functions . The WHO political guidance document on oral health highlights the importance of research to create and continuously update a local body of research evidence to inform oral health policies in the WHO African region . This essential body of evidence is noticeably lacking in most countries. Strengthening research capacity and training in population and public health in the WHO African region can only realize its full potential when bolstered by robust governmental and institutional backing. This support is crucial to create and sustain research hubs of excellence to conduct state-of-the-art, locally relevant African-led research initiatives. The results of this research should be disseminated in locally curated knowledge translation platforms that are easily accessible to stakeholders.
Our results suggest that the oral health research output of the WHO African region has remained constant in the past 10 y, and it has focused on the conduct of cross-sectional studies describing the magnitude of oral health issues. Other primary study designs are infrequently conducted. This paucity of research evidence is also apparent in secondary (evidence synthesis) studies, including systematic reviews and policy briefs, essential bricks to support the creation of national and regional evidence-informed clinical practice guidelines and oral health policies. Research studies informing the effect of interventions to create locally tailored solutions to oral health issues and the assessment of the implementation and evaluation of those solutions remain understudied. Significant variation in research capabilities across countries represents an opportunity for oral health researchers from academic entities in the WHO African area to engage in dialogues about establishing intra- and intercountry partnerships. These collaborations can serve as the knowledge engine to inform and define African oral health research agendas, producing the necessary direct evidence to accelerate the creation, dissemination, implementation, monitoring, and evaluation of oral health policies.
T.F. Labarca, D. Ortuño, contributed to conception, protocol writing, and research design, created screening, data extraction forms, and instructions, contributed to citation screening and data extraction, arbiter for defining final eligibility, analyzed data, designed and created tables and figures, produced the first draft of the manuscript, reviewed and provided input into the manuscript. L. Neira, F.J. Bravo, C.R. Cantarutti, M. Dallaserra, A. Gatarayiha, R.J. Kulchar, C.C. Martins-Pfeifer, L. Pilcher, S. Pahlke, C. Pirela, A. Song, J.P. Vargas, C. Véliz, F. Verdugo-Paiva, P. Vergara, V. Zaffiri, J. Zuñiga, N. Olivares, contributed to citation screening and data extraction, reviewed and provided input into the manuscript. G. Andrade, M. Res, contributed to conception, protocol writing, and research design, contributed to citation screening and data extraction, reviewed and provided input into the manuscript. J. Karajgikar, X. Liu, designed and created the data visualization tool for the collaboration map, reviewed and provided input into the manuscript. J.M. Sanchez, retrieved citations and affiliations to inform the collaboration map using APIs, contributed to citation screening and data extraction, reviewed and provided input into the manuscript. O. Urquhart, contributed to conception, protocol writing, and research design, created screening, data extraction forms, and instructions, analyzed data, designed and created tables and figures, reviewed and provided input into the manuscript. Y. Makino, contributed to conception, protocol writing, and research design, analyzed data, designed and created tables and figures, reviewed and provided input into the manuscript. M. Glick, A. Carrasco-Labra, contributed to conception, protocol writing, and research design, created screening, data extraction forms, and instructions, analyzed data, designed and created tables and figures, produced the first draft of the manuscript, reviewed and provided input into the manuscript. All authors gave final approval and agreed to be accountable for all aspects of the work.
sj-docx-1-jdr-10.1177_00220345241272024 – Supplemental material for Oral Health Research in the WHO African Region between 2011 and 2022: A Scoping Review Supplemental material, sj-docx-1-jdr-10.1177_00220345241272024 for Oral Health Research in the WHO African Region between 2011 and 2022: A Scoping Review by T.F. Labarca, D. Ortuño, L. Neira, G. Andrade, F.J. Bravo, C.R. Cantarutti, M. Dallaserra, A. Gatarayiha, J. Karajgikar, R.J. Kulchar, X. Liu, C.C. Martins-Pfeifer, N. Olivares, L. Pilcher, S. Pahlke, C. Pirela, J.M. Sanchez, A. Song, O. Urquhart, J.P. Vargas, C. Véliz, F. Verdugo-Paiva, P. Vergara, V. Zaffiri, J. Zuñiga, Y. Makino, M. Glick and A. Carrasco-Labra in Journal of Dental Research
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Advances for pediatricians in 2022: allergy, anesthesiology, cardiology, dermatology, endocrinology, gastroenterology, genetics, global health, infectious diseases, metabolism, neonatology, neurology, oncology, pulmonology | eefdf682-994a-429a-adff-b772e9b6f2ea | 10485969 | Pediatrics[mh] | The Italian Journal of Pediatrics in 2022 has published a high number of papers that can considerably ameliorate the management of several childhood illness. Among the most accessed papers of the past year, we present those thought to potentially have a significant impact. The selected articles highlight progresses in several fields: allergy, anesthesiology, cardiology, dermatology, endocrinology, gastroenterology, genetics, global health, metabolism, neonatology, neurology, oncology, pulmonology. Findings have been discussed and context of the studies has been provided with the aim of identifying changes in the clinical approach to the diseases. Allergy. 1‑ Food allergy; 2‑ Atopic eczema; 3‑ Asthma; 4- Hypersensitivity pneumonitis 1- Food allergy Food allergy is one of the most challenging field in pediatrics . It is common in developed countries resulting in an important problem for quality of life. Food allergy care has gone a long way. The Italian Society for Pediatric Gastroenterology Hepatology and Nutrition and the Italian Society for Pediatric Allergy and Immunology joint position paper on the management of food allergy including activities and responsibilities of general practitioners, emergency departments and tertiary centers, such as food challenge , has been published last year. The document provided information that will decrease delayed diagnosis, costs, and hazards for children with food allergy. 2- Atopic eczema Atopic eczema has several clinical phenotypes that needs a multidisciplinary approach . The review by Galli et al. reports current knowledge on treatment for moderate to severe cases. It has been highlighted the use of traditional treatments including trigger avoidance, emollients, bath, topical steroids, calcineurin inhibitors, immunosuppressive agents, anti-H1 antihistamines, phototherapy, education. Furthermore, it illustrates novel systemic drugs that acts on specific molecules involved in the immune mechanism that driven the pathogenetic pathways of AD, including biologics, janus kinase inhibitors and allergen specific immunotherapy . An algorithm for clinical use of topical and systemic treatments is also provided. 3- Asthma Chronic airway inflammation leads to occurrence of symptoms in asthmatics . Inhaled drugs are useful for asthma treatment since are effective and systemic adverse reactions are rare. In children with asthma, an inhaler both dry powder and pressurised metered-dose with and without the use of spacer should be used for delivering drugs to the lungs. So, a correct inhaler technique is one of the most important step for controlling asthma in children. McCrossan et al. review data from several studies and describe the pros and cons of educational methods used to improve inhaler technique. They underline that researches in this field are warranted to identify the best outcome measure for inhaler technique in children. Furthermore, trials comparing interventions for teaching inhaler technique at different ages are lacking. 4- Hypersensitivity pneumonitis Mastrorilli et al. address the ‘hot topic’ of hypersensitivity pneumonitis in children . They underline that on the basis of a suggestive history, the diagnosis relies on respiratory signs and symptoms, pulmonary function tests, characteristic HRCT nodular opacities, serum specific IgG levels, response to elimination of eliciting factor. Of note, aspergillus species are commonly considered the cause. However, other fungi such as Schizophyllum commune can be involved . Bronchoscopy with bronchoalveolar lavage, and the provocation test may be helpful. Besides supportive treatment, the resolution generally takes place with trigger avoidance and systemic corticosteroids. Hydroxychloroquine, azithromycin, immunosuppressants, rituximab, nintedanib might be considered. Anesthesiology. 1‑ Sedation 1- Sedation In infant and children, the optimal treatment and management of diseases sometimes require high quality diagnostic procedures for which a prolonged inactive state are necessary and sedation needed. Some of the desire characteristics for the sedation regimen are rapid onset of action, predictable duration, rapid cessation of effects, few side effects and no need of rescue drug. Cossovel et al. interestingly investigate the difference between two approach, combination of intranasal ketamine and intranasal dexmedetomidine compare with the the combination intranasal dexmedetomidine and oral midazolam. Compared with other studies in literature, the results look controversial, probably because of limits and confounding factors in the design of the studies . Prospective studies are needed. Cardiology. 1‑ Cardiovascular risk 1- Cardiovascular risk Noncommunicable diseases are the current challenge for the sustainability of the health system in industrialized countries and among them cardiovascular diseases remain the main issue, being the leading cause of death, handicap, and health expenditure . Atherosclerosis the matrix lesion, begins and can be detected in infancy, and its severity, extent and progression in childhood is related to exposure to risks factors such as dyslipidemia, hypertension, chronic kidney diseases, congenital heart and acquired heart diseases, cancer . Several studies have shown an association between exposure to cardiovascular risk factor in childhood and subclinical atherosclerosis in adulthood but to date there is still limited evidence a lack of direct link between these factors with adult clinical cardiovascular diseases . Dermatology. 1‑ Hemangioma; 2‑ Epidermolysis bullosa; 3‑ Congenital ichthyosis 1- Hemangioma Infantile hemangioma are benign tumors, quickly growing that are commonly located on the skin, but may also be in solid organ as brain or liver. They may go through gradual and spontaneous involution, but in some cases, depending on their location, they can lead to severe complications, ulceration, bleeding organ dysfunction or transient to permanent deformity which significantly impact patient and family quality of life. Propranolol has changed the history and the management of infantile hemangiomas , because of facility of administration and high success rate, becoming the first line treatment before glucocorticoids and surgery . Although the mechanism underlying its effect is still unclear and controversies about side effects and potential safety issues . Early and prompt diagnosis and treatment is essential . 2- Epidermolysis bullosa Epidermolysis bullosa is a chronic genodermatosis with different phenotypes and severity. Retrosi et al. reported on treatment in 3 groups of patients: < 5 years, > 5–12 years and > 12–18 years. In the first group, the family should change lifestyle and burden of caring on quality of life is high. alliance between the health professional and parents can facilitate relationship between all family members . In the second group, children have to be educated in accepting and understanding the disease and involved in the management to diminish complications. In the third group, adolescents have a deep knowledge of the disease. Denial of the disease and low adherence to treatment are common . Authors found that 27 (13%) out of 215 patients died. Eighteen (8%) patients died because of sepsis and/or respiratory failure during the first months of life. Nine (5%) died for squamous cell carcinoma in adulthood . 3- Congenital ichthyosis Congenital ichthyosis is a rare disease. Mutations of more than 50 genes have been found. Genotype is often correlated with distinct form of the disease . Serra et al. found new variants of the ABCA12, KRT1 and ST14 genes. Mutations of ABCA12 gene is significantly associated with neonatal mortality . Mutations of KRT1 gene can be hereditary or occur de novo. The epithelial barrier disruption is associated with infections and dyselectrolytemia. Mutations of ST14 gene are not clearly associated with a specific clinical picture. Authors highlight the importance of the molecular characterization to make an appropriate treatment, define the prognosis and the genetic counselling . Endocrinology. 1- Obesity; 2- Puberty; 3- Type 1 diabetes; 4- Hypothyroidism 1- Obesity Obesity is a chronic diseases with the greatest impact on public health with an increasing incidence over time. In adolescents, the reasons for obesity are various and among the most significant we might mention the wide prevalence of the obesity on a world scale with an increase also in low-income countries, the many complications that excess weight can cause at the level of various organs and systems and the high percentage of obese adolescents becoming obese adults. On the other hand, the therapeutic approach used in the last years that is based on the (difficult) attempt to change the lifestyle of obese adolescents has not yet given satisfactory results, especially in the long term. For this reason Nicolucci and Maffeis dedicate an in-depth review both to the anti-obesity medications available (with particular attention to the glucagon-like peptide-1 analog) and to the bariatric surgery. The results obtained with the combination of lifestyle changes and new drugs seem encouraging but there is a need to know the long-term results on the efficacy in determining and maintaining weight loss, on the action on possible complications (cardiovascular), on the quality of life and the long term side effects . Even with regards to bariatric surgery (currently, sleeve gastrectomy and “Rouxen-Y gastric bypass are two of the most popular bariatric procedures the results seem promising . Bariatric surgery is considered very selectively for example by the American Society of Metabolic and Bariatric Surgery in adolescents with body mass index (BMI) ≥ 35 kg/m2 with major comorbidities or with a BMI ≥ 40 kg/m2 with minor comorbidities, However, as discussed by Martinelli et al. in a recent review “the use of bariatric surgery in adolescent patients is still limited, with significant disparities among countries. Reasons include ethical concerns raised by performing an irreversible and invasive procedure in adolescence, with potential life-long alterations” . In conclusion, on the one hand there is an increasingly general agreement in treating adolescent obesity by deepening knowledge on the use of new generations of drugs and/or bariatric surgery combined with behavioral therapies that help change lifestyles and on the other hand, the undoubted difficulties of this path lead us to insist on programs for the global prevention of obesity. In childhood, drug dosing is often based on body weight. However pharmacokinetic is influenced by body weight . This issue should be considered in obese children, but it has not been deeply studied . So far, higher doses are required only for trimethoprim/sulfamethoxazole , among antibiotics, midazolam, inhaled corticosteroids. Data on dosing of low molecular weight heparins and vitamin D are unclear . So, further investigation on drug dosing are warranted in obese children. 2- Puberty Much attention has been given to early and delayed puberty. A worldwide growing decrease of puberty timing has been observed. Key features in the pathogenesis are nutrition in pregnancy, birth weight, dietary habits, physical activity, psychological factors, exposure to electromagnetic fields, endocrine-disruptors . Ferrari et al. showed in 577 girls that increased Z-score change from birth weight and higher BMI may predict earlier age at menarche and at thelarche. This underlines the role of modifiable factors in the onset of puberty. Among delayed puberty causes, constitutional delay of growth and puberty, a variant of normal growth is frequent. It may induce psychological difficulties, poor school performance and perhaps lower final height . So, therapeutic interventions have received interest in recent years. In adolescent > 14 years of age with psychological problems, a course of estradiol and progesterone in females or testosterone in males can be given. Importantly, despite clinical interest, the benefit of growth hormone on final height is uncertain . 3- Type 1 diabetes Type 1 diabetes is a disease that is heterogeneously growing in prevalence. Several studies have examined annual incidence that greatly varies from 0.1 to 100,000 person in China to 62.3 per 100,000 person in Scandinavia and from 19 to 45 per 100,000 person-years in different Italian regions. Passanisi et al. found an incidence of 20.6/100,000 person/years with an increase of 43% between 2019 and 2021 during Coronavirus Disease (COVID-19) pandemics in Calabria region, Italy. This supports the view that Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection may predispose to a diabetes onset . They also found an increase of children with severe ketoacidosis that can be justified by shutting of many health services during the pandemics. 4- Hypothyroidism Menorrhagia is frequent after menarche onset. It is a challenging issue that often depend on anovulatory cycles due to the immaturity of the hypothalamic-pituitary-ovarian axis , but it can also be related to coagulopathy, pregnancy, medications, endocrine disorders, trauma, infection, cancer, structural pathologies, gastrointestinal bleeding . Barbero et al. delineated a girl with menorrhagia associated with short stature, bradycardia, dry skin, thinning hair. Hypothyroidism was confirmed by ultrasound showing an expanded thyroid with non-homogeneous hypoechogenic structure and laboratory tests showing anemia, high thyrotropin releasing hormone level, low free thyroxin, high serum anti-thyroid peroxidase antibodies and antithyroglobulin antibodies levels. Levothyroxine treatment was associated with remission of symptoms and regulation of laboratory parameters. This case highlights that follow up of the pubertal development is of particular importance for timely identification of underlying disorders. Gastroenterology. 1‑ Diet; 2‑ Cyclic vomiting syndrome 1- Diet The effect of diet in the prevention and treatment of several conditions with gastrointestinal symptoms attracts constant attention. Pulvirenti et al. summarized studies on diseases that are cured by eliminating the offending food or need an appropriate dietary management to prevent malnutrition. A causal role of food has been found in food allergy, including eosinophil esophagitis and regurgitation. In infancy, early introduction into the diet of foods such as peanuts, seems to reduce the likelihood of developing food allergy compared with delayed introduction . In cystic fibrosis, the caloric intake should be 10–15% higher than in healthy children because of pancreatic insufficiency. So, diet should be supplemented with fat for energy requirements and pancreatic enzyme should be given . Breast-feeding and diet rich in fruit, vegetables and n-3 fatty acids are associated with a lower risk of developing inflammatory bowel disease. A specific diet is necessary for inflammatory bowel disease because of the risk of malnutrition due to low caloric intake, enteropathy, metabolic conditions, or steroid treatment. In active Crohn’s disease, enteral or parental nutrition with polymeric formulas, is the first option for 6–8 weeks . Short-bowel syndrome requires parental nutrition and gradually increase of enteral feeding that stimulates gastrointestinal functions. Breast milk or polymeric formulas are recommended in infants, oral or partially enteral solid foods in children. 2- Cyclic vomiting syndrome The Italian Society of Pediatric Gastroenterology, Hepatology and Nutrition and the Italian Society of Pediatric Neurology provided a timely survey on diagnosis and treatment of cyclic vomiting syndrome in 51 tertiary centers . They found a significant difference in diagnostic criteria between centers. Foods as triggers were more commonly identified by gastroenterologists and neuro-gastroenterologists than neurologist. Gastroenterologists more often diagnosed gastrointestinal diseases and checked ammonia and lactic acid levels. Besides supportive treatment, the most common medication given during vomiting, ondansetron followed by proton pump inhibitors, sedative, anti-H2 antihistamines, non steroidal antiinflammatories, steroids . Prophylactic treatment widely varied among centers. In about 50% of cases cyproheptadine was used, in 25% pizotifen, in 25% amitriptyline, in 15% mitochondrial supplements, in 10% anticonvulsants. Other drugs were aprepitant, propranolol, flunarizine, 5-hydroxytryptophan, proton pump inhibitors, paracetamol, magnesium supplementation. Probiotics have not been used . Overall, there is a need for a consensus to standardize the management of cyclic vomiting syndrome. Genetics. 1- Cat Eye syndrome; 2- Deletion of chromosome; 3- Hao-Fountain syndrome 1- Cat Eye syndrome Cat Eye syndrome is due to duplication and inversion of part of chromosome 22 . It is characterized by iris coloboma, anal atresia and ear anomalies in 40% of patients and it can have multiple malformations . Serra et al. described an infant with cat eye syndrome associated with persistent hypoglycemia and subsequent cholestasis, abnormal midline structures, including aplasia of the anterior pituitary gland, abnormal stalk, and ectopic neurohypophysis with hypopituitarism with cortisol, thyroxine, growth hormone deficits, corpus callosum hypoplasia. This report emphasizes that clinical presentation of cat eye syndrome is variable . So, the presence of additional malformations and/or abnormalities should always be considered. 2- Deletion of chromosome Contiguous gene syndromes are the result of a loss of multiple neighboring genes from a particular chromosomal segment. Contiguous gene syndromes have been identified in patients with various clinical features such as intellectual disability, developmental delay, and congenital anomalies . Serra et al. report on a female newborn with a 1p31.3p22.2 deletion of 20.7 Mb containing about ninety genes, inherited from the healthy mother with a smaller deletion (2.6 Mb) within the same centromeric region. Clinical phenotype included craniosynostosis, facial dysmorphisms with bilateral microphthalmia and coloboma, cleft palate, and a severe global developmental and growth delay . In this report, in view of a better characterization of a genomic and phenotypic profile, the authors paid attention to about 20 out of the involved genes, considered loss of function intolerant according to their haploinsuficiency score . A female term newborn with cardio-facio-cutaneous syndrome, a pathology belonging to RASopathies , a group of conditions caused by germline mutations in genes encoding components or regulators of the rat sarcoma/mitogen-activated protein kinase (RAS/MAPK) pathway was observed . Fetal macrosomia and a prenatal diagnosis of omphalocele suggested an array comparative genomic hybridization by amniocentesis that identified a 19p13.3 deletion including the MAP 2 K2 gene. Surgical correction of omphalocele took place on the second day of life. After discharge, at 1 month of age, she was readmitted for repeated episodes of vomiting, subtending a readily treated hypertrophic pyloric stenosis. The authors stress the need for integrated and individualized follow-up in all subjects with genetic syndromes, potentially carriers of abnormalities not evidentiable at birth . 3- Hao-Fountain syndrome Zampieri et al. report a case of Hao-Fountain syndrome (HAFOUS) a haploinsufficient condition caused by variants in the Ubiquitin-Specific Peptidase 7 gene (USP7), located on chromosome 16p13.2. USP7 is the largest family of deubiquitinating enzymes. Recent studies have shown that USP7 , as well as characterizing a complex neurodevelopmental disorder, plays a vital role in the regulation of various physiological and pathological processes related to endocrine complications, predisposition to autoimmune diseases, immunological deficit and a wide variety of cancers. The patient was a 15-year-old female patient with a complex neurodevelopmental disorder involving both cognitive and neurologic areas in which whole exome sequencing diagnosed a large de novo heterozygous deletions affecting USP7. The description in this patient of isolated tubal torsion expands the HAFOUS spectrum phenotype . Global Health. 1- Bullying; 2- Mobile phone; 3- Child abuse; 4- Burns; 5- Under-nutrition 1- Bullying A research carried out in Palermo, Italy, investigated the phenomenon of bullying , and includes an analysis of the data on bullying reported in the scientific literature . The authors have administered, to 22,455 school-aged children, attending one of the 58 secondary first-grade schools of Palermo during the school year 2017/18, a questionnaire of 30 items investigating the main areas related to bullying, such as physical, verbal and indirect bullying, observers of bullying, resiliency, and prosociality. Results showed an increase of bullying, reported in 2011 to be present in about 14% of young people, and involving up to 66% of young people in the area of verbal bullying. Bullying resulted more frequent in higher school classes, specifically in students attending third level classes in schools with lower socioeconomic index. The authors conclude that interventions and preventive measures in public health programs should be implemented to stem this phenomenon . 2- Mobile phone dependence Mobile phone dependence, considered a counterproductive use of mobile phone for interpersonal communication, information acquisition, self-expression, and leisure and entertainment, is a scourge of modern society that affects large segments of the youth population . In this report the authors analyse the correlations between self-core evaluation (SCE) (a personality trait that encompasses an individual’s subconscious, fundamental evaluations about themselves, their own abilities, and their own control), mental state and cell phone addiction in a sample of high school students . The results of the analysis, since SCE had a significant negative correlation with mobile phone dependence, show how SCE plays an important mediating role between mental health and mobile phone dependence . The authors conclude that improving high students’ SCE may be beneficial to improve the health status and reduce mobile phones dependence. 3- Child abuse Offidani et al. describe the process that led to the editing of the indicators of child abuse and the codification of three clinical pathways to apply in case of suspected child abuse at the Bambino Gesù Children’s Hospital . Field work has identified three areas of assessment that constitute a new screening tool (ST) created in 2009 and used in clinical practice from 2010. The fourteen items included in the ST have been grouped in three clusters: anamnestic declarations or incongruences, carelessness/neglect and evident lesions at physical examination. All items of the ST are reported in the text. Adopting the ST from 2010 to 2020, the authors report improvement in diagnostic accuracy resulting in reduction of under-diagnosed cases and confirm that females are more likely to experience sexual abuse, while males are more likely to experience physical abuse. It would be useful to share these indicators with non-paediatric hospitals, that do not have the same experience in recognizing cases of child abuse . 4- Burns Tiruneh et al. conducted this institutional-based cross-sectional study on children admitted for burns to South Gondar zone hospitals from 2015 to 2019. They used for data collection a checklist including sociodemographic characteristics, as nutritional status, comorbidity, availability of health insurance, medical history such as the place of the accident, the mode of occurrence, the timing of hospitalization, the degree of burn, the duration of hospitalization and the therapeutic measures taken . The mortality among burn victim children (8,5%) was higher than most of the studies conducted all over the world and similar to data from East Africa and Tanzania. No medical insurance, being malnourished, burnt by electrical and flame burn, having total body surface area burnt greater than 20%, and having poor clinical condition at admission increased mortality by four times compared to a good clinical condition. These results may suggest priority actions to reduce mortality and worst outcomes in children . 5- Undernutrition Undernutrition is a major public health issue especially in some countries of Africa and Asia . Inadequate feeding practices is a key factor associated with under-nutrition in children younger than 5 years of age . Bidira et al. educated caregivers of Ethiopian preschool children on healthy diet, nutrition, hygiene for 9 months. They showed that wasting and underweight significantly improved in the intervention group compared with controls. These findings highlight that the strategy of community-based nutritional education is effective in ameliorating nutritional status of young children . Infectious diseases. 1- Staphylococcus aureus; 2- Epstein-Barr virus 1- Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) can cause severe and highly prevalent diseases in the pediatric population. During the COVID-19 pandemic an increased rate of MRSA infections has been described in adults. In a study conducted in the period 2017–2020, which included the pandemic period, a similar epidemiologic trend in patients aged < 18 years, has not been confirmed . Moreover, the efficacy against MRSA of several antibiotics was tested and linezolid and vancomycin were identified as the only antibiotics to which MRSA did not develop resistance. Of particular interest to the reader are the in-depth analysis of the factors related to methicillin resistance, as well as the antimicrobial susceptibility pattern according to methicillin resistance . The authors conclude that surveillance of antimicrobial resistance is essential to improve infection control, antibiotic prescriptions, and preventions policies . 2- Epstein-Barr virus Accomando et al. report a child with symptomatic pancreatitis associated to Epstein-Barr virus infection. In this case, characterized by an enlarged pancreas visualized by abdominal ultrasound, the etiological diagnosis was only achieved by a positive serology for the presence of Epstein-Barr virus VCA IgM and IgG. The review of the few cases described in the literature draws attention to the importance of differential diagnosis that include specific serological research even in the absence of the classic clinical and haematological characteristics of Epstein-Barr virus infection . Generally, Epstein-Barr virus-associated acute pancreatitis is characterized by a favorable prognosis, with a spontaneous resolution . Metabolism. 1- Familial hypercholesterolemia; 2- Pompe disease 1- Familial hypercholesterolemia Familial hypercholesterolemia is the most common hereditary disorder of lipid metabolism causing life-long accumulation of low-density lipoprotein cholesterol . In this article, Banderali et al. highlight the main diagnostic strategies to identify this condition that, if left untreated, can lead to atherosclerosis and possible premature coronary heart disease and other vascular. Early detection of the condition is important to prevent complications and improve life expectancy. Cardiovascular events are rare among children and adolescents. Thus, identification of suggestive symptoms and family history is crucial. Some of the most typical signs of FH are: corneal arcus, xanthelasma and xanthomas . Currently, screening tools are not available for pediatric patients, especially younger than 2 years old. Performing a cascade screening of close relatives, starting by drawing a pedigree and then identifying the subjects with a cardiovascular event or hypercholesterolemia (clinical or genetically defined) would be suitable. If familial hypercholesterolemia is reported, hypercholesterolemia or a cardiovascular event is found in all generations. The gold standard diagnostic tool of familial hypercholesterolemia includes the detection of familial hypercholesterolemia -causing mutation . Further multidisciplinary team works are needed to identify optimal models of diagnosis and treatment of this life-threatening condition. 2- Pompe disease A review , enriched with the shared patient experience of the Italian Pediatric study group on immune response to enzymatic replacement therapy (ERT) with alglucosidase alfa in patients with Pompe disease, deals in depth with key elements of the complex and individualized management of immune response to ERT . Main objectives of the analysis, supported by useful diagrams, are identification of two types of immune reactions against ERT, quantification of risk factors for immunogenicity, choose of prophylaxis protocols in relation to cross reactive immunological material status, importance of immune-tolerance induction protocols in patients who develop antibodies during ERT, recommendations on the test to be performed before starting immunomodulation and tips on managing the frequent (up to 50%) hypersensitivity reactions during ERT. The authors conclude that further studies are needed to improve actual management protocols . Neonatology. 1- Preterm infant; 2- HIV infection; 3- Loeys-Dietz syndrome; 4- Hyperbilirubinemia; 5- Frenotomy; 6- Hypothermia 1- Preterm infant Parents of premature newborns admitted to neonatal intensive care unit (NICU) experience high levels of stress, determined both by the basic individual psychological well-being and the risk profile, the clinical complications, and the duration of the stay of their child. These conditions can have psychological consequences such as post-traumatic stress disorder . Salomè et al. examined how the state of parental psychological well-being and stress experienced affect the appearance of post-traumatic stress disorder in the year following discharge. The authors use an up-to-date and valuable set of psychological tests, described in the article and useful to the reader for planning similar studies. The results identify a higher risk of post-traumatic stress disorder in mothers compared to fathers (55% vs. 20%), with specific correlations between specific experiences and emotional reactions. In addition, they provide interesting research cues and identification of targeted intervention to reduce the risk of a post-traumatic stress disorder in parents of the preterm infants . In the position paper of the Italian scientific societies of pediatric area , recommendations are suggested regarding the important step of the introduction of complementary feeding in preterm infants. The main objectives are to reduce the risk of extrauterine growth restriction and the features of altered body composition with reduction of free mass and increased adiposity . Main recommendations include to start complementary feeding between 5 and 8 months of chronological age, consider the limit of 3 months corrected age to ensure the acquisition of developmental skills allowing the consumption of solid foods, consider multidisciplinary assessment before starting complementary feeding in case of oral dysfunction or comorbidities, type (included allergenic food), sequence and speed of introduction of food, vitamins, and micronutrients. Exclusive breastfeeding, mixed feeding or standard infant formula enriched with long chain polyunsaturated fatty acids should be preferred for infants without extrauterine growth restriction while extrauterine growth restriction infants or who is at risk of long-term growth failure may be fed with fortified human milk or formula adapted for preterm infants as long as to gain an optimal weight for corrected age. 2- HIV infection In West Amhara, Ethiopia an unmatched case–control study from 2016 to 2020, aimed to identify determinants of HIV infection among children born to HIV positive mothers on the prevention of the mother -child transmission program at referral hospitals . The region is characterized by an important HIV public health problem with a high transmission rate. The identified determinants were home delivery, mixed feeding, poor maternal antiretroviral drug adherence, advanced World Health Organization (WHO) clinical stage, poor nevirapine adherence and late enrollment of the infant. The authors conclude that Minister of Health and non-governmental organizations should work on mobilization of the community and awareness creation on the important of exclusive breast feeding, drugs adherence, on benefits of health institutional delivery as well as the risk of homedelivery. Public health interventions are very important tools to reduce mother-child transmission of HIV . 3- Loeys-Dietz syndrome Loeys-Dietz syndrome is a rare connective tissue disorder related to a pathogenic variant in TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2 or TGFB3 genes. It involves the aorta with progressive dilatation, craniofacial skeleton, joints, skin, and is associated with hypotonia and motor delay . In a first patient a trio based Whole Exome Sequencing found a novel, heterozygote, missense, de novo variant in the TGFBR2 gene. In a second patient , with a suspicious family history, a genetic panel for connective tissue disorders identified a pathogenetic variant in TGFB3 gene. Only in the first male patient the aortic aneurysm progressed despite of a low-dose angiotensin receptor blocker therapy, while the second female patient showed no aortic abnormalities. An early diagnosis of Loeys-Dietz syndrome implies a potential modification of the natural history of the disease with early interventions on its complications . 4- Hyperbilirubinemia A meta-analysis evaluates, in case of neonatal indirect hyperbilirubinemia in treatment with phototherapy, the use of ursodeoxycholic acid as adjunctive therapy . Ursodeoxycholic acid is classically used for the management of cholestatic liver pathology through increased production of bile, the removal of the most toxic components of bile acids and additional hepatoprotective and neuroprotective effects . The five selected studies from scientific literature suggest that the addition of ursodeoxycholic acid to phototherapy could reduce phototherapy duration by almost 18 h compared to phototherapy only. It also resulted in a lower mean of total serum bilirubin in the 48 h post-treatment, especially in Asian countries. Two infants with isolated severe unconiugated hyperbilirubinemia related to Gilbert syndrome have been reported . In both infants the prolonged indirect hyperbilirubinemia associated with the finding of a mutation p.Pro364Leu in the bilirubin uridine diphosphate-glucuronosyltransferase gene raised the initial suspicion of Crigler-Najjar syndrome type II. Both infants were treated with phototherapy and phenobarbital, achieving a normalization of bilirubin levels at 1 and 5 months respectively . The authors suggest that in these patients the concomitant breastfeeding jaundice was not a possible explanation of the clinical picture since a brief interruption of breastfeeding was not effective. 5- Frenotomy In a prospective observational cohort study, Dall’Olio et al. evaluated diode laser frenotomy in fifty-five newborns with ankyloglossia with or without difficult breastfeeding. Important functional consequences of ankyloglossia were manifested by 20 to 50% of cases adversely affecting the nutrition and growth of the newborn and the degree of adhesion of the mother to breastfeeding, her psychological well-being and bonding . The suggestions of the clinical tools used for evaluation of ankyloglossia, quality of breastfeeding, and perioperative pain of the newborn are useful. Short duration of the diode laser frenotomy, minimal reported complications, short hospital stay, and positive effects on breastfeeding, are elements in favor of the implementation of this procedure. 6- Hypothermia A single center, parallel-group, and no-blinded randomized controlled , has been conducted in a level III, and academic neonatal intensive care unit in China from 2020 to 2021, to evaluate the effect of early vs. delayed enteral nutrition on the incidence of feeding intolerance and secondary outcome including the incidences of late-onset bloodstream infection, hypoglycemia, survival at neonatal discharge, duration of parenteral nutrition, duration until attainment of full enteral feeds, length of hospital stay and body weight gain, in newborns treated with therapeutic hypothermia. Feeding strategy consisted of minimal enteral feeding and slow speed of increasing milk feeds. Early enteral nutrition was performed during therapeutic hypothermia /rewarming period while delayed enteral nutrition was performed after the therapeutic hypothermia phase. This study showed that the average time of parenteral nutrition, reaching full enteral feeds and hospital stay were shorter in the early enteral nutrition group compared with the delayed group with significant differences . Neurology. 1- Drooling; 2- Alternating hemiplegia; 3- Autism; 4- Pontocerebellar hypoplasia; 5- Vertigo; 6- Idiopathic intracranial hypertension; 7- Pressure palsies; 8- Epilepsy; 9- Diencephalic syndrome 1- Drooling Drooling is defined as the involuntary loss of saliva and oral content. While most children reach the salivary continence by age 15–18 months, the persistence of drooling is common in children with neurological disorders, like cerebral palsy (CP) and is associated with oral motor dysfunction, dysphagia, and/or intraoral sensitivity disorder . Several therapeutic interventions are used to reduce or eliminate drooling. These include surgery, drugs, botulinum toxin, physical therapies to improve sensory function, behavioral interventions, appliances placed in the oral cavity, and acupuncture . There is no consensus on which interventions are safe and effective in managing drooling in children with CP. As the salivary glands are controlled by the parasympathetic autonomic nervous system, the anticholinergic drugs are indeed widely used to reduce the volume of saliva. Glycopyrrolate is the only oral formulation including an anticholinergic agent approved by the United States Food and Drug Administration to treat drooling in children 3–16 years-old which inhibits the acetylcholine receptors on peripheral tissues and reduces the saliva rate production . Lovardi et al. described a case series of eighteen children (median age 17 months, range 2–36 months) with CP or genetic/malformative syndrome, who have been administered oral glycopyrrolate (0.065 mg/kg/die in 3 daily doses) for drooling control. The Drooling Impact Scale was administered at time O and after 1 month. Results showed a significant reduction of the Drooling Impact Scale after 1 month (89 versus 61; p < 0.001), with few adverse effects and an overall response to treatment equal to 94%. Further studies are needed to confirm these results. 2- Alternating hemiplegia Alternating hemiplegia of childhood is an uncommon complex disorder, which was first described in late 1960s by Verret and Steele . Alternating hemiplegia of childhood is characterized by paroxysmal episodes of repeated, transient paresis involving either or both sides of the body and usually presents before age 18 months. The diagnosis of alternating hemiplegia of childhood is mainly clinical but may be supported by molecular analysis. Although the pathophysiologic mechanism of the disorder is partially unclear, it is well known that a relevant role is played by mutations in ATP1A2 and ATP1A3 genes, which encode two different alpha subunits of the Na+/K + ATPase transmembrane ion pump, respectively . Pavone et al. reported on the clinical and genetic findings of a couple of twins and a couple of siblings with alternating hemiplegia of childhood from two different Italian families affected. In the twins a pathogenic variant in ATP1A3 gene (c.2318 A > G) was detected. In the siblings, the younger brother showed a novel GRIN2A variant (c.3175 T > A), while the older one carried the same GRIN2A variant, associated with two likely pathogenetic variants in SCNIB (c.632 > A) and KCNQ2 (1870 G > A) genes, which have been implicated in childhood epilepsies. This report provides additional information about alternating hemiplegia of childhood, showing that the variability of clinical features is mirrored by an unexpected genetic heterogeneity. Clinical signs of alternating hemiplegia of childhood usually follow a sequential pattern, in which the paroxysmal episodes are triggered by precipitating factors such as environmental stress, bathing or other events. Non-paroxysmal features include developmental delay/intellectual disability, epilepsy, autonomic dysfunctions, abnormal eye involvement, movement disorders, ataxia, dystonia, and choreoathetosis . 3- Autism Autism spectrum disorder is a term used to describe a set of social communication deficits and repetitive sensory–motor behaviors. It is a neurological developmental disorder, and is characterized by a complex etiology involving genetic, environmental, and biochemical factors . The autism spectrum includes several disorders, such as the Autistic disorder, the Rett disorder, the Asperger syndrome, and the pervasive developmental disorder. Although individuals with autism spectrum disorder are very different from one another, the disorder affects social interactions, verbal and nonverbal social communication skills, as well as intelligence and motor functions, usually originating unusual interests and repetitive behaviors . Epidemiological surveys have shown a rapid increase in autism spectrum disorder prevalence rate. Besides the real increase in prevalence, a variety of other reasons may contribute to the disorder, such as a broader definition of autism spectrum disorder, changes in diagnostic criteria and screening tools, and finally an increased awareness of autism spectrum disorder . In this scenario, Salari et al. conducted a systematic review and meta-analysis from 2008 to July 2021 finalized to clarify the global prevalence of autism spectrum disorder The results show that the world prevalence of autism spectrum disorder is 0.6% (95% confidence interval: 0.4;1%), with slight differences between Asia (0.4%), America (1%), Europe (0.5%), Africa (1%) or Australia (1.7%). Standardized screening for autism spectrum disorder with ongoing developmental surveillance continues to be recommended in primary care at 18 and 24 months of age, because autism spectrum disorder is common, can be diagnosed as young as 18 months of age, and medical interventions may significantly affect neurological compromise . These data should be disseminated by health policymakers in order to implement appropriate planning and interventions. 4- Pontocerebellar hypoplasia Pontocerebellar hypoplasia includes a heterogeneous group of neurodegenerative disorders with prenatal onset, characterized by severe microcephaly, global developmental delay and radiological manifestation, such as hypoplasia of pons and cerebellum . At least 21 pontocerebellar hypoplasia -related genes are currently listed in the OMIM-database and 15 types of pontocerebellar hypoplasia are known, all characterized by patients’ motor and cognitive impairment. However, the clinical presentation can be very different, ranging from lethal neonatal subtypes to milder variants with survival up to adolescence . The study from Bilge et al. consisted of the description of different clinical and radiological manifestations of six genetically diagnosed patients with pontocerebellar hypoplasia, highlighting the differences in onset and progression of the symptoms . The genetic mutations found in 4 out 6 cases were well known variants that had been previously reported in the literature, while an additional subject was homozygous for the TBC1D23 gene mutation (c.1263 + 1G > A), which was a novel variant never reported until now. Although the last case had the same clinical features, the whole-exome sequencing revealed compound heterozygous mutations in the BRF1 gene, which is also associated with cerebellofaciodental syndrome. Common problems in pontocerebellar hypoplasia include sleep apnea, feeding problems, epilepsy, movement disorders, rhabdomyolysis, and extremely elevated serum creatinine kinase, especially during infections . There is no definite treatment for any type of pontocerebellar hypoplasia, and management is supportive in all types and subtypes. 5- Vertigo Vertigo is a disorder of space sensitivity characterized by an illusory and unpleasant sensation of body movement toward the environment or vice versa. Vertigo is characterized by different clinical features, therefore it is often difficult for the single physician to identify its etiopathogenesis, especially when the vertigo is the only clinical presenting sign . Compared to adults, vertigo is not so common in children and adolescents. Indeed, while adults surveys reported a one-year prevalence of 23% for unspecified dizziness and 5% for vestibular vertigo, in pediatric patients a prevalence of only 0.4% for nonspecific dizziness, 0.03% for peripheral, and 0.02% for central vestibulopathy is reported . Nevertheless, it is a common reason for emergency department presentation, both alone or associated with other symptoms. Pellegrino et al. reported on the etiopathology of neurological vertigo in childhood and adulthood, highlighting the characteristics which may lead clinicians to a proper diagnosis, and proposed a diagnostic algorithm to support the approach to patients with isolated vertigo, both in pediatric and adult age. The most common cause of isolated vertigo in the pediatric population is vestibular migraine, followed by benign paroxysmal vertigo in childhood. Other causes of vertigo are orthostatic hypotension, vestibular neuritis and vestibular paroxysmia, neurovascular diseases, tumors and demyelinating . Authors underline that the outcome of most neurological vertigos in childhood is good, in contrary to what is reported in adults. 6- Idiopathic intracranial hypertension Idiopathic intracranial hypertension is very rare in the pediatric age, and etiology is still largely unknown. Del Monte et al. presented a case report of an 8-month old male affected by idiopathic intracranial hypertension, and provide discussion on what literature suggests about optimal management also including the therapeutic strategies in idiopathic intracranial hypertension. The presented case was admitted with vomit, anorexia, irritability and bulging anterior fontanel. Brain magnetic resonance and cerebrospinal fluid analysis were negative. After diagnosis and treatment with acetazolamide and corticosteroids, progressive resolution of symptoms was observed. Pseudotumor cerebri syndrome is a rare condition characterized by elevated intracranial pressure, normal cerebrospinal fluid analysis in the absence of intracranial lesions on neuroimaging. Most common type of pseudotumor cerebri syndrome in both children and adults is the primary type known as idiopathic intracranial hypertension . Secondary types can result from vascular malformations, underlying systemic conditions or drugs. Female gender, obesity, and polycystic ovary represent risk factors for idiopathic intracranial hypertension in adolescents, but not in younger ones . Clinical manifestations of idiopathic intracranial hypertension vary with age. In pediatric populations, symptoms are often unspecific, including vomiting, headache, irritability, hyporeactivity, anorexia, sleep disruption, head tilting, papilledema and bulging fontanelle in infants. Based on adults’ treatment guidelines, also in children the drug of choice is acetazolamide, that could be associated with intravenous corticosteroids if severe visual impairment is present as well. So far, the moment, acetazolamide alone is preferred because of fewer side effects. Most common complications of idiopathic intracranial hypertension are vision loss and relapse, especially in the first 18 months after diagnosis . Early diagnosis, treatment and strict follow-up of complications may help in reducing the risk of relapse and may prevent vision loss. 7- Pressure palsies Karlinsky et al. reported their experience about an unusual case of hereditary neuropathy with liability to pressure palsies (HNPP) who had limping as a major presenting symptom. HNPP is an autosomal dominant disease characterized by recurrent, episodic demyelinating neuropathy caused by 17p11.2 chromosomal deletion encompassing the PMP22 gene. The onset is acute, usually in adulthood, and involves a single nerve, most frequently peroneal and ulnar, with a non-painful focal sensory and motor neuropathy . The onset in children most commonly involves peroneal nerve palsy and brachial plexus palsy . Other signs are previous atrophy, focal weakness or sensory loss, reduction of tendon reflexes and mild to moderate pes cavus foot deformity . The definitive diagnosis is provided by genetic analysis, and treatment is mostly supportive. Prognosis is good with an expected full recovery within months or days in best case scenarios. In rare cases a persistence of severe symptoms is reported. Since presenting phenotypes can be extremely variable, the authors describe their experience that led to the identification of 3 additional cases within the pedigree, providing family members with an accurate genetic counseling. 8- Epilepsy Febrile infection-related Epilepsy Syndrome (FIRES) is an uncommon but severe disorder manifesting a prior febrile infection starting between 2 weeks and 24 h before the onset of the refractory status epilepticus with or without fever at the onset of status epilepticus. Symptoms of FIRES had been previously named as “acute encephalitis with refractory, repetitive partial seizures” or as “fever-induced refractory epileptic encephalopathy in school age children”. In this literature review, Pavone et al. presented two cases with FIRES and discussed about this rare even though severe disorder. Both cases presented with the classical FIRES symptoms and reported moderate-severe cognitive impairment and persistence of seizures. Clinical manifestations of FIRES are focal seizures in the acute phase with possible secondary generalization, sometimes associated with pallor, apnea, and cyanosis . Current guidelines for diagnosis of FIRES are not yet available . Different treatment options have been reported, usually suggested during the second phase of the disease. Most used drugs are intravenous benzodiazepines as midazolam, clonazepam, lorazepam and diazepam in association with standard anticonvulsant drugs, as oral levetiracetam, valproic acid, and lacosamide . Alternative therapeutic strategies are ketogenic diet immunomodulatory and intravenous steroids at high-dose or immunoglobulins, plasmapheresis and other agents . Clinical outcomes usually see the persistence of seizure episodes, intellectual disability or, in more severe cases, vegetative state . FIRES is an uncommon and not yet totally understood entity. Since many aspects of this disease such as pathogenesis and treatment still need to be clarified, further future studies are needed. Epilepsy is a common and serious multifactorial neurologic disease with a strong genetic component . Ghazala et al. investigated the SCN1A-A3184G polymorphism among Egyptian children and adolescents with non-lesional epilepsy conducting a prospective case-control observational study. Neuronal voltage-gated sodium channels are involved in the generation and propagation of the action potentials within the neurons, acting on membrane permeability to sodium ions that allows ions diffusion down an electrochemical gradient till the sodium equilibrium potential . There is evidence about the role of the neuronal voltage-gated sodium channels polymorphisms in the epilepsy pathogenesis that causes a spectrum of epilepsy syndromes. For this reason, sodium-channel blockers such as carbamazepine, oxcarbazepine, phenytoin, lamotrigine, lacosamide and lidocaine are some of the most common therapeutic options for epilepsies due to genetic channelopathies. The SCN1A-A3184G (p.Thr1067Ala) polymorphism has been suggested to be linked with the epilepsy risk in several non-Caucasian populations . In their study, the authors extracted and analyzed genomic DNA controls and cases. Results of the study report insignificant differences between epilepsy cases and the control group regarding the frequency of SCN1A-A3184G genotypes and allele. Authors also suggest that the identification of SCN1A-A3184G genotypes might help to choose the most suitable antiepileptic drugs. Further studies on a larger scale are needed to better clarify the matter. 9- Diencephalic syndrome Trapani et al. provide relevant insights on the diencephalic syndrome, a rare childhood disease causing failure to thrive. They presented three children who were admitted for progressive weight loss. Diencephalic syndrome is provoked by a hypothalamic dysfunction caused by a tumor that may involve thalamus/hypothalamus and optic chiasm . Presenting symptoms of diencephalic syndrome may be unspecific. Most common features are severe emaciation with normal caloric intake, locomotor hyperactivity and euphoria, pallor without anemia, hypoglycemia and hypotension . Neurological symptoms typically appear later and include nystagmus, strabismus and visual loss; intracranial hypertension causing recurrent vomiting can be also present, without signs of psychomotor impairment. Diagnosis can be tricky due to the non-specific signs of presentation. Gold standard for radiological detection of brain mass is magnetic resonance. Since surgical removal is not always feasible, chemotherapy based on vincristine, carboplatin and/or etoposide is one of the main therapeutic options . The first case is a 26-month-old boy with persistent failure to gain weight despite adequate caloric intake, no gastrointestinal or other symptoms and negative blood exams about possible malabsorption or vitamin deficiencies. Hormone evaluations and other organ functions were also normal. Brain magnetic resonance was finally performed and detected a large low-grade astrocytoma located in the supra-sellar region. The second case is a 14-month-old girl admitted for poor weight gain, with normal caloric intake. Same investigations were conducted, as well as a brain magnetic resonance showing supra-sellar mass involving hypothalamic-pituitary region, later identified as a low-grade astrocytoma. Finally, the third case is a 16-month-old male who was admitted for severe failure to thrive. After initial diagnostic workup, in the suspicion of diencephalic syndrome, brain imaging identified a large multilobate pseudo-cystic suprasellar lesion in the hypothalamic-pituitary and chiasmatic region. Biopsy of the lesion led to histological diagnosis of pilomyxoid astrocytoma. In this interesting series authors clarified useful elements for the identification of this uncommon syndrome in which subtle presenting symptoms can often lead to a delayed diagnosis. Oncology. 1- Care burden 1- Care burden X 400.000 children and adolescents between ages of 0 and 19 each year. The most common types of childhood cancers include leukemias, brain cancers, lymphomas and solid tumors, such as neuroblastoma and Wilms tumor . Scientific advancement has led to an increase in the survival rate of children with cancer and, as a result, living with cancer affects different aspects of life of these children and their caregivers . Chaghazardi et al. determined the level of care burden and the factors associated with it among the caregivers of children with cancer, and have highlighted that the majority of caregivers experience moderate to high care burden. Following the diagnosis, caregivers have to face several challenges such as ignorance, instability, anxiety, helplessness, confusion, and stress. High care burden can affect the quality of care provided to the patient and thus exacerbate their condition, leading to an increase of care burden in a vicious circle . For this reason, identifying and helping caregivers with care burden play a crucial role in improving the quality of care to affected children. Authorities need to take family-centered measures to reduce the caregiver burden, referring low-income caregivers to support organizations, providing counseling services, and holding training workshops on caring for children with cancer. Pneumology. 1- E-cigarettes; 2- Cystic fibrosis 1- E-cigarettes Electronic cigarettes are devices that heat liquid substances that may contain nicotine that can substitute burned cigarettes. Virgili et al. presented the effects of e-cigarette use on health and pointed out the potential damages arising also from second- and third-hand exposure to smoke. Even though e-cigarettes are considered to be less harmful than burned cigarettes, the Center for Disease Control and Prevention defines them as unsafe and potentially dangerous for brain development and increasement of risk for addiction . Damages of vaping on the respiratory system are analogous to the ones of smoking: chronic inflammation of bronchial mucosa and lung epithelium injury are typically observed. Due to the increasing popularity of vaping products, a new type of lung damage has been described: E-cigarette or Vaping Associated Lung Injury (EVALI). The diagnosis is made after the exclusion of all other potential causes of lung injury when pulmonary infiltrates are found at chest imaging in patients using e-cigarettes and related products 90 days prior to symptom onset . Symptoms of presentation are usually cough, dyspnea and tachycardia and chest imaging usually shows ground glass appearance or dense consolidations. Therapy is based on antibiotics and steroids and, when needed, high-flow oxygen therapy, noninvasive ventilation or mechanical ventilation . Effects on the cardiovascular system include myocardial infarction, hypertension and tachycardia. Neurological effects are the ones observed in nicotine users and include craving and withdrawal symptoms. Gastroenteric effects are an increased risk for gastro-esophageal reflux and for esophagitis exacerbations. Overall, due to its various harmful effects, vaping could be useful only if used as a smoking-cessation device. It is important for institutions to keep restrictions and prevention strategies and for educators and pediatricians to increase awareness in children and their families. During the last years the use of E-cigarettes has increased dramatically in Italy . Casamento Tumeo et al. described a case of E-cigarette or EVALI in a 15-year-old girl. The girl presented to the emergency room with severe dyspnea and a SatO2 of 75% and chest auscultation revealed bilateral wheezes with prolonged expiration. After admission, oxygen supplementation was started and a chest tomography was performed, showing central ground glass pattern with peripheral sparing. She was started on antibiotic therapy with clarithromycin and ceftriaxone. High Flow Nasal Cannula, systemic steroid and nebulized salbutamol, ipratropium and corticosteroids. The girl had a history of asthma and multiple allergies and she was a tobacco and E-cigarette smoker. Dyspnea started 4 days before the admission to the emergency department, but no fever or cough were present. Prior to admission, antibiotic therapy was prescribed with no improvements. After admission and initial treatments, further examinations were performed. Among them, pulmonary function tests showed a restrictive pattern, without bronchodilator reversibility. Diagnostic criteria for identification of EVALI include the use of e-cigarettes 90 days prior to the onset of symptoms accompanied by specific radiological findings (typically ground glass opacities at chest tomography) . EVALI is more common in patients with underlying asthma . Even if its cause is still not yet known, in recent years, Vitamin E has been reported in the broncho-alveolar lavage of patients affected by EVALI, thus identifying it as possible pathogen . Since its relatively recent spread, the use of E-cigarette still needs to be studied in order to better understand the impact and potential damages on health. 2- Cystic fibrosis Cystic fibrosis (CF) is a life-limiting autosomal recessive disorder due to variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR protein is responsible for chloride ion transport across apical epithelial cells in tissues of the airway, intestine, pancreas, kidney, sweat gland, and male reproductive tract . Nowadays, CFTR modulator therapies, targeting the basic CF molecular defect, have been developed for specific CFTR variants and are associated with an improvement in health outcomes, including respiratory function, nutritional status and enhanced quality of life. The quality of life and the survival of patients with CF significantly improved since these drugs have been available and several progresses are being made in the development of such drugs . Nevertheless, lung disease remains the most common cause of death in CF patients and symptomatic mucolytic drugs are crucial for reducing secretion build-up, preventing infections and slowing lung damage. Terlizzi et al. have summarized the current knowledge on dornase alfa (DNAse) in the treatment of CF lung disease, showing the positive effects of this drug on lung ventilation homogeneity. To date DNAse is the only mucus degrading agent that has proven efficacy in CF , by reducing the number of pulmonary exacerbations and improving Forced Expiratory Volume in the 1st second and lung clearance index in patients with CF, in the absence of significant side effects. That is why an early use would be desirable in CF children from 6 years of age, especially in the presence of an abnormal lung clearance index. 1- Food allergy Food allergy is one of the most challenging field in pediatrics . It is common in developed countries resulting in an important problem for quality of life. Food allergy care has gone a long way. The Italian Society for Pediatric Gastroenterology Hepatology and Nutrition and the Italian Society for Pediatric Allergy and Immunology joint position paper on the management of food allergy including activities and responsibilities of general practitioners, emergency departments and tertiary centers, such as food challenge , has been published last year. The document provided information that will decrease delayed diagnosis, costs, and hazards for children with food allergy. 2- Atopic eczema Atopic eczema has several clinical phenotypes that needs a multidisciplinary approach . The review by Galli et al. reports current knowledge on treatment for moderate to severe cases. It has been highlighted the use of traditional treatments including trigger avoidance, emollients, bath, topical steroids, calcineurin inhibitors, immunosuppressive agents, anti-H1 antihistamines, phototherapy, education. Furthermore, it illustrates novel systemic drugs that acts on specific molecules involved in the immune mechanism that driven the pathogenetic pathways of AD, including biologics, janus kinase inhibitors and allergen specific immunotherapy . An algorithm for clinical use of topical and systemic treatments is also provided. 3- Asthma Chronic airway inflammation leads to occurrence of symptoms in asthmatics . Inhaled drugs are useful for asthma treatment since are effective and systemic adverse reactions are rare. In children with asthma, an inhaler both dry powder and pressurised metered-dose with and without the use of spacer should be used for delivering drugs to the lungs. So, a correct inhaler technique is one of the most important step for controlling asthma in children. McCrossan et al. review data from several studies and describe the pros and cons of educational methods used to improve inhaler technique. They underline that researches in this field are warranted to identify the best outcome measure for inhaler technique in children. Furthermore, trials comparing interventions for teaching inhaler technique at different ages are lacking. 4- Hypersensitivity pneumonitis Mastrorilli et al. address the ‘hot topic’ of hypersensitivity pneumonitis in children . They underline that on the basis of a suggestive history, the diagnosis relies on respiratory signs and symptoms, pulmonary function tests, characteristic HRCT nodular opacities, serum specific IgG levels, response to elimination of eliciting factor. Of note, aspergillus species are commonly considered the cause. However, other fungi such as Schizophyllum commune can be involved . Bronchoscopy with bronchoalveolar lavage, and the provocation test may be helpful. Besides supportive treatment, the resolution generally takes place with trigger avoidance and systemic corticosteroids. Hydroxychloroquine, azithromycin, immunosuppressants, rituximab, nintedanib might be considered. Food allergy is one of the most challenging field in pediatrics . It is common in developed countries resulting in an important problem for quality of life. Food allergy care has gone a long way. The Italian Society for Pediatric Gastroenterology Hepatology and Nutrition and the Italian Society for Pediatric Allergy and Immunology joint position paper on the management of food allergy including activities and responsibilities of general practitioners, emergency departments and tertiary centers, such as food challenge , has been published last year. The document provided information that will decrease delayed diagnosis, costs, and hazards for children with food allergy. Atopic eczema has several clinical phenotypes that needs a multidisciplinary approach . The review by Galli et al. reports current knowledge on treatment for moderate to severe cases. It has been highlighted the use of traditional treatments including trigger avoidance, emollients, bath, topical steroids, calcineurin inhibitors, immunosuppressive agents, anti-H1 antihistamines, phototherapy, education. Furthermore, it illustrates novel systemic drugs that acts on specific molecules involved in the immune mechanism that driven the pathogenetic pathways of AD, including biologics, janus kinase inhibitors and allergen specific immunotherapy . An algorithm for clinical use of topical and systemic treatments is also provided. Chronic airway inflammation leads to occurrence of symptoms in asthmatics . Inhaled drugs are useful for asthma treatment since are effective and systemic adverse reactions are rare. In children with asthma, an inhaler both dry powder and pressurised metered-dose with and without the use of spacer should be used for delivering drugs to the lungs. So, a correct inhaler technique is one of the most important step for controlling asthma in children. McCrossan et al. review data from several studies and describe the pros and cons of educational methods used to improve inhaler technique. They underline that researches in this field are warranted to identify the best outcome measure for inhaler technique in children. Furthermore, trials comparing interventions for teaching inhaler technique at different ages are lacking. Mastrorilli et al. address the ‘hot topic’ of hypersensitivity pneumonitis in children . They underline that on the basis of a suggestive history, the diagnosis relies on respiratory signs and symptoms, pulmonary function tests, characteristic HRCT nodular opacities, serum specific IgG levels, response to elimination of eliciting factor. Of note, aspergillus species are commonly considered the cause. However, other fungi such as Schizophyllum commune can be involved . Bronchoscopy with bronchoalveolar lavage, and the provocation test may be helpful. Besides supportive treatment, the resolution generally takes place with trigger avoidance and systemic corticosteroids. Hydroxychloroquine, azithromycin, immunosuppressants, rituximab, nintedanib might be considered. 1- Sedation In infant and children, the optimal treatment and management of diseases sometimes require high quality diagnostic procedures for which a prolonged inactive state are necessary and sedation needed. Some of the desire characteristics for the sedation regimen are rapid onset of action, predictable duration, rapid cessation of effects, few side effects and no need of rescue drug. Cossovel et al. interestingly investigate the difference between two approach, combination of intranasal ketamine and intranasal dexmedetomidine compare with the the combination intranasal dexmedetomidine and oral midazolam. Compared with other studies in literature, the results look controversial, probably because of limits and confounding factors in the design of the studies . Prospective studies are needed. In infant and children, the optimal treatment and management of diseases sometimes require high quality diagnostic procedures for which a prolonged inactive state are necessary and sedation needed. Some of the desire characteristics for the sedation regimen are rapid onset of action, predictable duration, rapid cessation of effects, few side effects and no need of rescue drug. Cossovel et al. interestingly investigate the difference between two approach, combination of intranasal ketamine and intranasal dexmedetomidine compare with the the combination intranasal dexmedetomidine and oral midazolam. Compared with other studies in literature, the results look controversial, probably because of limits and confounding factors in the design of the studies . Prospective studies are needed. 1- Cardiovascular risk Noncommunicable diseases are the current challenge for the sustainability of the health system in industrialized countries and among them cardiovascular diseases remain the main issue, being the leading cause of death, handicap, and health expenditure . Atherosclerosis the matrix lesion, begins and can be detected in infancy, and its severity, extent and progression in childhood is related to exposure to risks factors such as dyslipidemia, hypertension, chronic kidney diseases, congenital heart and acquired heart diseases, cancer . Several studies have shown an association between exposure to cardiovascular risk factor in childhood and subclinical atherosclerosis in adulthood but to date there is still limited evidence a lack of direct link between these factors with adult clinical cardiovascular diseases . Noncommunicable diseases are the current challenge for the sustainability of the health system in industrialized countries and among them cardiovascular diseases remain the main issue, being the leading cause of death, handicap, and health expenditure . Atherosclerosis the matrix lesion, begins and can be detected in infancy, and its severity, extent and progression in childhood is related to exposure to risks factors such as dyslipidemia, hypertension, chronic kidney diseases, congenital heart and acquired heart diseases, cancer . Several studies have shown an association between exposure to cardiovascular risk factor in childhood and subclinical atherosclerosis in adulthood but to date there is still limited evidence a lack of direct link between these factors with adult clinical cardiovascular diseases . 1- Hemangioma Infantile hemangioma are benign tumors, quickly growing that are commonly located on the skin, but may also be in solid organ as brain or liver. They may go through gradual and spontaneous involution, but in some cases, depending on their location, they can lead to severe complications, ulceration, bleeding organ dysfunction or transient to permanent deformity which significantly impact patient and family quality of life. Propranolol has changed the history and the management of infantile hemangiomas , because of facility of administration and high success rate, becoming the first line treatment before glucocorticoids and surgery . Although the mechanism underlying its effect is still unclear and controversies about side effects and potential safety issues . Early and prompt diagnosis and treatment is essential . 2- Epidermolysis bullosa Epidermolysis bullosa is a chronic genodermatosis with different phenotypes and severity. Retrosi et al. reported on treatment in 3 groups of patients: < 5 years, > 5–12 years and > 12–18 years. In the first group, the family should change lifestyle and burden of caring on quality of life is high. alliance between the health professional and parents can facilitate relationship between all family members . In the second group, children have to be educated in accepting and understanding the disease and involved in the management to diminish complications. In the third group, adolescents have a deep knowledge of the disease. Denial of the disease and low adherence to treatment are common . Authors found that 27 (13%) out of 215 patients died. Eighteen (8%) patients died because of sepsis and/or respiratory failure during the first months of life. Nine (5%) died for squamous cell carcinoma in adulthood . 3- Congenital ichthyosis Congenital ichthyosis is a rare disease. Mutations of more than 50 genes have been found. Genotype is often correlated with distinct form of the disease . Serra et al. found new variants of the ABCA12, KRT1 and ST14 genes. Mutations of ABCA12 gene is significantly associated with neonatal mortality . Mutations of KRT1 gene can be hereditary or occur de novo. The epithelial barrier disruption is associated with infections and dyselectrolytemia. Mutations of ST14 gene are not clearly associated with a specific clinical picture. Authors highlight the importance of the molecular characterization to make an appropriate treatment, define the prognosis and the genetic counselling . Infantile hemangioma are benign tumors, quickly growing that are commonly located on the skin, but may also be in solid organ as brain or liver. They may go through gradual and spontaneous involution, but in some cases, depending on their location, they can lead to severe complications, ulceration, bleeding organ dysfunction or transient to permanent deformity which significantly impact patient and family quality of life. Propranolol has changed the history and the management of infantile hemangiomas , because of facility of administration and high success rate, becoming the first line treatment before glucocorticoids and surgery . Although the mechanism underlying its effect is still unclear and controversies about side effects and potential safety issues . Early and prompt diagnosis and treatment is essential . Epidermolysis bullosa is a chronic genodermatosis with different phenotypes and severity. Retrosi et al. reported on treatment in 3 groups of patients: < 5 years, > 5–12 years and > 12–18 years. In the first group, the family should change lifestyle and burden of caring on quality of life is high. alliance between the health professional and parents can facilitate relationship between all family members . In the second group, children have to be educated in accepting and understanding the disease and involved in the management to diminish complications. In the third group, adolescents have a deep knowledge of the disease. Denial of the disease and low adherence to treatment are common . Authors found that 27 (13%) out of 215 patients died. Eighteen (8%) patients died because of sepsis and/or respiratory failure during the first months of life. Nine (5%) died for squamous cell carcinoma in adulthood . Congenital ichthyosis is a rare disease. Mutations of more than 50 genes have been found. Genotype is often correlated with distinct form of the disease . Serra et al. found new variants of the ABCA12, KRT1 and ST14 genes. Mutations of ABCA12 gene is significantly associated with neonatal mortality . Mutations of KRT1 gene can be hereditary or occur de novo. The epithelial barrier disruption is associated with infections and dyselectrolytemia. Mutations of ST14 gene are not clearly associated with a specific clinical picture. Authors highlight the importance of the molecular characterization to make an appropriate treatment, define the prognosis and the genetic counselling . 1- Obesity Obesity is a chronic diseases with the greatest impact on public health with an increasing incidence over time. In adolescents, the reasons for obesity are various and among the most significant we might mention the wide prevalence of the obesity on a world scale with an increase also in low-income countries, the many complications that excess weight can cause at the level of various organs and systems and the high percentage of obese adolescents becoming obese adults. On the other hand, the therapeutic approach used in the last years that is based on the (difficult) attempt to change the lifestyle of obese adolescents has not yet given satisfactory results, especially in the long term. For this reason Nicolucci and Maffeis dedicate an in-depth review both to the anti-obesity medications available (with particular attention to the glucagon-like peptide-1 analog) and to the bariatric surgery. The results obtained with the combination of lifestyle changes and new drugs seem encouraging but there is a need to know the long-term results on the efficacy in determining and maintaining weight loss, on the action on possible complications (cardiovascular), on the quality of life and the long term side effects . Even with regards to bariatric surgery (currently, sleeve gastrectomy and “Rouxen-Y gastric bypass are two of the most popular bariatric procedures the results seem promising . Bariatric surgery is considered very selectively for example by the American Society of Metabolic and Bariatric Surgery in adolescents with body mass index (BMI) ≥ 35 kg/m2 with major comorbidities or with a BMI ≥ 40 kg/m2 with minor comorbidities, However, as discussed by Martinelli et al. in a recent review “the use of bariatric surgery in adolescent patients is still limited, with significant disparities among countries. Reasons include ethical concerns raised by performing an irreversible and invasive procedure in adolescence, with potential life-long alterations” . In conclusion, on the one hand there is an increasingly general agreement in treating adolescent obesity by deepening knowledge on the use of new generations of drugs and/or bariatric surgery combined with behavioral therapies that help change lifestyles and on the other hand, the undoubted difficulties of this path lead us to insist on programs for the global prevention of obesity. In childhood, drug dosing is often based on body weight. However pharmacokinetic is influenced by body weight . This issue should be considered in obese children, but it has not been deeply studied . So far, higher doses are required only for trimethoprim/sulfamethoxazole , among antibiotics, midazolam, inhaled corticosteroids. Data on dosing of low molecular weight heparins and vitamin D are unclear . So, further investigation on drug dosing are warranted in obese children. 2- Puberty Much attention has been given to early and delayed puberty. A worldwide growing decrease of puberty timing has been observed. Key features in the pathogenesis are nutrition in pregnancy, birth weight, dietary habits, physical activity, psychological factors, exposure to electromagnetic fields, endocrine-disruptors . Ferrari et al. showed in 577 girls that increased Z-score change from birth weight and higher BMI may predict earlier age at menarche and at thelarche. This underlines the role of modifiable factors in the onset of puberty. Among delayed puberty causes, constitutional delay of growth and puberty, a variant of normal growth is frequent. It may induce psychological difficulties, poor school performance and perhaps lower final height . So, therapeutic interventions have received interest in recent years. In adolescent > 14 years of age with psychological problems, a course of estradiol and progesterone in females or testosterone in males can be given. Importantly, despite clinical interest, the benefit of growth hormone on final height is uncertain . 3- Type 1 diabetes Type 1 diabetes is a disease that is heterogeneously growing in prevalence. Several studies have examined annual incidence that greatly varies from 0.1 to 100,000 person in China to 62.3 per 100,000 person in Scandinavia and from 19 to 45 per 100,000 person-years in different Italian regions. Passanisi et al. found an incidence of 20.6/100,000 person/years with an increase of 43% between 2019 and 2021 during Coronavirus Disease (COVID-19) pandemics in Calabria region, Italy. This supports the view that Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection may predispose to a diabetes onset . They also found an increase of children with severe ketoacidosis that can be justified by shutting of many health services during the pandemics. 4- Hypothyroidism Menorrhagia is frequent after menarche onset. It is a challenging issue that often depend on anovulatory cycles due to the immaturity of the hypothalamic-pituitary-ovarian axis , but it can also be related to coagulopathy, pregnancy, medications, endocrine disorders, trauma, infection, cancer, structural pathologies, gastrointestinal bleeding . Barbero et al. delineated a girl with menorrhagia associated with short stature, bradycardia, dry skin, thinning hair. Hypothyroidism was confirmed by ultrasound showing an expanded thyroid with non-homogeneous hypoechogenic structure and laboratory tests showing anemia, high thyrotropin releasing hormone level, low free thyroxin, high serum anti-thyroid peroxidase antibodies and antithyroglobulin antibodies levels. Levothyroxine treatment was associated with remission of symptoms and regulation of laboratory parameters. This case highlights that follow up of the pubertal development is of particular importance for timely identification of underlying disorders. Obesity is a chronic diseases with the greatest impact on public health with an increasing incidence over time. In adolescents, the reasons for obesity are various and among the most significant we might mention the wide prevalence of the obesity on a world scale with an increase also in low-income countries, the many complications that excess weight can cause at the level of various organs and systems and the high percentage of obese adolescents becoming obese adults. On the other hand, the therapeutic approach used in the last years that is based on the (difficult) attempt to change the lifestyle of obese adolescents has not yet given satisfactory results, especially in the long term. For this reason Nicolucci and Maffeis dedicate an in-depth review both to the anti-obesity medications available (with particular attention to the glucagon-like peptide-1 analog) and to the bariatric surgery. The results obtained with the combination of lifestyle changes and new drugs seem encouraging but there is a need to know the long-term results on the efficacy in determining and maintaining weight loss, on the action on possible complications (cardiovascular), on the quality of life and the long term side effects . Even with regards to bariatric surgery (currently, sleeve gastrectomy and “Rouxen-Y gastric bypass are two of the most popular bariatric procedures the results seem promising . Bariatric surgery is considered very selectively for example by the American Society of Metabolic and Bariatric Surgery in adolescents with body mass index (BMI) ≥ 35 kg/m2 with major comorbidities or with a BMI ≥ 40 kg/m2 with minor comorbidities, However, as discussed by Martinelli et al. in a recent review “the use of bariatric surgery in adolescent patients is still limited, with significant disparities among countries. Reasons include ethical concerns raised by performing an irreversible and invasive procedure in adolescence, with potential life-long alterations” . In conclusion, on the one hand there is an increasingly general agreement in treating adolescent obesity by deepening knowledge on the use of new generations of drugs and/or bariatric surgery combined with behavioral therapies that help change lifestyles and on the other hand, the undoubted difficulties of this path lead us to insist on programs for the global prevention of obesity. In childhood, drug dosing is often based on body weight. However pharmacokinetic is influenced by body weight . This issue should be considered in obese children, but it has not been deeply studied . So far, higher doses are required only for trimethoprim/sulfamethoxazole , among antibiotics, midazolam, inhaled corticosteroids. Data on dosing of low molecular weight heparins and vitamin D are unclear . So, further investigation on drug dosing are warranted in obese children. Much attention has been given to early and delayed puberty. A worldwide growing decrease of puberty timing has been observed. Key features in the pathogenesis are nutrition in pregnancy, birth weight, dietary habits, physical activity, psychological factors, exposure to electromagnetic fields, endocrine-disruptors . Ferrari et al. showed in 577 girls that increased Z-score change from birth weight and higher BMI may predict earlier age at menarche and at thelarche. This underlines the role of modifiable factors in the onset of puberty. Among delayed puberty causes, constitutional delay of growth and puberty, a variant of normal growth is frequent. It may induce psychological difficulties, poor school performance and perhaps lower final height . So, therapeutic interventions have received interest in recent years. In adolescent > 14 years of age with psychological problems, a course of estradiol and progesterone in females or testosterone in males can be given. Importantly, despite clinical interest, the benefit of growth hormone on final height is uncertain . Type 1 diabetes is a disease that is heterogeneously growing in prevalence. Several studies have examined annual incidence that greatly varies from 0.1 to 100,000 person in China to 62.3 per 100,000 person in Scandinavia and from 19 to 45 per 100,000 person-years in different Italian regions. Passanisi et al. found an incidence of 20.6/100,000 person/years with an increase of 43% between 2019 and 2021 during Coronavirus Disease (COVID-19) pandemics in Calabria region, Italy. This supports the view that Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection may predispose to a diabetes onset . They also found an increase of children with severe ketoacidosis that can be justified by shutting of many health services during the pandemics. Menorrhagia is frequent after menarche onset. It is a challenging issue that often depend on anovulatory cycles due to the immaturity of the hypothalamic-pituitary-ovarian axis , but it can also be related to coagulopathy, pregnancy, medications, endocrine disorders, trauma, infection, cancer, structural pathologies, gastrointestinal bleeding . Barbero et al. delineated a girl with menorrhagia associated with short stature, bradycardia, dry skin, thinning hair. Hypothyroidism was confirmed by ultrasound showing an expanded thyroid with non-homogeneous hypoechogenic structure and laboratory tests showing anemia, high thyrotropin releasing hormone level, low free thyroxin, high serum anti-thyroid peroxidase antibodies and antithyroglobulin antibodies levels. Levothyroxine treatment was associated with remission of symptoms and regulation of laboratory parameters. This case highlights that follow up of the pubertal development is of particular importance for timely identification of underlying disorders. 1- Diet The effect of diet in the prevention and treatment of several conditions with gastrointestinal symptoms attracts constant attention. Pulvirenti et al. summarized studies on diseases that are cured by eliminating the offending food or need an appropriate dietary management to prevent malnutrition. A causal role of food has been found in food allergy, including eosinophil esophagitis and regurgitation. In infancy, early introduction into the diet of foods such as peanuts, seems to reduce the likelihood of developing food allergy compared with delayed introduction . In cystic fibrosis, the caloric intake should be 10–15% higher than in healthy children because of pancreatic insufficiency. So, diet should be supplemented with fat for energy requirements and pancreatic enzyme should be given . Breast-feeding and diet rich in fruit, vegetables and n-3 fatty acids are associated with a lower risk of developing inflammatory bowel disease. A specific diet is necessary for inflammatory bowel disease because of the risk of malnutrition due to low caloric intake, enteropathy, metabolic conditions, or steroid treatment. In active Crohn’s disease, enteral or parental nutrition with polymeric formulas, is the first option for 6–8 weeks . Short-bowel syndrome requires parental nutrition and gradually increase of enteral feeding that stimulates gastrointestinal functions. Breast milk or polymeric formulas are recommended in infants, oral or partially enteral solid foods in children. 2- Cyclic vomiting syndrome The Italian Society of Pediatric Gastroenterology, Hepatology and Nutrition and the Italian Society of Pediatric Neurology provided a timely survey on diagnosis and treatment of cyclic vomiting syndrome in 51 tertiary centers . They found a significant difference in diagnostic criteria between centers. Foods as triggers were more commonly identified by gastroenterologists and neuro-gastroenterologists than neurologist. Gastroenterologists more often diagnosed gastrointestinal diseases and checked ammonia and lactic acid levels. Besides supportive treatment, the most common medication given during vomiting, ondansetron followed by proton pump inhibitors, sedative, anti-H2 antihistamines, non steroidal antiinflammatories, steroids . Prophylactic treatment widely varied among centers. In about 50% of cases cyproheptadine was used, in 25% pizotifen, in 25% amitriptyline, in 15% mitochondrial supplements, in 10% anticonvulsants. Other drugs were aprepitant, propranolol, flunarizine, 5-hydroxytryptophan, proton pump inhibitors, paracetamol, magnesium supplementation. Probiotics have not been used . Overall, there is a need for a consensus to standardize the management of cyclic vomiting syndrome. The effect of diet in the prevention and treatment of several conditions with gastrointestinal symptoms attracts constant attention. Pulvirenti et al. summarized studies on diseases that are cured by eliminating the offending food or need an appropriate dietary management to prevent malnutrition. A causal role of food has been found in food allergy, including eosinophil esophagitis and regurgitation. In infancy, early introduction into the diet of foods such as peanuts, seems to reduce the likelihood of developing food allergy compared with delayed introduction . In cystic fibrosis, the caloric intake should be 10–15% higher than in healthy children because of pancreatic insufficiency. So, diet should be supplemented with fat for energy requirements and pancreatic enzyme should be given . Breast-feeding and diet rich in fruit, vegetables and n-3 fatty acids are associated with a lower risk of developing inflammatory bowel disease. A specific diet is necessary for inflammatory bowel disease because of the risk of malnutrition due to low caloric intake, enteropathy, metabolic conditions, or steroid treatment. In active Crohn’s disease, enteral or parental nutrition with polymeric formulas, is the first option for 6–8 weeks . Short-bowel syndrome requires parental nutrition and gradually increase of enteral feeding that stimulates gastrointestinal functions. Breast milk or polymeric formulas are recommended in infants, oral or partially enteral solid foods in children. The Italian Society of Pediatric Gastroenterology, Hepatology and Nutrition and the Italian Society of Pediatric Neurology provided a timely survey on diagnosis and treatment of cyclic vomiting syndrome in 51 tertiary centers . They found a significant difference in diagnostic criteria between centers. Foods as triggers were more commonly identified by gastroenterologists and neuro-gastroenterologists than neurologist. Gastroenterologists more often diagnosed gastrointestinal diseases and checked ammonia and lactic acid levels. Besides supportive treatment, the most common medication given during vomiting, ondansetron followed by proton pump inhibitors, sedative, anti-H2 antihistamines, non steroidal antiinflammatories, steroids . Prophylactic treatment widely varied among centers. In about 50% of cases cyproheptadine was used, in 25% pizotifen, in 25% amitriptyline, in 15% mitochondrial supplements, in 10% anticonvulsants. Other drugs were aprepitant, propranolol, flunarizine, 5-hydroxytryptophan, proton pump inhibitors, paracetamol, magnesium supplementation. Probiotics have not been used . Overall, there is a need for a consensus to standardize the management of cyclic vomiting syndrome. 1- Cat Eye syndrome Cat Eye syndrome is due to duplication and inversion of part of chromosome 22 . It is characterized by iris coloboma, anal atresia and ear anomalies in 40% of patients and it can have multiple malformations . Serra et al. described an infant with cat eye syndrome associated with persistent hypoglycemia and subsequent cholestasis, abnormal midline structures, including aplasia of the anterior pituitary gland, abnormal stalk, and ectopic neurohypophysis with hypopituitarism with cortisol, thyroxine, growth hormone deficits, corpus callosum hypoplasia. This report emphasizes that clinical presentation of cat eye syndrome is variable . So, the presence of additional malformations and/or abnormalities should always be considered. 2- Deletion of chromosome Contiguous gene syndromes are the result of a loss of multiple neighboring genes from a particular chromosomal segment. Contiguous gene syndromes have been identified in patients with various clinical features such as intellectual disability, developmental delay, and congenital anomalies . Serra et al. report on a female newborn with a 1p31.3p22.2 deletion of 20.7 Mb containing about ninety genes, inherited from the healthy mother with a smaller deletion (2.6 Mb) within the same centromeric region. Clinical phenotype included craniosynostosis, facial dysmorphisms with bilateral microphthalmia and coloboma, cleft palate, and a severe global developmental and growth delay . In this report, in view of a better characterization of a genomic and phenotypic profile, the authors paid attention to about 20 out of the involved genes, considered loss of function intolerant according to their haploinsuficiency score . A female term newborn with cardio-facio-cutaneous syndrome, a pathology belonging to RASopathies , a group of conditions caused by germline mutations in genes encoding components or regulators of the rat sarcoma/mitogen-activated protein kinase (RAS/MAPK) pathway was observed . Fetal macrosomia and a prenatal diagnosis of omphalocele suggested an array comparative genomic hybridization by amniocentesis that identified a 19p13.3 deletion including the MAP 2 K2 gene. Surgical correction of omphalocele took place on the second day of life. After discharge, at 1 month of age, she was readmitted for repeated episodes of vomiting, subtending a readily treated hypertrophic pyloric stenosis. The authors stress the need for integrated and individualized follow-up in all subjects with genetic syndromes, potentially carriers of abnormalities not evidentiable at birth . 3- Hao-Fountain syndrome Zampieri et al. report a case of Hao-Fountain syndrome (HAFOUS) a haploinsufficient condition caused by variants in the Ubiquitin-Specific Peptidase 7 gene (USP7), located on chromosome 16p13.2. USP7 is the largest family of deubiquitinating enzymes. Recent studies have shown that USP7 , as well as characterizing a complex neurodevelopmental disorder, plays a vital role in the regulation of various physiological and pathological processes related to endocrine complications, predisposition to autoimmune diseases, immunological deficit and a wide variety of cancers. The patient was a 15-year-old female patient with a complex neurodevelopmental disorder involving both cognitive and neurologic areas in which whole exome sequencing diagnosed a large de novo heterozygous deletions affecting USP7. The description in this patient of isolated tubal torsion expands the HAFOUS spectrum phenotype . Cat Eye syndrome is due to duplication and inversion of part of chromosome 22 . It is characterized by iris coloboma, anal atresia and ear anomalies in 40% of patients and it can have multiple malformations . Serra et al. described an infant with cat eye syndrome associated with persistent hypoglycemia and subsequent cholestasis, abnormal midline structures, including aplasia of the anterior pituitary gland, abnormal stalk, and ectopic neurohypophysis with hypopituitarism with cortisol, thyroxine, growth hormone deficits, corpus callosum hypoplasia. This report emphasizes that clinical presentation of cat eye syndrome is variable . So, the presence of additional malformations and/or abnormalities should always be considered. Contiguous gene syndromes are the result of a loss of multiple neighboring genes from a particular chromosomal segment. Contiguous gene syndromes have been identified in patients with various clinical features such as intellectual disability, developmental delay, and congenital anomalies . Serra et al. report on a female newborn with a 1p31.3p22.2 deletion of 20.7 Mb containing about ninety genes, inherited from the healthy mother with a smaller deletion (2.6 Mb) within the same centromeric region. Clinical phenotype included craniosynostosis, facial dysmorphisms with bilateral microphthalmia and coloboma, cleft palate, and a severe global developmental and growth delay . In this report, in view of a better characterization of a genomic and phenotypic profile, the authors paid attention to about 20 out of the involved genes, considered loss of function intolerant according to their haploinsuficiency score . A female term newborn with cardio-facio-cutaneous syndrome, a pathology belonging to RASopathies , a group of conditions caused by germline mutations in genes encoding components or regulators of the rat sarcoma/mitogen-activated protein kinase (RAS/MAPK) pathway was observed . Fetal macrosomia and a prenatal diagnosis of omphalocele suggested an array comparative genomic hybridization by amniocentesis that identified a 19p13.3 deletion including the MAP 2 K2 gene. Surgical correction of omphalocele took place on the second day of life. After discharge, at 1 month of age, she was readmitted for repeated episodes of vomiting, subtending a readily treated hypertrophic pyloric stenosis. The authors stress the need for integrated and individualized follow-up in all subjects with genetic syndromes, potentially carriers of abnormalities not evidentiable at birth . Zampieri et al. report a case of Hao-Fountain syndrome (HAFOUS) a haploinsufficient condition caused by variants in the Ubiquitin-Specific Peptidase 7 gene (USP7), located on chromosome 16p13.2. USP7 is the largest family of deubiquitinating enzymes. Recent studies have shown that USP7 , as well as characterizing a complex neurodevelopmental disorder, plays a vital role in the regulation of various physiological and pathological processes related to endocrine complications, predisposition to autoimmune diseases, immunological deficit and a wide variety of cancers. The patient was a 15-year-old female patient with a complex neurodevelopmental disorder involving both cognitive and neurologic areas in which whole exome sequencing diagnosed a large de novo heterozygous deletions affecting USP7. The description in this patient of isolated tubal torsion expands the HAFOUS spectrum phenotype . 1- Bullying A research carried out in Palermo, Italy, investigated the phenomenon of bullying , and includes an analysis of the data on bullying reported in the scientific literature . The authors have administered, to 22,455 school-aged children, attending one of the 58 secondary first-grade schools of Palermo during the school year 2017/18, a questionnaire of 30 items investigating the main areas related to bullying, such as physical, verbal and indirect bullying, observers of bullying, resiliency, and prosociality. Results showed an increase of bullying, reported in 2011 to be present in about 14% of young people, and involving up to 66% of young people in the area of verbal bullying. Bullying resulted more frequent in higher school classes, specifically in students attending third level classes in schools with lower socioeconomic index. The authors conclude that interventions and preventive measures in public health programs should be implemented to stem this phenomenon . 2- Mobile phone dependence Mobile phone dependence, considered a counterproductive use of mobile phone for interpersonal communication, information acquisition, self-expression, and leisure and entertainment, is a scourge of modern society that affects large segments of the youth population . In this report the authors analyse the correlations between self-core evaluation (SCE) (a personality trait that encompasses an individual’s subconscious, fundamental evaluations about themselves, their own abilities, and their own control), mental state and cell phone addiction in a sample of high school students . The results of the analysis, since SCE had a significant negative correlation with mobile phone dependence, show how SCE plays an important mediating role between mental health and mobile phone dependence . The authors conclude that improving high students’ SCE may be beneficial to improve the health status and reduce mobile phones dependence. 3- Child abuse Offidani et al. describe the process that led to the editing of the indicators of child abuse and the codification of three clinical pathways to apply in case of suspected child abuse at the Bambino Gesù Children’s Hospital . Field work has identified three areas of assessment that constitute a new screening tool (ST) created in 2009 and used in clinical practice from 2010. The fourteen items included in the ST have been grouped in three clusters: anamnestic declarations or incongruences, carelessness/neglect and evident lesions at physical examination. All items of the ST are reported in the text. Adopting the ST from 2010 to 2020, the authors report improvement in diagnostic accuracy resulting in reduction of under-diagnosed cases and confirm that females are more likely to experience sexual abuse, while males are more likely to experience physical abuse. It would be useful to share these indicators with non-paediatric hospitals, that do not have the same experience in recognizing cases of child abuse . 4- Burns Tiruneh et al. conducted this institutional-based cross-sectional study on children admitted for burns to South Gondar zone hospitals from 2015 to 2019. They used for data collection a checklist including sociodemographic characteristics, as nutritional status, comorbidity, availability of health insurance, medical history such as the place of the accident, the mode of occurrence, the timing of hospitalization, the degree of burn, the duration of hospitalization and the therapeutic measures taken . The mortality among burn victim children (8,5%) was higher than most of the studies conducted all over the world and similar to data from East Africa and Tanzania. No medical insurance, being malnourished, burnt by electrical and flame burn, having total body surface area burnt greater than 20%, and having poor clinical condition at admission increased mortality by four times compared to a good clinical condition. These results may suggest priority actions to reduce mortality and worst outcomes in children . 5- Undernutrition Undernutrition is a major public health issue especially in some countries of Africa and Asia . Inadequate feeding practices is a key factor associated with under-nutrition in children younger than 5 years of age . Bidira et al. educated caregivers of Ethiopian preschool children on healthy diet, nutrition, hygiene for 9 months. They showed that wasting and underweight significantly improved in the intervention group compared with controls. These findings highlight that the strategy of community-based nutritional education is effective in ameliorating nutritional status of young children . A research carried out in Palermo, Italy, investigated the phenomenon of bullying , and includes an analysis of the data on bullying reported in the scientific literature . The authors have administered, to 22,455 school-aged children, attending one of the 58 secondary first-grade schools of Palermo during the school year 2017/18, a questionnaire of 30 items investigating the main areas related to bullying, such as physical, verbal and indirect bullying, observers of bullying, resiliency, and prosociality. Results showed an increase of bullying, reported in 2011 to be present in about 14% of young people, and involving up to 66% of young people in the area of verbal bullying. Bullying resulted more frequent in higher school classes, specifically in students attending third level classes in schools with lower socioeconomic index. The authors conclude that interventions and preventive measures in public health programs should be implemented to stem this phenomenon . Mobile phone dependence, considered a counterproductive use of mobile phone for interpersonal communication, information acquisition, self-expression, and leisure and entertainment, is a scourge of modern society that affects large segments of the youth population . In this report the authors analyse the correlations between self-core evaluation (SCE) (a personality trait that encompasses an individual’s subconscious, fundamental evaluations about themselves, their own abilities, and their own control), mental state and cell phone addiction in a sample of high school students . The results of the analysis, since SCE had a significant negative correlation with mobile phone dependence, show how SCE plays an important mediating role between mental health and mobile phone dependence . The authors conclude that improving high students’ SCE may be beneficial to improve the health status and reduce mobile phones dependence. Offidani et al. describe the process that led to the editing of the indicators of child abuse and the codification of three clinical pathways to apply in case of suspected child abuse at the Bambino Gesù Children’s Hospital . Field work has identified three areas of assessment that constitute a new screening tool (ST) created in 2009 and used in clinical practice from 2010. The fourteen items included in the ST have been grouped in three clusters: anamnestic declarations or incongruences, carelessness/neglect and evident lesions at physical examination. All items of the ST are reported in the text. Adopting the ST from 2010 to 2020, the authors report improvement in diagnostic accuracy resulting in reduction of under-diagnosed cases and confirm that females are more likely to experience sexual abuse, while males are more likely to experience physical abuse. It would be useful to share these indicators with non-paediatric hospitals, that do not have the same experience in recognizing cases of child abuse . Tiruneh et al. conducted this institutional-based cross-sectional study on children admitted for burns to South Gondar zone hospitals from 2015 to 2019. They used for data collection a checklist including sociodemographic characteristics, as nutritional status, comorbidity, availability of health insurance, medical history such as the place of the accident, the mode of occurrence, the timing of hospitalization, the degree of burn, the duration of hospitalization and the therapeutic measures taken . The mortality among burn victim children (8,5%) was higher than most of the studies conducted all over the world and similar to data from East Africa and Tanzania. No medical insurance, being malnourished, burnt by electrical and flame burn, having total body surface area burnt greater than 20%, and having poor clinical condition at admission increased mortality by four times compared to a good clinical condition. These results may suggest priority actions to reduce mortality and worst outcomes in children . Undernutrition is a major public health issue especially in some countries of Africa and Asia . Inadequate feeding practices is a key factor associated with under-nutrition in children younger than 5 years of age . Bidira et al. educated caregivers of Ethiopian preschool children on healthy diet, nutrition, hygiene for 9 months. They showed that wasting and underweight significantly improved in the intervention group compared with controls. These findings highlight that the strategy of community-based nutritional education is effective in ameliorating nutritional status of young children . 1- Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) can cause severe and highly prevalent diseases in the pediatric population. During the COVID-19 pandemic an increased rate of MRSA infections has been described in adults. In a study conducted in the period 2017–2020, which included the pandemic period, a similar epidemiologic trend in patients aged < 18 years, has not been confirmed . Moreover, the efficacy against MRSA of several antibiotics was tested and linezolid and vancomycin were identified as the only antibiotics to which MRSA did not develop resistance. Of particular interest to the reader are the in-depth analysis of the factors related to methicillin resistance, as well as the antimicrobial susceptibility pattern according to methicillin resistance . The authors conclude that surveillance of antimicrobial resistance is essential to improve infection control, antibiotic prescriptions, and preventions policies . 2- Epstein-Barr virus Accomando et al. report a child with symptomatic pancreatitis associated to Epstein-Barr virus infection. In this case, characterized by an enlarged pancreas visualized by abdominal ultrasound, the etiological diagnosis was only achieved by a positive serology for the presence of Epstein-Barr virus VCA IgM and IgG. The review of the few cases described in the literature draws attention to the importance of differential diagnosis that include specific serological research even in the absence of the classic clinical and haematological characteristics of Epstein-Barr virus infection . Generally, Epstein-Barr virus-associated acute pancreatitis is characterized by a favorable prognosis, with a spontaneous resolution . Methicillin-resistant Staphylococcus aureus (MRSA) can cause severe and highly prevalent diseases in the pediatric population. During the COVID-19 pandemic an increased rate of MRSA infections has been described in adults. In a study conducted in the period 2017–2020, which included the pandemic period, a similar epidemiologic trend in patients aged < 18 years, has not been confirmed . Moreover, the efficacy against MRSA of several antibiotics was tested and linezolid and vancomycin were identified as the only antibiotics to which MRSA did not develop resistance. Of particular interest to the reader are the in-depth analysis of the factors related to methicillin resistance, as well as the antimicrobial susceptibility pattern according to methicillin resistance . The authors conclude that surveillance of antimicrobial resistance is essential to improve infection control, antibiotic prescriptions, and preventions policies . Accomando et al. report a child with symptomatic pancreatitis associated to Epstein-Barr virus infection. In this case, characterized by an enlarged pancreas visualized by abdominal ultrasound, the etiological diagnosis was only achieved by a positive serology for the presence of Epstein-Barr virus VCA IgM and IgG. The review of the few cases described in the literature draws attention to the importance of differential diagnosis that include specific serological research even in the absence of the classic clinical and haematological characteristics of Epstein-Barr virus infection . Generally, Epstein-Barr virus-associated acute pancreatitis is characterized by a favorable prognosis, with a spontaneous resolution . 1- Familial hypercholesterolemia Familial hypercholesterolemia is the most common hereditary disorder of lipid metabolism causing life-long accumulation of low-density lipoprotein cholesterol . In this article, Banderali et al. highlight the main diagnostic strategies to identify this condition that, if left untreated, can lead to atherosclerosis and possible premature coronary heart disease and other vascular. Early detection of the condition is important to prevent complications and improve life expectancy. Cardiovascular events are rare among children and adolescents. Thus, identification of suggestive symptoms and family history is crucial. Some of the most typical signs of FH are: corneal arcus, xanthelasma and xanthomas . Currently, screening tools are not available for pediatric patients, especially younger than 2 years old. Performing a cascade screening of close relatives, starting by drawing a pedigree and then identifying the subjects with a cardiovascular event or hypercholesterolemia (clinical or genetically defined) would be suitable. If familial hypercholesterolemia is reported, hypercholesterolemia or a cardiovascular event is found in all generations. The gold standard diagnostic tool of familial hypercholesterolemia includes the detection of familial hypercholesterolemia -causing mutation . Further multidisciplinary team works are needed to identify optimal models of diagnosis and treatment of this life-threatening condition. 2- Pompe disease A review , enriched with the shared patient experience of the Italian Pediatric study group on immune response to enzymatic replacement therapy (ERT) with alglucosidase alfa in patients with Pompe disease, deals in depth with key elements of the complex and individualized management of immune response to ERT . Main objectives of the analysis, supported by useful diagrams, are identification of two types of immune reactions against ERT, quantification of risk factors for immunogenicity, choose of prophylaxis protocols in relation to cross reactive immunological material status, importance of immune-tolerance induction protocols in patients who develop antibodies during ERT, recommendations on the test to be performed before starting immunomodulation and tips on managing the frequent (up to 50%) hypersensitivity reactions during ERT. The authors conclude that further studies are needed to improve actual management protocols . Familial hypercholesterolemia is the most common hereditary disorder of lipid metabolism causing life-long accumulation of low-density lipoprotein cholesterol . In this article, Banderali et al. highlight the main diagnostic strategies to identify this condition that, if left untreated, can lead to atherosclerosis and possible premature coronary heart disease and other vascular. Early detection of the condition is important to prevent complications and improve life expectancy. Cardiovascular events are rare among children and adolescents. Thus, identification of suggestive symptoms and family history is crucial. Some of the most typical signs of FH are: corneal arcus, xanthelasma and xanthomas . Currently, screening tools are not available for pediatric patients, especially younger than 2 years old. Performing a cascade screening of close relatives, starting by drawing a pedigree and then identifying the subjects with a cardiovascular event or hypercholesterolemia (clinical or genetically defined) would be suitable. If familial hypercholesterolemia is reported, hypercholesterolemia or a cardiovascular event is found in all generations. The gold standard diagnostic tool of familial hypercholesterolemia includes the detection of familial hypercholesterolemia -causing mutation . Further multidisciplinary team works are needed to identify optimal models of diagnosis and treatment of this life-threatening condition. A review , enriched with the shared patient experience of the Italian Pediatric study group on immune response to enzymatic replacement therapy (ERT) with alglucosidase alfa in patients with Pompe disease, deals in depth with key elements of the complex and individualized management of immune response to ERT . Main objectives of the analysis, supported by useful diagrams, are identification of two types of immune reactions against ERT, quantification of risk factors for immunogenicity, choose of prophylaxis protocols in relation to cross reactive immunological material status, importance of immune-tolerance induction protocols in patients who develop antibodies during ERT, recommendations on the test to be performed before starting immunomodulation and tips on managing the frequent (up to 50%) hypersensitivity reactions during ERT. The authors conclude that further studies are needed to improve actual management protocols . 1- Preterm infant Parents of premature newborns admitted to neonatal intensive care unit (NICU) experience high levels of stress, determined both by the basic individual psychological well-being and the risk profile, the clinical complications, and the duration of the stay of their child. These conditions can have psychological consequences such as post-traumatic stress disorder . Salomè et al. examined how the state of parental psychological well-being and stress experienced affect the appearance of post-traumatic stress disorder in the year following discharge. The authors use an up-to-date and valuable set of psychological tests, described in the article and useful to the reader for planning similar studies. The results identify a higher risk of post-traumatic stress disorder in mothers compared to fathers (55% vs. 20%), with specific correlations between specific experiences and emotional reactions. In addition, they provide interesting research cues and identification of targeted intervention to reduce the risk of a post-traumatic stress disorder in parents of the preterm infants . In the position paper of the Italian scientific societies of pediatric area , recommendations are suggested regarding the important step of the introduction of complementary feeding in preterm infants. The main objectives are to reduce the risk of extrauterine growth restriction and the features of altered body composition with reduction of free mass and increased adiposity . Main recommendations include to start complementary feeding between 5 and 8 months of chronological age, consider the limit of 3 months corrected age to ensure the acquisition of developmental skills allowing the consumption of solid foods, consider multidisciplinary assessment before starting complementary feeding in case of oral dysfunction or comorbidities, type (included allergenic food), sequence and speed of introduction of food, vitamins, and micronutrients. Exclusive breastfeeding, mixed feeding or standard infant formula enriched with long chain polyunsaturated fatty acids should be preferred for infants without extrauterine growth restriction while extrauterine growth restriction infants or who is at risk of long-term growth failure may be fed with fortified human milk or formula adapted for preterm infants as long as to gain an optimal weight for corrected age. 2- HIV infection In West Amhara, Ethiopia an unmatched case–control study from 2016 to 2020, aimed to identify determinants of HIV infection among children born to HIV positive mothers on the prevention of the mother -child transmission program at referral hospitals . The region is characterized by an important HIV public health problem with a high transmission rate. The identified determinants were home delivery, mixed feeding, poor maternal antiretroviral drug adherence, advanced World Health Organization (WHO) clinical stage, poor nevirapine adherence and late enrollment of the infant. The authors conclude that Minister of Health and non-governmental organizations should work on mobilization of the community and awareness creation on the important of exclusive breast feeding, drugs adherence, on benefits of health institutional delivery as well as the risk of homedelivery. Public health interventions are very important tools to reduce mother-child transmission of HIV . 3- Loeys-Dietz syndrome Loeys-Dietz syndrome is a rare connective tissue disorder related to a pathogenic variant in TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2 or TGFB3 genes. It involves the aorta with progressive dilatation, craniofacial skeleton, joints, skin, and is associated with hypotonia and motor delay . In a first patient a trio based Whole Exome Sequencing found a novel, heterozygote, missense, de novo variant in the TGFBR2 gene. In a second patient , with a suspicious family history, a genetic panel for connective tissue disorders identified a pathogenetic variant in TGFB3 gene. Only in the first male patient the aortic aneurysm progressed despite of a low-dose angiotensin receptor blocker therapy, while the second female patient showed no aortic abnormalities. An early diagnosis of Loeys-Dietz syndrome implies a potential modification of the natural history of the disease with early interventions on its complications . 4- Hyperbilirubinemia A meta-analysis evaluates, in case of neonatal indirect hyperbilirubinemia in treatment with phototherapy, the use of ursodeoxycholic acid as adjunctive therapy . Ursodeoxycholic acid is classically used for the management of cholestatic liver pathology through increased production of bile, the removal of the most toxic components of bile acids and additional hepatoprotective and neuroprotective effects . The five selected studies from scientific literature suggest that the addition of ursodeoxycholic acid to phototherapy could reduce phototherapy duration by almost 18 h compared to phototherapy only. It also resulted in a lower mean of total serum bilirubin in the 48 h post-treatment, especially in Asian countries. Two infants with isolated severe unconiugated hyperbilirubinemia related to Gilbert syndrome have been reported . In both infants the prolonged indirect hyperbilirubinemia associated with the finding of a mutation p.Pro364Leu in the bilirubin uridine diphosphate-glucuronosyltransferase gene raised the initial suspicion of Crigler-Najjar syndrome type II. Both infants were treated with phototherapy and phenobarbital, achieving a normalization of bilirubin levels at 1 and 5 months respectively . The authors suggest that in these patients the concomitant breastfeeding jaundice was not a possible explanation of the clinical picture since a brief interruption of breastfeeding was not effective. 5- Frenotomy In a prospective observational cohort study, Dall’Olio et al. evaluated diode laser frenotomy in fifty-five newborns with ankyloglossia with or without difficult breastfeeding. Important functional consequences of ankyloglossia were manifested by 20 to 50% of cases adversely affecting the nutrition and growth of the newborn and the degree of adhesion of the mother to breastfeeding, her psychological well-being and bonding . The suggestions of the clinical tools used for evaluation of ankyloglossia, quality of breastfeeding, and perioperative pain of the newborn are useful. Short duration of the diode laser frenotomy, minimal reported complications, short hospital stay, and positive effects on breastfeeding, are elements in favor of the implementation of this procedure. 6- Hypothermia A single center, parallel-group, and no-blinded randomized controlled , has been conducted in a level III, and academic neonatal intensive care unit in China from 2020 to 2021, to evaluate the effect of early vs. delayed enteral nutrition on the incidence of feeding intolerance and secondary outcome including the incidences of late-onset bloodstream infection, hypoglycemia, survival at neonatal discharge, duration of parenteral nutrition, duration until attainment of full enteral feeds, length of hospital stay and body weight gain, in newborns treated with therapeutic hypothermia. Feeding strategy consisted of minimal enteral feeding and slow speed of increasing milk feeds. Early enteral nutrition was performed during therapeutic hypothermia /rewarming period while delayed enteral nutrition was performed after the therapeutic hypothermia phase. This study showed that the average time of parenteral nutrition, reaching full enteral feeds and hospital stay were shorter in the early enteral nutrition group compared with the delayed group with significant differences . Parents of premature newborns admitted to neonatal intensive care unit (NICU) experience high levels of stress, determined both by the basic individual psychological well-being and the risk profile, the clinical complications, and the duration of the stay of their child. These conditions can have psychological consequences such as post-traumatic stress disorder . Salomè et al. examined how the state of parental psychological well-being and stress experienced affect the appearance of post-traumatic stress disorder in the year following discharge. The authors use an up-to-date and valuable set of psychological tests, described in the article and useful to the reader for planning similar studies. The results identify a higher risk of post-traumatic stress disorder in mothers compared to fathers (55% vs. 20%), with specific correlations between specific experiences and emotional reactions. In addition, they provide interesting research cues and identification of targeted intervention to reduce the risk of a post-traumatic stress disorder in parents of the preterm infants . In the position paper of the Italian scientific societies of pediatric area , recommendations are suggested regarding the important step of the introduction of complementary feeding in preterm infants. The main objectives are to reduce the risk of extrauterine growth restriction and the features of altered body composition with reduction of free mass and increased adiposity . Main recommendations include to start complementary feeding between 5 and 8 months of chronological age, consider the limit of 3 months corrected age to ensure the acquisition of developmental skills allowing the consumption of solid foods, consider multidisciplinary assessment before starting complementary feeding in case of oral dysfunction or comorbidities, type (included allergenic food), sequence and speed of introduction of food, vitamins, and micronutrients. Exclusive breastfeeding, mixed feeding or standard infant formula enriched with long chain polyunsaturated fatty acids should be preferred for infants without extrauterine growth restriction while extrauterine growth restriction infants or who is at risk of long-term growth failure may be fed with fortified human milk or formula adapted for preterm infants as long as to gain an optimal weight for corrected age. In West Amhara, Ethiopia an unmatched case–control study from 2016 to 2020, aimed to identify determinants of HIV infection among children born to HIV positive mothers on the prevention of the mother -child transmission program at referral hospitals . The region is characterized by an important HIV public health problem with a high transmission rate. The identified determinants were home delivery, mixed feeding, poor maternal antiretroviral drug adherence, advanced World Health Organization (WHO) clinical stage, poor nevirapine adherence and late enrollment of the infant. The authors conclude that Minister of Health and non-governmental organizations should work on mobilization of the community and awareness creation on the important of exclusive breast feeding, drugs adherence, on benefits of health institutional delivery as well as the risk of homedelivery. Public health interventions are very important tools to reduce mother-child transmission of HIV . Loeys-Dietz syndrome is a rare connective tissue disorder related to a pathogenic variant in TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2 or TGFB3 genes. It involves the aorta with progressive dilatation, craniofacial skeleton, joints, skin, and is associated with hypotonia and motor delay . In a first patient a trio based Whole Exome Sequencing found a novel, heterozygote, missense, de novo variant in the TGFBR2 gene. In a second patient , with a suspicious family history, a genetic panel for connective tissue disorders identified a pathogenetic variant in TGFB3 gene. Only in the first male patient the aortic aneurysm progressed despite of a low-dose angiotensin receptor blocker therapy, while the second female patient showed no aortic abnormalities. An early diagnosis of Loeys-Dietz syndrome implies a potential modification of the natural history of the disease with early interventions on its complications . A meta-analysis evaluates, in case of neonatal indirect hyperbilirubinemia in treatment with phototherapy, the use of ursodeoxycholic acid as adjunctive therapy . Ursodeoxycholic acid is classically used for the management of cholestatic liver pathology through increased production of bile, the removal of the most toxic components of bile acids and additional hepatoprotective and neuroprotective effects . The five selected studies from scientific literature suggest that the addition of ursodeoxycholic acid to phototherapy could reduce phototherapy duration by almost 18 h compared to phototherapy only. It also resulted in a lower mean of total serum bilirubin in the 48 h post-treatment, especially in Asian countries. Two infants with isolated severe unconiugated hyperbilirubinemia related to Gilbert syndrome have been reported . In both infants the prolonged indirect hyperbilirubinemia associated with the finding of a mutation p.Pro364Leu in the bilirubin uridine diphosphate-glucuronosyltransferase gene raised the initial suspicion of Crigler-Najjar syndrome type II. Both infants were treated with phototherapy and phenobarbital, achieving a normalization of bilirubin levels at 1 and 5 months respectively . The authors suggest that in these patients the concomitant breastfeeding jaundice was not a possible explanation of the clinical picture since a brief interruption of breastfeeding was not effective. In a prospective observational cohort study, Dall’Olio et al. evaluated diode laser frenotomy in fifty-five newborns with ankyloglossia with or without difficult breastfeeding. Important functional consequences of ankyloglossia were manifested by 20 to 50% of cases adversely affecting the nutrition and growth of the newborn and the degree of adhesion of the mother to breastfeeding, her psychological well-being and bonding . The suggestions of the clinical tools used for evaluation of ankyloglossia, quality of breastfeeding, and perioperative pain of the newborn are useful. Short duration of the diode laser frenotomy, minimal reported complications, short hospital stay, and positive effects on breastfeeding, are elements in favor of the implementation of this procedure. A single center, parallel-group, and no-blinded randomized controlled , has been conducted in a level III, and academic neonatal intensive care unit in China from 2020 to 2021, to evaluate the effect of early vs. delayed enteral nutrition on the incidence of feeding intolerance and secondary outcome including the incidences of late-onset bloodstream infection, hypoglycemia, survival at neonatal discharge, duration of parenteral nutrition, duration until attainment of full enteral feeds, length of hospital stay and body weight gain, in newborns treated with therapeutic hypothermia. Feeding strategy consisted of minimal enteral feeding and slow speed of increasing milk feeds. Early enteral nutrition was performed during therapeutic hypothermia /rewarming period while delayed enteral nutrition was performed after the therapeutic hypothermia phase. This study showed that the average time of parenteral nutrition, reaching full enteral feeds and hospital stay were shorter in the early enteral nutrition group compared with the delayed group with significant differences . 1- Drooling Drooling is defined as the involuntary loss of saliva and oral content. While most children reach the salivary continence by age 15–18 months, the persistence of drooling is common in children with neurological disorders, like cerebral palsy (CP) and is associated with oral motor dysfunction, dysphagia, and/or intraoral sensitivity disorder . Several therapeutic interventions are used to reduce or eliminate drooling. These include surgery, drugs, botulinum toxin, physical therapies to improve sensory function, behavioral interventions, appliances placed in the oral cavity, and acupuncture . There is no consensus on which interventions are safe and effective in managing drooling in children with CP. As the salivary glands are controlled by the parasympathetic autonomic nervous system, the anticholinergic drugs are indeed widely used to reduce the volume of saliva. Glycopyrrolate is the only oral formulation including an anticholinergic agent approved by the United States Food and Drug Administration to treat drooling in children 3–16 years-old which inhibits the acetylcholine receptors on peripheral tissues and reduces the saliva rate production . Lovardi et al. described a case series of eighteen children (median age 17 months, range 2–36 months) with CP or genetic/malformative syndrome, who have been administered oral glycopyrrolate (0.065 mg/kg/die in 3 daily doses) for drooling control. The Drooling Impact Scale was administered at time O and after 1 month. Results showed a significant reduction of the Drooling Impact Scale after 1 month (89 versus 61; p < 0.001), with few adverse effects and an overall response to treatment equal to 94%. Further studies are needed to confirm these results. 2- Alternating hemiplegia Alternating hemiplegia of childhood is an uncommon complex disorder, which was first described in late 1960s by Verret and Steele . Alternating hemiplegia of childhood is characterized by paroxysmal episodes of repeated, transient paresis involving either or both sides of the body and usually presents before age 18 months. The diagnosis of alternating hemiplegia of childhood is mainly clinical but may be supported by molecular analysis. Although the pathophysiologic mechanism of the disorder is partially unclear, it is well known that a relevant role is played by mutations in ATP1A2 and ATP1A3 genes, which encode two different alpha subunits of the Na+/K + ATPase transmembrane ion pump, respectively . Pavone et al. reported on the clinical and genetic findings of a couple of twins and a couple of siblings with alternating hemiplegia of childhood from two different Italian families affected. In the twins a pathogenic variant in ATP1A3 gene (c.2318 A > G) was detected. In the siblings, the younger brother showed a novel GRIN2A variant (c.3175 T > A), while the older one carried the same GRIN2A variant, associated with two likely pathogenetic variants in SCNIB (c.632 > A) and KCNQ2 (1870 G > A) genes, which have been implicated in childhood epilepsies. This report provides additional information about alternating hemiplegia of childhood, showing that the variability of clinical features is mirrored by an unexpected genetic heterogeneity. Clinical signs of alternating hemiplegia of childhood usually follow a sequential pattern, in which the paroxysmal episodes are triggered by precipitating factors such as environmental stress, bathing or other events. Non-paroxysmal features include developmental delay/intellectual disability, epilepsy, autonomic dysfunctions, abnormal eye involvement, movement disorders, ataxia, dystonia, and choreoathetosis . 3- Autism Autism spectrum disorder is a term used to describe a set of social communication deficits and repetitive sensory–motor behaviors. It is a neurological developmental disorder, and is characterized by a complex etiology involving genetic, environmental, and biochemical factors . The autism spectrum includes several disorders, such as the Autistic disorder, the Rett disorder, the Asperger syndrome, and the pervasive developmental disorder. Although individuals with autism spectrum disorder are very different from one another, the disorder affects social interactions, verbal and nonverbal social communication skills, as well as intelligence and motor functions, usually originating unusual interests and repetitive behaviors . Epidemiological surveys have shown a rapid increase in autism spectrum disorder prevalence rate. Besides the real increase in prevalence, a variety of other reasons may contribute to the disorder, such as a broader definition of autism spectrum disorder, changes in diagnostic criteria and screening tools, and finally an increased awareness of autism spectrum disorder . In this scenario, Salari et al. conducted a systematic review and meta-analysis from 2008 to July 2021 finalized to clarify the global prevalence of autism spectrum disorder The results show that the world prevalence of autism spectrum disorder is 0.6% (95% confidence interval: 0.4;1%), with slight differences between Asia (0.4%), America (1%), Europe (0.5%), Africa (1%) or Australia (1.7%). Standardized screening for autism spectrum disorder with ongoing developmental surveillance continues to be recommended in primary care at 18 and 24 months of age, because autism spectrum disorder is common, can be diagnosed as young as 18 months of age, and medical interventions may significantly affect neurological compromise . These data should be disseminated by health policymakers in order to implement appropriate planning and interventions. 4- Pontocerebellar hypoplasia Pontocerebellar hypoplasia includes a heterogeneous group of neurodegenerative disorders with prenatal onset, characterized by severe microcephaly, global developmental delay and radiological manifestation, such as hypoplasia of pons and cerebellum . At least 21 pontocerebellar hypoplasia -related genes are currently listed in the OMIM-database and 15 types of pontocerebellar hypoplasia are known, all characterized by patients’ motor and cognitive impairment. However, the clinical presentation can be very different, ranging from lethal neonatal subtypes to milder variants with survival up to adolescence . The study from Bilge et al. consisted of the description of different clinical and radiological manifestations of six genetically diagnosed patients with pontocerebellar hypoplasia, highlighting the differences in onset and progression of the symptoms . The genetic mutations found in 4 out 6 cases were well known variants that had been previously reported in the literature, while an additional subject was homozygous for the TBC1D23 gene mutation (c.1263 + 1G > A), which was a novel variant never reported until now. Although the last case had the same clinical features, the whole-exome sequencing revealed compound heterozygous mutations in the BRF1 gene, which is also associated with cerebellofaciodental syndrome. Common problems in pontocerebellar hypoplasia include sleep apnea, feeding problems, epilepsy, movement disorders, rhabdomyolysis, and extremely elevated serum creatinine kinase, especially during infections . There is no definite treatment for any type of pontocerebellar hypoplasia, and management is supportive in all types and subtypes. 5- Vertigo Vertigo is a disorder of space sensitivity characterized by an illusory and unpleasant sensation of body movement toward the environment or vice versa. Vertigo is characterized by different clinical features, therefore it is often difficult for the single physician to identify its etiopathogenesis, especially when the vertigo is the only clinical presenting sign . Compared to adults, vertigo is not so common in children and adolescents. Indeed, while adults surveys reported a one-year prevalence of 23% for unspecified dizziness and 5% for vestibular vertigo, in pediatric patients a prevalence of only 0.4% for nonspecific dizziness, 0.03% for peripheral, and 0.02% for central vestibulopathy is reported . Nevertheless, it is a common reason for emergency department presentation, both alone or associated with other symptoms. Pellegrino et al. reported on the etiopathology of neurological vertigo in childhood and adulthood, highlighting the characteristics which may lead clinicians to a proper diagnosis, and proposed a diagnostic algorithm to support the approach to patients with isolated vertigo, both in pediatric and adult age. The most common cause of isolated vertigo in the pediatric population is vestibular migraine, followed by benign paroxysmal vertigo in childhood. Other causes of vertigo are orthostatic hypotension, vestibular neuritis and vestibular paroxysmia, neurovascular diseases, tumors and demyelinating . Authors underline that the outcome of most neurological vertigos in childhood is good, in contrary to what is reported in adults. 6- Idiopathic intracranial hypertension Idiopathic intracranial hypertension is very rare in the pediatric age, and etiology is still largely unknown. Del Monte et al. presented a case report of an 8-month old male affected by idiopathic intracranial hypertension, and provide discussion on what literature suggests about optimal management also including the therapeutic strategies in idiopathic intracranial hypertension. The presented case was admitted with vomit, anorexia, irritability and bulging anterior fontanel. Brain magnetic resonance and cerebrospinal fluid analysis were negative. After diagnosis and treatment with acetazolamide and corticosteroids, progressive resolution of symptoms was observed. Pseudotumor cerebri syndrome is a rare condition characterized by elevated intracranial pressure, normal cerebrospinal fluid analysis in the absence of intracranial lesions on neuroimaging. Most common type of pseudotumor cerebri syndrome in both children and adults is the primary type known as idiopathic intracranial hypertension . Secondary types can result from vascular malformations, underlying systemic conditions or drugs. Female gender, obesity, and polycystic ovary represent risk factors for idiopathic intracranial hypertension in adolescents, but not in younger ones . Clinical manifestations of idiopathic intracranial hypertension vary with age. In pediatric populations, symptoms are often unspecific, including vomiting, headache, irritability, hyporeactivity, anorexia, sleep disruption, head tilting, papilledema and bulging fontanelle in infants. Based on adults’ treatment guidelines, also in children the drug of choice is acetazolamide, that could be associated with intravenous corticosteroids if severe visual impairment is present as well. So far, the moment, acetazolamide alone is preferred because of fewer side effects. Most common complications of idiopathic intracranial hypertension are vision loss and relapse, especially in the first 18 months after diagnosis . Early diagnosis, treatment and strict follow-up of complications may help in reducing the risk of relapse and may prevent vision loss. 7- Pressure palsies Karlinsky et al. reported their experience about an unusual case of hereditary neuropathy with liability to pressure palsies (HNPP) who had limping as a major presenting symptom. HNPP is an autosomal dominant disease characterized by recurrent, episodic demyelinating neuropathy caused by 17p11.2 chromosomal deletion encompassing the PMP22 gene. The onset is acute, usually in adulthood, and involves a single nerve, most frequently peroneal and ulnar, with a non-painful focal sensory and motor neuropathy . The onset in children most commonly involves peroneal nerve palsy and brachial plexus palsy . Other signs are previous atrophy, focal weakness or sensory loss, reduction of tendon reflexes and mild to moderate pes cavus foot deformity . The definitive diagnosis is provided by genetic analysis, and treatment is mostly supportive. Prognosis is good with an expected full recovery within months or days in best case scenarios. In rare cases a persistence of severe symptoms is reported. Since presenting phenotypes can be extremely variable, the authors describe their experience that led to the identification of 3 additional cases within the pedigree, providing family members with an accurate genetic counseling. 8- Epilepsy Febrile infection-related Epilepsy Syndrome (FIRES) is an uncommon but severe disorder manifesting a prior febrile infection starting between 2 weeks and 24 h before the onset of the refractory status epilepticus with or without fever at the onset of status epilepticus. Symptoms of FIRES had been previously named as “acute encephalitis with refractory, repetitive partial seizures” or as “fever-induced refractory epileptic encephalopathy in school age children”. In this literature review, Pavone et al. presented two cases with FIRES and discussed about this rare even though severe disorder. Both cases presented with the classical FIRES symptoms and reported moderate-severe cognitive impairment and persistence of seizures. Clinical manifestations of FIRES are focal seizures in the acute phase with possible secondary generalization, sometimes associated with pallor, apnea, and cyanosis . Current guidelines for diagnosis of FIRES are not yet available . Different treatment options have been reported, usually suggested during the second phase of the disease. Most used drugs are intravenous benzodiazepines as midazolam, clonazepam, lorazepam and diazepam in association with standard anticonvulsant drugs, as oral levetiracetam, valproic acid, and lacosamide . Alternative therapeutic strategies are ketogenic diet immunomodulatory and intravenous steroids at high-dose or immunoglobulins, plasmapheresis and other agents . Clinical outcomes usually see the persistence of seizure episodes, intellectual disability or, in more severe cases, vegetative state . FIRES is an uncommon and not yet totally understood entity. Since many aspects of this disease such as pathogenesis and treatment still need to be clarified, further future studies are needed. Epilepsy is a common and serious multifactorial neurologic disease with a strong genetic component . Ghazala et al. investigated the SCN1A-A3184G polymorphism among Egyptian children and adolescents with non-lesional epilepsy conducting a prospective case-control observational study. Neuronal voltage-gated sodium channels are involved in the generation and propagation of the action potentials within the neurons, acting on membrane permeability to sodium ions that allows ions diffusion down an electrochemical gradient till the sodium equilibrium potential . There is evidence about the role of the neuronal voltage-gated sodium channels polymorphisms in the epilepsy pathogenesis that causes a spectrum of epilepsy syndromes. For this reason, sodium-channel blockers such as carbamazepine, oxcarbazepine, phenytoin, lamotrigine, lacosamide and lidocaine are some of the most common therapeutic options for epilepsies due to genetic channelopathies. The SCN1A-A3184G (p.Thr1067Ala) polymorphism has been suggested to be linked with the epilepsy risk in several non-Caucasian populations . In their study, the authors extracted and analyzed genomic DNA controls and cases. Results of the study report insignificant differences between epilepsy cases and the control group regarding the frequency of SCN1A-A3184G genotypes and allele. Authors also suggest that the identification of SCN1A-A3184G genotypes might help to choose the most suitable antiepileptic drugs. Further studies on a larger scale are needed to better clarify the matter. 9- Diencephalic syndrome Trapani et al. provide relevant insights on the diencephalic syndrome, a rare childhood disease causing failure to thrive. They presented three children who were admitted for progressive weight loss. Diencephalic syndrome is provoked by a hypothalamic dysfunction caused by a tumor that may involve thalamus/hypothalamus and optic chiasm . Presenting symptoms of diencephalic syndrome may be unspecific. Most common features are severe emaciation with normal caloric intake, locomotor hyperactivity and euphoria, pallor without anemia, hypoglycemia and hypotension . Neurological symptoms typically appear later and include nystagmus, strabismus and visual loss; intracranial hypertension causing recurrent vomiting can be also present, without signs of psychomotor impairment. Diagnosis can be tricky due to the non-specific signs of presentation. Gold standard for radiological detection of brain mass is magnetic resonance. Since surgical removal is not always feasible, chemotherapy based on vincristine, carboplatin and/or etoposide is one of the main therapeutic options . The first case is a 26-month-old boy with persistent failure to gain weight despite adequate caloric intake, no gastrointestinal or other symptoms and negative blood exams about possible malabsorption or vitamin deficiencies. Hormone evaluations and other organ functions were also normal. Brain magnetic resonance was finally performed and detected a large low-grade astrocytoma located in the supra-sellar region. The second case is a 14-month-old girl admitted for poor weight gain, with normal caloric intake. Same investigations were conducted, as well as a brain magnetic resonance showing supra-sellar mass involving hypothalamic-pituitary region, later identified as a low-grade astrocytoma. Finally, the third case is a 16-month-old male who was admitted for severe failure to thrive. After initial diagnostic workup, in the suspicion of diencephalic syndrome, brain imaging identified a large multilobate pseudo-cystic suprasellar lesion in the hypothalamic-pituitary and chiasmatic region. Biopsy of the lesion led to histological diagnosis of pilomyxoid astrocytoma. In this interesting series authors clarified useful elements for the identification of this uncommon syndrome in which subtle presenting symptoms can often lead to a delayed diagnosis. Drooling is defined as the involuntary loss of saliva and oral content. While most children reach the salivary continence by age 15–18 months, the persistence of drooling is common in children with neurological disorders, like cerebral palsy (CP) and is associated with oral motor dysfunction, dysphagia, and/or intraoral sensitivity disorder . Several therapeutic interventions are used to reduce or eliminate drooling. These include surgery, drugs, botulinum toxin, physical therapies to improve sensory function, behavioral interventions, appliances placed in the oral cavity, and acupuncture . There is no consensus on which interventions are safe and effective in managing drooling in children with CP. As the salivary glands are controlled by the parasympathetic autonomic nervous system, the anticholinergic drugs are indeed widely used to reduce the volume of saliva. Glycopyrrolate is the only oral formulation including an anticholinergic agent approved by the United States Food and Drug Administration to treat drooling in children 3–16 years-old which inhibits the acetylcholine receptors on peripheral tissues and reduces the saliva rate production . Lovardi et al. described a case series of eighteen children (median age 17 months, range 2–36 months) with CP or genetic/malformative syndrome, who have been administered oral glycopyrrolate (0.065 mg/kg/die in 3 daily doses) for drooling control. The Drooling Impact Scale was administered at time O and after 1 month. Results showed a significant reduction of the Drooling Impact Scale after 1 month (89 versus 61; p < 0.001), with few adverse effects and an overall response to treatment equal to 94%. Further studies are needed to confirm these results. Alternating hemiplegia of childhood is an uncommon complex disorder, which was first described in late 1960s by Verret and Steele . Alternating hemiplegia of childhood is characterized by paroxysmal episodes of repeated, transient paresis involving either or both sides of the body and usually presents before age 18 months. The diagnosis of alternating hemiplegia of childhood is mainly clinical but may be supported by molecular analysis. Although the pathophysiologic mechanism of the disorder is partially unclear, it is well known that a relevant role is played by mutations in ATP1A2 and ATP1A3 genes, which encode two different alpha subunits of the Na+/K + ATPase transmembrane ion pump, respectively . Pavone et al. reported on the clinical and genetic findings of a couple of twins and a couple of siblings with alternating hemiplegia of childhood from two different Italian families affected. In the twins a pathogenic variant in ATP1A3 gene (c.2318 A > G) was detected. In the siblings, the younger brother showed a novel GRIN2A variant (c.3175 T > A), while the older one carried the same GRIN2A variant, associated with two likely pathogenetic variants in SCNIB (c.632 > A) and KCNQ2 (1870 G > A) genes, which have been implicated in childhood epilepsies. This report provides additional information about alternating hemiplegia of childhood, showing that the variability of clinical features is mirrored by an unexpected genetic heterogeneity. Clinical signs of alternating hemiplegia of childhood usually follow a sequential pattern, in which the paroxysmal episodes are triggered by precipitating factors such as environmental stress, bathing or other events. Non-paroxysmal features include developmental delay/intellectual disability, epilepsy, autonomic dysfunctions, abnormal eye involvement, movement disorders, ataxia, dystonia, and choreoathetosis . Autism spectrum disorder is a term used to describe a set of social communication deficits and repetitive sensory–motor behaviors. It is a neurological developmental disorder, and is characterized by a complex etiology involving genetic, environmental, and biochemical factors . The autism spectrum includes several disorders, such as the Autistic disorder, the Rett disorder, the Asperger syndrome, and the pervasive developmental disorder. Although individuals with autism spectrum disorder are very different from one another, the disorder affects social interactions, verbal and nonverbal social communication skills, as well as intelligence and motor functions, usually originating unusual interests and repetitive behaviors . Epidemiological surveys have shown a rapid increase in autism spectrum disorder prevalence rate. Besides the real increase in prevalence, a variety of other reasons may contribute to the disorder, such as a broader definition of autism spectrum disorder, changes in diagnostic criteria and screening tools, and finally an increased awareness of autism spectrum disorder . In this scenario, Salari et al. conducted a systematic review and meta-analysis from 2008 to July 2021 finalized to clarify the global prevalence of autism spectrum disorder The results show that the world prevalence of autism spectrum disorder is 0.6% (95% confidence interval: 0.4;1%), with slight differences between Asia (0.4%), America (1%), Europe (0.5%), Africa (1%) or Australia (1.7%). Standardized screening for autism spectrum disorder with ongoing developmental surveillance continues to be recommended in primary care at 18 and 24 months of age, because autism spectrum disorder is common, can be diagnosed as young as 18 months of age, and medical interventions may significantly affect neurological compromise . These data should be disseminated by health policymakers in order to implement appropriate planning and interventions. Pontocerebellar hypoplasia includes a heterogeneous group of neurodegenerative disorders with prenatal onset, characterized by severe microcephaly, global developmental delay and radiological manifestation, such as hypoplasia of pons and cerebellum . At least 21 pontocerebellar hypoplasia -related genes are currently listed in the OMIM-database and 15 types of pontocerebellar hypoplasia are known, all characterized by patients’ motor and cognitive impairment. However, the clinical presentation can be very different, ranging from lethal neonatal subtypes to milder variants with survival up to adolescence . The study from Bilge et al. consisted of the description of different clinical and radiological manifestations of six genetically diagnosed patients with pontocerebellar hypoplasia, highlighting the differences in onset and progression of the symptoms . The genetic mutations found in 4 out 6 cases were well known variants that had been previously reported in the literature, while an additional subject was homozygous for the TBC1D23 gene mutation (c.1263 + 1G > A), which was a novel variant never reported until now. Although the last case had the same clinical features, the whole-exome sequencing revealed compound heterozygous mutations in the BRF1 gene, which is also associated with cerebellofaciodental syndrome. Common problems in pontocerebellar hypoplasia include sleep apnea, feeding problems, epilepsy, movement disorders, rhabdomyolysis, and extremely elevated serum creatinine kinase, especially during infections . There is no definite treatment for any type of pontocerebellar hypoplasia, and management is supportive in all types and subtypes. Vertigo is a disorder of space sensitivity characterized by an illusory and unpleasant sensation of body movement toward the environment or vice versa. Vertigo is characterized by different clinical features, therefore it is often difficult for the single physician to identify its etiopathogenesis, especially when the vertigo is the only clinical presenting sign . Compared to adults, vertigo is not so common in children and adolescents. Indeed, while adults surveys reported a one-year prevalence of 23% for unspecified dizziness and 5% for vestibular vertigo, in pediatric patients a prevalence of only 0.4% for nonspecific dizziness, 0.03% for peripheral, and 0.02% for central vestibulopathy is reported . Nevertheless, it is a common reason for emergency department presentation, both alone or associated with other symptoms. Pellegrino et al. reported on the etiopathology of neurological vertigo in childhood and adulthood, highlighting the characteristics which may lead clinicians to a proper diagnosis, and proposed a diagnostic algorithm to support the approach to patients with isolated vertigo, both in pediatric and adult age. The most common cause of isolated vertigo in the pediatric population is vestibular migraine, followed by benign paroxysmal vertigo in childhood. Other causes of vertigo are orthostatic hypotension, vestibular neuritis and vestibular paroxysmia, neurovascular diseases, tumors and demyelinating . Authors underline that the outcome of most neurological vertigos in childhood is good, in contrary to what is reported in adults. Idiopathic intracranial hypertension is very rare in the pediatric age, and etiology is still largely unknown. Del Monte et al. presented a case report of an 8-month old male affected by idiopathic intracranial hypertension, and provide discussion on what literature suggests about optimal management also including the therapeutic strategies in idiopathic intracranial hypertension. The presented case was admitted with vomit, anorexia, irritability and bulging anterior fontanel. Brain magnetic resonance and cerebrospinal fluid analysis were negative. After diagnosis and treatment with acetazolamide and corticosteroids, progressive resolution of symptoms was observed. Pseudotumor cerebri syndrome is a rare condition characterized by elevated intracranial pressure, normal cerebrospinal fluid analysis in the absence of intracranial lesions on neuroimaging. Most common type of pseudotumor cerebri syndrome in both children and adults is the primary type known as idiopathic intracranial hypertension . Secondary types can result from vascular malformations, underlying systemic conditions or drugs. Female gender, obesity, and polycystic ovary represent risk factors for idiopathic intracranial hypertension in adolescents, but not in younger ones . Clinical manifestations of idiopathic intracranial hypertension vary with age. In pediatric populations, symptoms are often unspecific, including vomiting, headache, irritability, hyporeactivity, anorexia, sleep disruption, head tilting, papilledema and bulging fontanelle in infants. Based on adults’ treatment guidelines, also in children the drug of choice is acetazolamide, that could be associated with intravenous corticosteroids if severe visual impairment is present as well. So far, the moment, acetazolamide alone is preferred because of fewer side effects. Most common complications of idiopathic intracranial hypertension are vision loss and relapse, especially in the first 18 months after diagnosis . Early diagnosis, treatment and strict follow-up of complications may help in reducing the risk of relapse and may prevent vision loss. Karlinsky et al. reported their experience about an unusual case of hereditary neuropathy with liability to pressure palsies (HNPP) who had limping as a major presenting symptom. HNPP is an autosomal dominant disease characterized by recurrent, episodic demyelinating neuropathy caused by 17p11.2 chromosomal deletion encompassing the PMP22 gene. The onset is acute, usually in adulthood, and involves a single nerve, most frequently peroneal and ulnar, with a non-painful focal sensory and motor neuropathy . The onset in children most commonly involves peroneal nerve palsy and brachial plexus palsy . Other signs are previous atrophy, focal weakness or sensory loss, reduction of tendon reflexes and mild to moderate pes cavus foot deformity . The definitive diagnosis is provided by genetic analysis, and treatment is mostly supportive. Prognosis is good with an expected full recovery within months or days in best case scenarios. In rare cases a persistence of severe symptoms is reported. Since presenting phenotypes can be extremely variable, the authors describe their experience that led to the identification of 3 additional cases within the pedigree, providing family members with an accurate genetic counseling. Febrile infection-related Epilepsy Syndrome (FIRES) is an uncommon but severe disorder manifesting a prior febrile infection starting between 2 weeks and 24 h before the onset of the refractory status epilepticus with or without fever at the onset of status epilepticus. Symptoms of FIRES had been previously named as “acute encephalitis with refractory, repetitive partial seizures” or as “fever-induced refractory epileptic encephalopathy in school age children”. In this literature review, Pavone et al. presented two cases with FIRES and discussed about this rare even though severe disorder. Both cases presented with the classical FIRES symptoms and reported moderate-severe cognitive impairment and persistence of seizures. Clinical manifestations of FIRES are focal seizures in the acute phase with possible secondary generalization, sometimes associated with pallor, apnea, and cyanosis . Current guidelines for diagnosis of FIRES are not yet available . Different treatment options have been reported, usually suggested during the second phase of the disease. Most used drugs are intravenous benzodiazepines as midazolam, clonazepam, lorazepam and diazepam in association with standard anticonvulsant drugs, as oral levetiracetam, valproic acid, and lacosamide . Alternative therapeutic strategies are ketogenic diet immunomodulatory and intravenous steroids at high-dose or immunoglobulins, plasmapheresis and other agents . Clinical outcomes usually see the persistence of seizure episodes, intellectual disability or, in more severe cases, vegetative state . FIRES is an uncommon and not yet totally understood entity. Since many aspects of this disease such as pathogenesis and treatment still need to be clarified, further future studies are needed. Epilepsy is a common and serious multifactorial neurologic disease with a strong genetic component . Ghazala et al. investigated the SCN1A-A3184G polymorphism among Egyptian children and adolescents with non-lesional epilepsy conducting a prospective case-control observational study. Neuronal voltage-gated sodium channels are involved in the generation and propagation of the action potentials within the neurons, acting on membrane permeability to sodium ions that allows ions diffusion down an electrochemical gradient till the sodium equilibrium potential . There is evidence about the role of the neuronal voltage-gated sodium channels polymorphisms in the epilepsy pathogenesis that causes a spectrum of epilepsy syndromes. For this reason, sodium-channel blockers such as carbamazepine, oxcarbazepine, phenytoin, lamotrigine, lacosamide and lidocaine are some of the most common therapeutic options for epilepsies due to genetic channelopathies. The SCN1A-A3184G (p.Thr1067Ala) polymorphism has been suggested to be linked with the epilepsy risk in several non-Caucasian populations . In their study, the authors extracted and analyzed genomic DNA controls and cases. Results of the study report insignificant differences between epilepsy cases and the control group regarding the frequency of SCN1A-A3184G genotypes and allele. Authors also suggest that the identification of SCN1A-A3184G genotypes might help to choose the most suitable antiepileptic drugs. Further studies on a larger scale are needed to better clarify the matter. Trapani et al. provide relevant insights on the diencephalic syndrome, a rare childhood disease causing failure to thrive. They presented three children who were admitted for progressive weight loss. Diencephalic syndrome is provoked by a hypothalamic dysfunction caused by a tumor that may involve thalamus/hypothalamus and optic chiasm . Presenting symptoms of diencephalic syndrome may be unspecific. Most common features are severe emaciation with normal caloric intake, locomotor hyperactivity and euphoria, pallor without anemia, hypoglycemia and hypotension . Neurological symptoms typically appear later and include nystagmus, strabismus and visual loss; intracranial hypertension causing recurrent vomiting can be also present, without signs of psychomotor impairment. Diagnosis can be tricky due to the non-specific signs of presentation. Gold standard for radiological detection of brain mass is magnetic resonance. Since surgical removal is not always feasible, chemotherapy based on vincristine, carboplatin and/or etoposide is one of the main therapeutic options . The first case is a 26-month-old boy with persistent failure to gain weight despite adequate caloric intake, no gastrointestinal or other symptoms and negative blood exams about possible malabsorption or vitamin deficiencies. Hormone evaluations and other organ functions were also normal. Brain magnetic resonance was finally performed and detected a large low-grade astrocytoma located in the supra-sellar region. The second case is a 14-month-old girl admitted for poor weight gain, with normal caloric intake. Same investigations were conducted, as well as a brain magnetic resonance showing supra-sellar mass involving hypothalamic-pituitary region, later identified as a low-grade astrocytoma. Finally, the third case is a 16-month-old male who was admitted for severe failure to thrive. After initial diagnostic workup, in the suspicion of diencephalic syndrome, brain imaging identified a large multilobate pseudo-cystic suprasellar lesion in the hypothalamic-pituitary and chiasmatic region. Biopsy of the lesion led to histological diagnosis of pilomyxoid astrocytoma. In this interesting series authors clarified useful elements for the identification of this uncommon syndrome in which subtle presenting symptoms can often lead to a delayed diagnosis. 1- Care burden X 400.000 children and adolescents between ages of 0 and 19 each year. The most common types of childhood cancers include leukemias, brain cancers, lymphomas and solid tumors, such as neuroblastoma and Wilms tumor . Scientific advancement has led to an increase in the survival rate of children with cancer and, as a result, living with cancer affects different aspects of life of these children and their caregivers . Chaghazardi et al. determined the level of care burden and the factors associated with it among the caregivers of children with cancer, and have highlighted that the majority of caregivers experience moderate to high care burden. Following the diagnosis, caregivers have to face several challenges such as ignorance, instability, anxiety, helplessness, confusion, and stress. High care burden can affect the quality of care provided to the patient and thus exacerbate their condition, leading to an increase of care burden in a vicious circle . For this reason, identifying and helping caregivers with care burden play a crucial role in improving the quality of care to affected children. Authorities need to take family-centered measures to reduce the caregiver burden, referring low-income caregivers to support organizations, providing counseling services, and holding training workshops on caring for children with cancer. X 400.000 children and adolescents between ages of 0 and 19 each year. The most common types of childhood cancers include leukemias, brain cancers, lymphomas and solid tumors, such as neuroblastoma and Wilms tumor . Scientific advancement has led to an increase in the survival rate of children with cancer and, as a result, living with cancer affects different aspects of life of these children and their caregivers . Chaghazardi et al. determined the level of care burden and the factors associated with it among the caregivers of children with cancer, and have highlighted that the majority of caregivers experience moderate to high care burden. Following the diagnosis, caregivers have to face several challenges such as ignorance, instability, anxiety, helplessness, confusion, and stress. High care burden can affect the quality of care provided to the patient and thus exacerbate their condition, leading to an increase of care burden in a vicious circle . For this reason, identifying and helping caregivers with care burden play a crucial role in improving the quality of care to affected children. Authorities need to take family-centered measures to reduce the caregiver burden, referring low-income caregivers to support organizations, providing counseling services, and holding training workshops on caring for children with cancer. 1- E-cigarettes Electronic cigarettes are devices that heat liquid substances that may contain nicotine that can substitute burned cigarettes. Virgili et al. presented the effects of e-cigarette use on health and pointed out the potential damages arising also from second- and third-hand exposure to smoke. Even though e-cigarettes are considered to be less harmful than burned cigarettes, the Center for Disease Control and Prevention defines them as unsafe and potentially dangerous for brain development and increasement of risk for addiction . Damages of vaping on the respiratory system are analogous to the ones of smoking: chronic inflammation of bronchial mucosa and lung epithelium injury are typically observed. Due to the increasing popularity of vaping products, a new type of lung damage has been described: E-cigarette or Vaping Associated Lung Injury (EVALI). The diagnosis is made after the exclusion of all other potential causes of lung injury when pulmonary infiltrates are found at chest imaging in patients using e-cigarettes and related products 90 days prior to symptom onset . Symptoms of presentation are usually cough, dyspnea and tachycardia and chest imaging usually shows ground glass appearance or dense consolidations. Therapy is based on antibiotics and steroids and, when needed, high-flow oxygen therapy, noninvasive ventilation or mechanical ventilation . Effects on the cardiovascular system include myocardial infarction, hypertension and tachycardia. Neurological effects are the ones observed in nicotine users and include craving and withdrawal symptoms. Gastroenteric effects are an increased risk for gastro-esophageal reflux and for esophagitis exacerbations. Overall, due to its various harmful effects, vaping could be useful only if used as a smoking-cessation device. It is important for institutions to keep restrictions and prevention strategies and for educators and pediatricians to increase awareness in children and their families. During the last years the use of E-cigarettes has increased dramatically in Italy . Casamento Tumeo et al. described a case of E-cigarette or EVALI in a 15-year-old girl. The girl presented to the emergency room with severe dyspnea and a SatO2 of 75% and chest auscultation revealed bilateral wheezes with prolonged expiration. After admission, oxygen supplementation was started and a chest tomography was performed, showing central ground glass pattern with peripheral sparing. She was started on antibiotic therapy with clarithromycin and ceftriaxone. High Flow Nasal Cannula, systemic steroid and nebulized salbutamol, ipratropium and corticosteroids. The girl had a history of asthma and multiple allergies and she was a tobacco and E-cigarette smoker. Dyspnea started 4 days before the admission to the emergency department, but no fever or cough were present. Prior to admission, antibiotic therapy was prescribed with no improvements. After admission and initial treatments, further examinations were performed. Among them, pulmonary function tests showed a restrictive pattern, without bronchodilator reversibility. Diagnostic criteria for identification of EVALI include the use of e-cigarettes 90 days prior to the onset of symptoms accompanied by specific radiological findings (typically ground glass opacities at chest tomography) . EVALI is more common in patients with underlying asthma . Even if its cause is still not yet known, in recent years, Vitamin E has been reported in the broncho-alveolar lavage of patients affected by EVALI, thus identifying it as possible pathogen . Since its relatively recent spread, the use of E-cigarette still needs to be studied in order to better understand the impact and potential damages on health. 2- Cystic fibrosis Cystic fibrosis (CF) is a life-limiting autosomal recessive disorder due to variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR protein is responsible for chloride ion transport across apical epithelial cells in tissues of the airway, intestine, pancreas, kidney, sweat gland, and male reproductive tract . Nowadays, CFTR modulator therapies, targeting the basic CF molecular defect, have been developed for specific CFTR variants and are associated with an improvement in health outcomes, including respiratory function, nutritional status and enhanced quality of life. The quality of life and the survival of patients with CF significantly improved since these drugs have been available and several progresses are being made in the development of such drugs . Nevertheless, lung disease remains the most common cause of death in CF patients and symptomatic mucolytic drugs are crucial for reducing secretion build-up, preventing infections and slowing lung damage. Terlizzi et al. have summarized the current knowledge on dornase alfa (DNAse) in the treatment of CF lung disease, showing the positive effects of this drug on lung ventilation homogeneity. To date DNAse is the only mucus degrading agent that has proven efficacy in CF , by reducing the number of pulmonary exacerbations and improving Forced Expiratory Volume in the 1st second and lung clearance index in patients with CF, in the absence of significant side effects. That is why an early use would be desirable in CF children from 6 years of age, especially in the presence of an abnormal lung clearance index. Electronic cigarettes are devices that heat liquid substances that may contain nicotine that can substitute burned cigarettes. Virgili et al. presented the effects of e-cigarette use on health and pointed out the potential damages arising also from second- and third-hand exposure to smoke. Even though e-cigarettes are considered to be less harmful than burned cigarettes, the Center for Disease Control and Prevention defines them as unsafe and potentially dangerous for brain development and increasement of risk for addiction . Damages of vaping on the respiratory system are analogous to the ones of smoking: chronic inflammation of bronchial mucosa and lung epithelium injury are typically observed. Due to the increasing popularity of vaping products, a new type of lung damage has been described: E-cigarette or Vaping Associated Lung Injury (EVALI). The diagnosis is made after the exclusion of all other potential causes of lung injury when pulmonary infiltrates are found at chest imaging in patients using e-cigarettes and related products 90 days prior to symptom onset . Symptoms of presentation are usually cough, dyspnea and tachycardia and chest imaging usually shows ground glass appearance or dense consolidations. Therapy is based on antibiotics and steroids and, when needed, high-flow oxygen therapy, noninvasive ventilation or mechanical ventilation . Effects on the cardiovascular system include myocardial infarction, hypertension and tachycardia. Neurological effects are the ones observed in nicotine users and include craving and withdrawal symptoms. Gastroenteric effects are an increased risk for gastro-esophageal reflux and for esophagitis exacerbations. Overall, due to its various harmful effects, vaping could be useful only if used as a smoking-cessation device. It is important for institutions to keep restrictions and prevention strategies and for educators and pediatricians to increase awareness in children and their families. During the last years the use of E-cigarettes has increased dramatically in Italy . Casamento Tumeo et al. described a case of E-cigarette or EVALI in a 15-year-old girl. The girl presented to the emergency room with severe dyspnea and a SatO2 of 75% and chest auscultation revealed bilateral wheezes with prolonged expiration. After admission, oxygen supplementation was started and a chest tomography was performed, showing central ground glass pattern with peripheral sparing. She was started on antibiotic therapy with clarithromycin and ceftriaxone. High Flow Nasal Cannula, systemic steroid and nebulized salbutamol, ipratropium and corticosteroids. The girl had a history of asthma and multiple allergies and she was a tobacco and E-cigarette smoker. Dyspnea started 4 days before the admission to the emergency department, but no fever or cough were present. Prior to admission, antibiotic therapy was prescribed with no improvements. After admission and initial treatments, further examinations were performed. Among them, pulmonary function tests showed a restrictive pattern, without bronchodilator reversibility. Diagnostic criteria for identification of EVALI include the use of e-cigarettes 90 days prior to the onset of symptoms accompanied by specific radiological findings (typically ground glass opacities at chest tomography) . EVALI is more common in patients with underlying asthma . Even if its cause is still not yet known, in recent years, Vitamin E has been reported in the broncho-alveolar lavage of patients affected by EVALI, thus identifying it as possible pathogen . Since its relatively recent spread, the use of E-cigarette still needs to be studied in order to better understand the impact and potential damages on health. Cystic fibrosis (CF) is a life-limiting autosomal recessive disorder due to variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR protein is responsible for chloride ion transport across apical epithelial cells in tissues of the airway, intestine, pancreas, kidney, sweat gland, and male reproductive tract . Nowadays, CFTR modulator therapies, targeting the basic CF molecular defect, have been developed for specific CFTR variants and are associated with an improvement in health outcomes, including respiratory function, nutritional status and enhanced quality of life. The quality of life and the survival of patients with CF significantly improved since these drugs have been available and several progresses are being made in the development of such drugs . Nevertheless, lung disease remains the most common cause of death in CF patients and symptomatic mucolytic drugs are crucial for reducing secretion build-up, preventing infections and slowing lung damage. Terlizzi et al. have summarized the current knowledge on dornase alfa (DNAse) in the treatment of CF lung disease, showing the positive effects of this drug on lung ventilation homogeneity. To date DNAse is the only mucus degrading agent that has proven efficacy in CF , by reducing the number of pulmonary exacerbations and improving Forced Expiratory Volume in the 1st second and lung clearance index in patients with CF, in the absence of significant side effects. That is why an early use would be desirable in CF children from 6 years of age, especially in the presence of an abnormal lung clearance index. Over the past year, advances in epidemiology, pathophysiologic mechanisms and prevention, have deepened our understanding of pediatric diseases. We think that developments achieved in 2022 can guide to improve and expand the horizon of care for children. |
Finite element analysis of retrograde superior ramus screw of pubis for the treament of pelvic anterior ring fracture | 9f206377-350e-4232-bedc-dd6a78337d22 | 11899371 | Surgical Procedures, Operative[mh] | The pelvis is a circular structure with complex anatomy. Compared with the posterior ring, the anterior ring of the pelvis has a weaker structure and is prone to fracture .The incidence of isolated pubic rami fractures after pelvic trauma is about 3-46% , and this type of fracture is often classified as stability injury. Fractures that included both anterior and posterior ring are more common, and about 96.8% of patients with rami pubis fractures were found to have concurrent injuries to the posterior pelvic rings . In addition, 78% of the high-energy injuries have trans-pubic instability . Indicating that in unstable pelvic fractures, anterior ring injury has a non-negligible proportion. With the continuous development of surgical technology and the popularization of minimally-invasive concept in recent years, pelvic anterior ring fixation has also entered the era of minimally-invasive instead of the traditional metal plate fixation. At present, the mature treatment techniques for anterior ring fracture mainly include external fixation, screw fixation, internal stent fixation and percutaneous minimally invasive plate fixation, ect. However, most of them are unsatisfactory and scholars have never stopped exploring new internal fixation methods. To comply with the demand of the concept of minimally invasive, by using the principle of the intramedullary central fixing and combining the anatomical characteristics of the pubic symphysis, our research team designed and developed the retrograde superior ramus screw of pubis (SRSP), and has obtained the patent for utility model. In our study, a 3D finite element model of TileC1.3 pelvic fracture model was constructed. We compared the stability of different internal fixation models and provide a theoretical basis for the new device -- SRSP.
Pelvic model reconstruction The pelvic computed tomography (CT) scan data in the Digital Imaging and Communications in Medicine (DICOM) format of a healthy adult volunteer (male, 25 years old, 175 cm height, 70 kg weight, and without previous traumatic or surgical history) were imported into Mimics21.0 (Materialise, Belgium). This study was approved by the Institutional Review Committee of our hospital with the informed consent of the patient. The 3D skeletal models including sacrum, bilateral ilium and the upper middle segment of femur were reconstructed. The STL files of bone were imported into Geomagic Wrap2021 software. The models were smoothed through remesh and local smoothing functions. Then the cancellous bone was created using integral migration function. The STP files of previous NURBS camber of skeletal models were imported into Solidworks2020 software to merge all those pelvic structures with the same origin. The articular cartilage locating at the sacroiliac joint, symphysis pubis, and hip joint were established using curve and stretching functions. Boolean operation was used to inspect there was no interference between every part. Imported into Ansys software, the pelvic model was meshed again. Internal fixation models of anterior ring fracture The healthy pelvic model was imported into the solidworks software, and the TileC1.3 pelvic fracture model which got left anterior pubic rami and ipsilateral sacral zone I fracture was established by cutting fracture line. Retrograde superior ramus screw of pubis, anterior ring plate, internal fixator (INFIX) and hollow screw for rami pubis (HSRP) were established in SOLIDWORKS software according to the location of the fracture line. Four different internal fixation models were established: (1) SRSP group: the anterior ring was fixed by retrograde superior ramus screw of pubis. (2) Plate group: the anterior ring was fixed by a 5-hole reconstruction plate. (3) INFIX group: the anterior ring was fixed by the minimal invasive subcutaneous internal fixator. (4) HSRP group: the anterior ring was fixed by a hollow screw of rami pubis. The posterior rings of the above models were uniformly fixed with two transsacral screws. (Fig. ) Meshing and material parameter settings All four fixation models were meshed in Ansys software. The statistics of the four assembly elements and the total numbers of nodes are shown in Table . It was assumed that the cortical bone, cancellous bone, plate, and screw were all continuous, isotropic, and uniform linear elastic materials. Based on previous research and our experience, the elastic modulus and Poisson’s ratio of various structural materials are shown in Table . Boundary and loading conditions Spring link was used to simulate the pelvic ligament reconstruction, and the parameters of the ligaments are shown in Table . The following three engineering conditions were set for the four groups of fixation models. (1) Load in vertical direction (engineering condition 1): To mimic the standing position, the lower cross section of the femur on both sides were fixed and the pelvis was restricted in 6 directions of freedom. A vertical downward load of 500 N was imposed on the upper surface of the S1 vertebra to simulate the gravity of the upper part of the body. (2) Anterior-posterior(A-P) compression (engineering condition 2): Back of bilateral iliac spines were fixed and 250 N horizontal loads from forward to backward were applied to each side of the anterior superior iliac spine to simulate the effect of anterior-posterior compression. (3) Load in horizontal lateral direction (engineering condition 3): Bilateral acetabular fossas were fixed and a 500 N horizontal lateral load was imposed to the left iliac tubercle to simulate the effect of lateral compression. Furthermore, a sensitivity analysis was systematically conducted to evaluate the accuracy and efficiency of FE models by adjusting the optimal element size. Element sizes of 1 mm, 2 mm, and 3 mm for the bone were examined. The maximum von Mises stress on bone was set to be tested and the used convergence criterion was a change of < 5% , which was considered to be mesh convergence. After the convergence measurement, the mesh size was determined to be 2 mm. Evaluation criteria Following data of total pelvic model and internal fixation were measured which comprised (1) the maximum displacements (2) the stress distribution and Von Mises (V-M) peak stress (3) displacements on both sides of the pubic fracture line (4) absolute value of separation in the horizontal direction of the fracture surfaces.
The pelvic computed tomography (CT) scan data in the Digital Imaging and Communications in Medicine (DICOM) format of a healthy adult volunteer (male, 25 years old, 175 cm height, 70 kg weight, and without previous traumatic or surgical history) were imported into Mimics21.0 (Materialise, Belgium). This study was approved by the Institutional Review Committee of our hospital with the informed consent of the patient. The 3D skeletal models including sacrum, bilateral ilium and the upper middle segment of femur were reconstructed. The STL files of bone were imported into Geomagic Wrap2021 software. The models were smoothed through remesh and local smoothing functions. Then the cancellous bone was created using integral migration function. The STP files of previous NURBS camber of skeletal models were imported into Solidworks2020 software to merge all those pelvic structures with the same origin. The articular cartilage locating at the sacroiliac joint, symphysis pubis, and hip joint were established using curve and stretching functions. Boolean operation was used to inspect there was no interference between every part. Imported into Ansys software, the pelvic model was meshed again.
The healthy pelvic model was imported into the solidworks software, and the TileC1.3 pelvic fracture model which got left anterior pubic rami and ipsilateral sacral zone I fracture was established by cutting fracture line. Retrograde superior ramus screw of pubis, anterior ring plate, internal fixator (INFIX) and hollow screw for rami pubis (HSRP) were established in SOLIDWORKS software according to the location of the fracture line. Four different internal fixation models were established: (1) SRSP group: the anterior ring was fixed by retrograde superior ramus screw of pubis. (2) Plate group: the anterior ring was fixed by a 5-hole reconstruction plate. (3) INFIX group: the anterior ring was fixed by the minimal invasive subcutaneous internal fixator. (4) HSRP group: the anterior ring was fixed by a hollow screw of rami pubis. The posterior rings of the above models were uniformly fixed with two transsacral screws. (Fig. )
All four fixation models were meshed in Ansys software. The statistics of the four assembly elements and the total numbers of nodes are shown in Table . It was assumed that the cortical bone, cancellous bone, plate, and screw were all continuous, isotropic, and uniform linear elastic materials. Based on previous research and our experience, the elastic modulus and Poisson’s ratio of various structural materials are shown in Table .
Spring link was used to simulate the pelvic ligament reconstruction, and the parameters of the ligaments are shown in Table . The following three engineering conditions were set for the four groups of fixation models. (1) Load in vertical direction (engineering condition 1): To mimic the standing position, the lower cross section of the femur on both sides were fixed and the pelvis was restricted in 6 directions of freedom. A vertical downward load of 500 N was imposed on the upper surface of the S1 vertebra to simulate the gravity of the upper part of the body. (2) Anterior-posterior(A-P) compression (engineering condition 2): Back of bilateral iliac spines were fixed and 250 N horizontal loads from forward to backward were applied to each side of the anterior superior iliac spine to simulate the effect of anterior-posterior compression. (3) Load in horizontal lateral direction (engineering condition 3): Bilateral acetabular fossas were fixed and a 500 N horizontal lateral load was imposed to the left iliac tubercle to simulate the effect of lateral compression. Furthermore, a sensitivity analysis was systematically conducted to evaluate the accuracy and efficiency of FE models by adjusting the optimal element size. Element sizes of 1 mm, 2 mm, and 3 mm for the bone were examined. The maximum von Mises stress on bone was set to be tested and the used convergence criterion was a change of < 5% , which was considered to be mesh convergence. After the convergence measurement, the mesh size was determined to be 2 mm.
Following data of total pelvic model and internal fixation were measured which comprised (1) the maximum displacements (2) the stress distribution and Von Mises (V-M) peak stress (3) displacements on both sides of the pubic fracture line (4) absolute value of separation in the horizontal direction of the fracture surfaces.
It can be seen that the stress of the pelvic ring is transmitted from the top to bottom and the middle to sides, with a maximum stress of 39.431 MPa. The displacement (deformation) of the pelvis is roughly symmetrical from left to right and weakened in a wavy pattern along the iliac crest, with a maximum displacement of 0.08923 mm. The overall displacement of the pelvic model and the stress cloud map are essentially similar to that of the description in the relevant literature , which also shows the validity of the pelvic finite element model. (Fig. ) Engineering condition 1 When simulating the vertical force of self-weight, it can be seen that the maximum displacement of overall model in each group, of which the highest value is 0.23161 mm (Fig. A), located in model INFIX. And the lowest is 0.045158 mm (Fig. A), located in model SRSP (Fig. ). Among the maximum displacement of internal fixation in each group, the highest is 0.07138 mm (Fig. C) located in model INFIX. The lowest is 0.026625 mm (Fig. C), located in model SRSP. The maximum space of pubic bone broken ends is 36.8032 × 10 − 3 mm in model INFIX and the minimum space is 1.5289 × 10 − 3 mm in model HSRP (Table ). Among the maximum V-M stress value of overall model in each group, of which the highest is 526.77 MPa (Fig. B) located in model INFIX. And the minimum is 22.419 MPa (Fig. B), located in model HSRP. Among the maximum V-M stress value of internal fixation, the highest is 185.63 MPa (Fig. D) located in model INFIX. The minimum is 12.685 MPa (Fig. D), located in model SRSP. According to the comparison of stress cloud figure in engineering condition 1, the stress distribution of SRSP is more uniform than others groups. Combining all groups of data (Table ), who has the better biomechanical effect are model SRSP and model HSRP, while the worse one is model INFIX (Fig. ). Engineering condition 2 When simulating to face the A-P compression, it can be seen that the maximum displacement of overall model in each group, of which the highest value is 0.26266 mm, located in model SRSP. And the lowest is 0.23279 mm, located in model HSRP. Among the maximum displacement of internal fixation in each group, the highest is 0.18505 mm located in model INFIX. The lowest is 0.14732 mm, located in model HSRP. The maximum space of pubic bone broken ends is 6.4647 × 10 − 3 mm in model INFIX and the minimum space is 0.7488 × 10 − 3 mm in model (Table ). Among the maximum V-M stress value of overall model in each group, of which the highest is 44.334 MPa located in model INFIX. And the lowest is 29.668 MPa, located in model Plate. Among the maximum V-M stress value of internal fixation, the highest is 44.334 MPa located in model INFIX. The lowest is 18.316 MPa, located in model HSRP. According to the comparison of stress cloud figure in engineering condition 2, the stress distribution of HSRP is more uniform than others groups. (See Supplementary Figs. – , Additional File ) Combining all groups of data(Table ), who has the better biomechanical effect are model SRSP and model HSRP (Fig. ). Engineering condition 3 When simulating the force in horizontal lateral direction, it can be seen that the maximum displacement of overall model in each group, of which the highest value is 1.055 mm, located in model INFIX. And the lowest is 0.88487 mm, located in model HSRP. Among the maximum displacement of internal fixation in each group, the highest is 0.32588 mm located in model SRSP. The lowest is 0.17156 mm, located in model Plate. The maximum space of the pubic bone broken ends is 30.334 × 10 − 3 mm in model INFIX and the minimum space is 0.271 × 10 − 3 mm in model HSRP (Table ). Among the maximum V-M stress value of overall model in each group, of which the highest is 533.62 MPa located in model INFIX. And the lowest value is 79.129 MPa, located in model SRSP. Among the maximum V-M stress value of internal fixation, the highest is 247.68 MPa located in model INFIX. The lowest is 79.129 MPa, located in model SRSP. According to the comparison of stress cloud images in engineering condition 3, the stress distribution of SRSP is more uniform than others groups. (See Supplementary Figs. – , Additional File ). Combining all groups of data (Table ), who has the better biomechanical effect are model HSRP and model SRSP (Fig. ).
When simulating the vertical force of self-weight, it can be seen that the maximum displacement of overall model in each group, of which the highest value is 0.23161 mm (Fig. A), located in model INFIX. And the lowest is 0.045158 mm (Fig. A), located in model SRSP (Fig. ). Among the maximum displacement of internal fixation in each group, the highest is 0.07138 mm (Fig. C) located in model INFIX. The lowest is 0.026625 mm (Fig. C), located in model SRSP. The maximum space of pubic bone broken ends is 36.8032 × 10 − 3 mm in model INFIX and the minimum space is 1.5289 × 10 − 3 mm in model HSRP (Table ). Among the maximum V-M stress value of overall model in each group, of which the highest is 526.77 MPa (Fig. B) located in model INFIX. And the minimum is 22.419 MPa (Fig. B), located in model HSRP. Among the maximum V-M stress value of internal fixation, the highest is 185.63 MPa (Fig. D) located in model INFIX. The minimum is 12.685 MPa (Fig. D), located in model SRSP. According to the comparison of stress cloud figure in engineering condition 1, the stress distribution of SRSP is more uniform than others groups. Combining all groups of data (Table ), who has the better biomechanical effect are model SRSP and model HSRP, while the worse one is model INFIX (Fig. ).
When simulating to face the A-P compression, it can be seen that the maximum displacement of overall model in each group, of which the highest value is 0.26266 mm, located in model SRSP. And the lowest is 0.23279 mm, located in model HSRP. Among the maximum displacement of internal fixation in each group, the highest is 0.18505 mm located in model INFIX. The lowest is 0.14732 mm, located in model HSRP. The maximum space of pubic bone broken ends is 6.4647 × 10 − 3 mm in model INFIX and the minimum space is 0.7488 × 10 − 3 mm in model (Table ). Among the maximum V-M stress value of overall model in each group, of which the highest is 44.334 MPa located in model INFIX. And the lowest is 29.668 MPa, located in model Plate. Among the maximum V-M stress value of internal fixation, the highest is 44.334 MPa located in model INFIX. The lowest is 18.316 MPa, located in model HSRP. According to the comparison of stress cloud figure in engineering condition 2, the stress distribution of HSRP is more uniform than others groups. (See Supplementary Figs. – , Additional File ) Combining all groups of data(Table ), who has the better biomechanical effect are model SRSP and model HSRP (Fig. ).
When simulating the force in horizontal lateral direction, it can be seen that the maximum displacement of overall model in each group, of which the highest value is 1.055 mm, located in model INFIX. And the lowest is 0.88487 mm, located in model HSRP. Among the maximum displacement of internal fixation in each group, the highest is 0.32588 mm located in model SRSP. The lowest is 0.17156 mm, located in model Plate. The maximum space of the pubic bone broken ends is 30.334 × 10 − 3 mm in model INFIX and the minimum space is 0.271 × 10 − 3 mm in model HSRP (Table ). Among the maximum V-M stress value of overall model in each group, of which the highest is 533.62 MPa located in model INFIX. And the lowest value is 79.129 MPa, located in model SRSP. Among the maximum V-M stress value of internal fixation, the highest is 247.68 MPa located in model INFIX. The lowest is 79.129 MPa, located in model SRSP. According to the comparison of stress cloud images in engineering condition 3, the stress distribution of SRSP is more uniform than others groups. (See Supplementary Figs. – , Additional File ). Combining all groups of data (Table ), who has the better biomechanical effect are model HSRP and model SRSP (Fig. ).
The pelvis is a complete ring composed of sacrum and iliums on both sides connected by strong ligaments and fibrocartilage, which plays an important role in transmitting the upper and lower stress of the axial bones. It was widely believed that injury to the posterior pelvic ring would cause significant instability of the overall pelvic ring. There is no consensus on whether simultaneous fixing anterior and posterior rings is as important to provide adequate stability to the pelvic ring as doing posterior rings alone . However, with the development of the research on anterior ring in recent years, researchers have found that the stability of anterior ring may influence the stress of posterior ring or the deformation of broken ends . More attention has been paid to the reconstruction of the stability of anterior pelvic ring . For the instability of the ring, restoring anatomical structure and solid fixation are the prerequisites for early functional rehabilitation, in order to meet the demands of more and more patients for a better prognosis. It is known from the literature that the incidence of simple isolated pubic branch fractures is relatively low. Scheyerer et al. ’s study showed that about 96.8% of patients with pubic branch fractures were found to be accompanied with injuries to the posterior pelvic ring. Therefore, our study chose to establish the finite element model of C1.3 fracture, which is a representative of unstable pelvic fractures. In our study, percutaneous sacroiliac screws were used to fix the posterior pelvic ring, which is a reliable and minimally invasive central fixation method, providing sufficient stability and effectively resist vertical shear and torsion forces . In this study, two sacroiliac screws were used to fix the posterior ring in all fracture models, which not only conformed to the concept of minimally invasive treatment, but also reduced the calculation error that may be caused by the unstable fixation of the posterior ring. It is generally considered that the growth of callus is greatly affected by movement between the broken ends of fracture clinically. And it is difficult to heal when the distance between ends is more than 2 cm, which usually means the failure of internal fixation . The controlled micromovements between the fracture ends can promote callus formation and fracture healing, which are important mechanical parameters in the healing process . Long-term follow-up by some scholars has shown that the fracture healing prognosis is better when the displacement of the broken ends of pelvic posterior ring is between 0.2 and 1 mm. Movements exceeding this range may have a negative effect on fracture healing . In our study four groups of the maximum displacement of overall finite element model and movement of broken ends of pubis in three engineering conditions were all less than 1 mm. Under the vertical force of self-weight, the maximum displacement of overall finite element model are compared as: INFIX model > Plate model > HSRP model > SRSP model. The movement of broken ends of pubis are compared as: INFIX model > Plate model > SRSP model > HSRP model. Under the force in horizontal from forward to backward direction, the maximum displacement of overall model are compared as: SRSP model > INFIX model > Plate model > HSRP model. The movement of broken ends of pubis are compared as: INFIX model > Plate model > HSRP model > SRSP model. Under the force in horizontal lateral direction, the maximum displacement of overall model are compared as: INFIX model > SRSP model > Plate model > HSRP model. The movement of broken ends of pubis are compared as: INFIX model > Plate model > SRSP model > HSRP. Smaller displacement strain in each part of pelvis represents better biomechanical stability. In summary, it can be seen that four internal fixation schemes can effectively fix TileC1.3 pelvic fractures and provide stability. And also there was no obvious stress concentration in the four internal fixation models. Comparison between groups shows the SRSP and HSRP are superior to others. In general, the risk of endoplant failure arises from stress load and travel distance. When there is no obvious stress concentration, the lower the maximum stress of internal fixation, the less the risk of breaking. The smaller the movement distance of internal fixation, the less the risk of loosening. Under the vertical force of self-weight, the maximum displacement of internal fixation are compared as: INFIX model > Plate model > HSRP model > SRSP model. The maximum stress of internal fixation are compared as: INFIX model > Plate model > HSRP model > SRSP model. Under the force in horizontal from forward to backward direction, the maximum displacement of internal fixation are compared as: INFIX model > SRSP model > Plate model > HSRP model. The maximum stress of internal fixation are compared as: INFIX model > SRSP model > Plate model > HSRP model. Under the force in horizontal lateral direction, the maximum displacement of internal fixation are compared as: SRSP model > INFIX model > HSRP model > Plate model. The maximum stress of internal fixation are compared as: INFIX model > HSRP model > Plate model > SRSP model. And we could discover from the stress cloud image that and the stress distribution of SRSP and HSRP were more uniform than others. In the above engineering conditions, the maximum stress subjected to internal fixation is lower than the yield stress of titanium metal (1050Mpa), thus basically no fracture will occur . In comparison, the SRSP is more advantageous in the field of less loosening. In this study, no obvious fracture end separation, fracture or obvious deformation, or loosening of the inner plant were found in each model. And the displacement and stress changes showed similar distribution rules to the normal model, indicating that all four anterior internal fixation schemes have effectively fixed and restored the load-bearing function for TileC1.3 pelvic fractures. In addition, the data of the reconstructed plate fixation group were satisfactory in various engineering conditions, which also verified that plate internal fixation is the potential treatment standard. Suprapubic branch hollow screw fixation is currently a commonly used method for minimally invasive treatment of pelvic fractures. However, due to the arcuated anatomy of the pubic branch, it is difficult to place nails because of the narrow bone channel in some patients, requiring multiple X-ray during operation. For some patients, the screw placement may can’t be done. And nails removement may happen to middle-aged and elderly female patients with osteoporosis . The SRSP was independently designed by our team based on anatomical data of the normal pelvic structure in the Chinese population (Chinese Patent No.: ZL 2020 2 1969847.8). It features a curved configuration with an approximately 15° arc, incorporating a bullet-shaped tip and an expanded cylindrical tail. (See Supplementary Figs. – , Additional File ). In contrast to the hollow screws, the arc-shaped structure of SRSP is more in line with the anatomical structure of the anterior pelvic ring. The solid bullet-shaped tip simultaneously, provides self-drilling and guidance capabilities during clinical implantation. This design facilitates reduced insertion resistance and enhanced procedural efficiency compared to traditional hollow screw systems. Combined with the uniform stress distribution and good overall control, SRSP fixation scheme can be used as a new choice for the treatment of pelvic ring injury. This study also has limitations that should be considered. One limitation of this study is that only sketetal and ligament systems of the pelvis was employed to develop finite element models, excluding the effect muscles. Although this is a common approach in similar finite element analyses, the muscle forces which were neglected may make the stress on the broken ends more complicated. Additionally, the bone was set as an isotropic material, while bone is in fact an anisotropic material. While our fracture models cannot cover all possible real-life situations, they still offer valuable insights. Given these limitations, our conclusions would benefit from further confirmation through clinical retrospective studies and cadaveric biomechanical experiments.
In our study, we explored the biomechanical outcome of the new device for treating pubis rami fracture by finite element analysis. Based on the established 3D finite element model of the pelvis, four internal fixation models for TileC1.3 fractures were built and compared by biomechanical finite element analysis. With sufficient biomechanical stability, the new device SRSP can be used as a reliable fixation for the treatment of pelvic anterior ring fracture. The study provides a good theoretical basis for subsequent clinical investigations of the various therapeutic procedures in the future.
Below is the link to the electronic supplementary material. Supplementary Material 1: Additional file: The file is PPT. The data in additional file are the results of the infite element analysis and more details about the design of SRSP. Considering the restriction of the space on printed page, we put more figures in additonal file.
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Atherogenic index of plasma and cardiovascular outcomes in female patients undergoing percutaneous coronary intervention: insights from a retrospective cohort study | 26d8816d-92cd-4db6-8c57-ad8911c0fbea | 11760380 | Surgical Procedures, Operative[mh] | CAD is a significant global health problem, being a major cause of morbidity and mortality, including 17.8 million deaths annually . Despite the accelerated advancement and widespread implementation of PCI leading to a significant decrease in cardiovascular events, the prospects for patients diagnosed with ACS are still not ideal . Though the significant efficacy of DES in reducing restenosis and advancing healthcare, some individuals still face a considerable risk of cardiovascular event recurrence . Therefore, improving risk stratification to promote the management of CAD patients has important clinical significance, which is a leading challenge in clinical practice . Dyslipidemia, defined as unusual levels of lipids in the blood, is popular in patients with ACS. Increasing levels of TC, TG and LDL-C or decreased levels of HDL-C are characteristics of dyslipidemia. This condition is an vital routine risk factor for ACS, leading to poor prognosis . The present guidelines suggest that the main goal of lipid-lowering intervention for ACS patients should be to reduce LDL-C . Nevertheless, despite achieving best LDL-C levels through intensified lipid-lowering drugs, there are still some patients having an increased risk of recurrent cardiovascular events . This emphasizes the need for a more comprehensive approach that goes beyond LDL-C levels. People are increasingly focusing on identifying risk factors related to residual lipids, with the aim of more accurately predicting cardiovascular outcomes and improving clinical management. AIP is defined as the logarithm of the ratio of TG to HDL-C. Because of its correlation with lipoprotein particle size, AIP has been identified as an independent cardiovascular risk factor . Numerous studies provide ample evidence to suggest a strong correlation between AIP and increased risk of stroke, heart failure, and CAD – , and it is independently related to poor prognosis in CAD patients , . Though only a few researches have examined the impact of AIP on cardiovascular outcomes in patients who underwent PCI with DESs. The aim of this study is to investigate the association between AIP and MACCESs in patients undergoing percutaneous coronary intervention.
Study population This study was a secondary analysis of an observational cohort study. The research population was sourced from the publicly available dataset provided by Yao H-M et al. , which is accessible through Dryad (10.5061/dryad.13d31) . This research was conducted with the approval of the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (approval number: 2023-KY-0327) in compliance with the principles outlined in the Helsinki Declaration. Informed consent was waived by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Furthermore, the research adhered to the guidelines of the Strengthening the Reporting of STROBE statement. In summary, between July 2009 and August 2011, a total of 2533 patients who underwent continuous PCI with DES were recruited. The median follow-up period for participants was 29.8 months (25.6–34.0 months). The PCI procedure is performed by experienced surgeons according to established protocols. Before PCI, patients should take loading doses of aspirin (300 mg) and clopidogrel (300 mg) unless they have already taken standard antiplatelet drugs. The medication administration after discharge was carried out according to the clinical guidelines at that time. As shown in Fig. , after excluding data that were considered incomplete or unclear, a total of 2098 patients were included in this study. Data collection and definitions At admission, data related to demographics, medical history, and clinical manifestations were recorded. Collect fasting peripheral venous blood samples before PCI to obtain laboratory data, including blood glucose, TC, LDL-C, creatinine, UA, TG, and HDL-C. Angiography and surgical data, including the location of diseased blood vessels, characteristics of lesions, length and diameter of stents, are extracted from medical records. In addition, data related to the medication regimen of the subjects was also collected. The purpose of designing standardized spreadsheets is to collect the aforementioned retrospective data. History of hypertension is defined as taking antihypertensive medication or self-reported diagnosis of hypertension. A history of diabetes mellitus is defined as taking diabetes drugs or self-reported diagnosis of diabetes mellitus. Smokers refer to individuals who have smoked within the past decade. BMI was calculated as: BMI (kg/m 2 ) = weight/height 2 . Repeat revascularization was defined as revascularization with PCI or CABG surgery during the follow-up period. Endpoints The objective of this study was to ascertain the incidence of MACCEs at the 29.8-month follow-up. The MACCEs included all-cause mortality, AMI, repeat revascularization and stroke. Statistical analysis Use multivariate logistic regression analysis to evaluate the independent correlation between AIP and MACCEs. Descriptive analysis was conducted on all patients. Perform subgroup analysis based on correlated covariates. Continuous data were expressed as either the mean and SD or the median and IQR, depending on whether the data set was normally distributed. Categorical variables are expressed in absolute numbers and percentages. The comparison of variables was conducted using the chi-square test (for categorical variables) and the one-way ANOVA (for variables with a normal distribution), as well as the Kruskal–Wallis test (for variables with a skewed distribution). A restricted cubic spline model was employed for the purpose of conducting a dose–response analysis. The analysis was conducted using R 4.2.1 ( http://www.Rproject.org ; The R Foundation, Vienna, Austria) and the Free Statistics software (version 1.9.2; Beijing Free Clinical Medical Technology Co., Ltd, Beijing, China). A two-tailed test was conducted, and p-value < 0.05 was considered statistically significant. The aim was to ascertain whether the correlation between AIP and MACCEs in female patients undergoing PCI was influenced by two models. Model 1 was adjusted for age. Model 2 was adjusted for covariates that demonstrated a statistically significant relationship between AIP and MACCEs in the female patients.
This study was a secondary analysis of an observational cohort study. The research population was sourced from the publicly available dataset provided by Yao H-M et al. , which is accessible through Dryad (10.5061/dryad.13d31) . This research was conducted with the approval of the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (approval number: 2023-KY-0327) in compliance with the principles outlined in the Helsinki Declaration. Informed consent was waived by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Furthermore, the research adhered to the guidelines of the Strengthening the Reporting of STROBE statement. In summary, between July 2009 and August 2011, a total of 2533 patients who underwent continuous PCI with DES were recruited. The median follow-up period for participants was 29.8 months (25.6–34.0 months). The PCI procedure is performed by experienced surgeons according to established protocols. Before PCI, patients should take loading doses of aspirin (300 mg) and clopidogrel (300 mg) unless they have already taken standard antiplatelet drugs. The medication administration after discharge was carried out according to the clinical guidelines at that time. As shown in Fig. , after excluding data that were considered incomplete or unclear, a total of 2098 patients were included in this study.
At admission, data related to demographics, medical history, and clinical manifestations were recorded. Collect fasting peripheral venous blood samples before PCI to obtain laboratory data, including blood glucose, TC, LDL-C, creatinine, UA, TG, and HDL-C. Angiography and surgical data, including the location of diseased blood vessels, characteristics of lesions, length and diameter of stents, are extracted from medical records. In addition, data related to the medication regimen of the subjects was also collected. The purpose of designing standardized spreadsheets is to collect the aforementioned retrospective data. History of hypertension is defined as taking antihypertensive medication or self-reported diagnosis of hypertension. A history of diabetes mellitus is defined as taking diabetes drugs or self-reported diagnosis of diabetes mellitus. Smokers refer to individuals who have smoked within the past decade. BMI was calculated as: BMI (kg/m 2 ) = weight/height 2 . Repeat revascularization was defined as revascularization with PCI or CABG surgery during the follow-up period.
The objective of this study was to ascertain the incidence of MACCEs at the 29.8-month follow-up. The MACCEs included all-cause mortality, AMI, repeat revascularization and stroke.
Use multivariate logistic regression analysis to evaluate the independent correlation between AIP and MACCEs. Descriptive analysis was conducted on all patients. Perform subgroup analysis based on correlated covariates. Continuous data were expressed as either the mean and SD or the median and IQR, depending on whether the data set was normally distributed. Categorical variables are expressed in absolute numbers and percentages. The comparison of variables was conducted using the chi-square test (for categorical variables) and the one-way ANOVA (for variables with a normal distribution), as well as the Kruskal–Wallis test (for variables with a skewed distribution). A restricted cubic spline model was employed for the purpose of conducting a dose–response analysis. The analysis was conducted using R 4.2.1 ( http://www.Rproject.org ; The R Foundation, Vienna, Austria) and the Free Statistics software (version 1.9.2; Beijing Free Clinical Medical Technology Co., Ltd, Beijing, China). A two-tailed test was conducted, and p-value < 0.05 was considered statistically significant. The aim was to ascertain whether the correlation between AIP and MACCEs in female patients undergoing PCI was influenced by two models. Model 1 was adjusted for age. Model 2 was adjusted for covariates that demonstrated a statistically significant relationship between AIP and MACCEs in the female patients.
Characteristics of the study population by gender The study population comprised 2533 patients at a single PCI clinical centre with long-term follow-up between July 2009 and August 2011. Following the exclusion of data deemed to be of an insufficient quality, as well as data pertaining to TG, HDL-C and other values, a total of 2098 participants were included in the subsequent analyses (Fig. , Table ), among them, 675 are female. Compared with male patients, female patients are older, have higher BMI, poorer metabolic status (Glycemia, TC, LDL-C), lower medical history and smoking frequency, and lower HR, creatinine, uric acid, and bilirubin. The distribution of drug therapy was found to be generally balanced, although female patients prescribed clopidogrel and calcium channel blockers more frequently. Regarding blood vessels, the incidence of occlusion and restenosis is lower in women, and the diameter of stents is also smaller. The number of diseased blood vessels, target lesion location, and stent length are similar between genders. During the follow-up period, a total of 343 participants experienced at least one MACCEs, of which 106 were females. AIP and clinical outcomes in the overall population A summary of the occurrence of MACCEs and individual events is provided in Table . A total of 343 patients (16.4%) experienced at least one MACCE during the follow-up period. Of these, 106 (15.8%) were female and 267 (16.7%) were male. The incidence of MACCEs and the individual events demonstrated no statistically significant difference between the two groups. To ascertain the predictors for MACCEs in the overall population, univariate and multivariate logistic regression analyses were conducted (see Table ). Univariate analysis showed that age, heart failure history, previous AMI, diabetes, LVEF, DBP, creatinine, UA were associated with MACCEs. The treatment of ACEIs, the number of diseased vessels, target lesion location, CTO, stent length, and stent diameter were correlated with MACCEs in patients receiving DESs percutaneous coronary intervention (p < 0.05). However, no significant association was observed between AIP and MACCEs. In addition, after adjusting for possible confounding factors based on univariate analysis, multivariate analysis found a significant correlation between AIP and MACCEs. Sex difference of AIP and MACCEs Given the inherent complexity of the clinical efficacy of AIP, particularly considering potential gender differences, a stratified analysis was conducted to elucidate the association between AIP and MACCE, with a particular focus on gender based differences. As shown in Table , there was a significant correlation between AIP and MACCEs in female patients during the 29.8-month follow-up period, as indicated by univariate analysis (OR = 2.31, 95% CI 1.12–4.73, p = 0.023). After adjusting for potential confounding factors in multivariate analysis, a significant correlation was also observed between AIP and MACCEs (OR = 2.45, 95% CI 1.17–5.11, p = 0.018). However, regardless of whether adjustments were made, no significant correlation was observed between AIP and MACCEs in the male patients. Table illustrates the correlation between AIP and MACCEs in female patients. In the univariate logistic regression model, the OR of MACCEs was higher for every unit increase in AIP, at 2.31 (95% CI: 1.12 ~ 4.73, p = 0.023). After adjusting for several covariates, including age, heart failure, previous AMI, DBP, creatinine, UA, number of diseased vessels, and target lesion location, this relationship remained statistically significant in two multivariate logistic regression models. (Model 1: OR = 2.45, 95% CI: 1.17–5.11, p = 0.018; Model 2: OR = 2.15, 95% CI: 1.01–4.58, p = 0.047). Subgroup analysis on the association of AIP and MACCEs Furthermore, subgroup analyses were conducted to ascertain the influence of potential confounding factors, including gender, age, hypertension, and diabetes mellitus (Fig. ). Significant interactions were identified between the gender-stratified subgroups and the effect of AIP on MACCEs (p-value for interaction = 0.01). While no significant associations were observed in other subgroups, a trend was evident.
The study population comprised 2533 patients at a single PCI clinical centre with long-term follow-up between July 2009 and August 2011. Following the exclusion of data deemed to be of an insufficient quality, as well as data pertaining to TG, HDL-C and other values, a total of 2098 participants were included in the subsequent analyses (Fig. , Table ), among them, 675 are female. Compared with male patients, female patients are older, have higher BMI, poorer metabolic status (Glycemia, TC, LDL-C), lower medical history and smoking frequency, and lower HR, creatinine, uric acid, and bilirubin. The distribution of drug therapy was found to be generally balanced, although female patients prescribed clopidogrel and calcium channel blockers more frequently. Regarding blood vessels, the incidence of occlusion and restenosis is lower in women, and the diameter of stents is also smaller. The number of diseased blood vessels, target lesion location, and stent length are similar between genders. During the follow-up period, a total of 343 participants experienced at least one MACCEs, of which 106 were females.
A summary of the occurrence of MACCEs and individual events is provided in Table . A total of 343 patients (16.4%) experienced at least one MACCE during the follow-up period. Of these, 106 (15.8%) were female and 267 (16.7%) were male. The incidence of MACCEs and the individual events demonstrated no statistically significant difference between the two groups. To ascertain the predictors for MACCEs in the overall population, univariate and multivariate logistic regression analyses were conducted (see Table ). Univariate analysis showed that age, heart failure history, previous AMI, diabetes, LVEF, DBP, creatinine, UA were associated with MACCEs. The treatment of ACEIs, the number of diseased vessels, target lesion location, CTO, stent length, and stent diameter were correlated with MACCEs in patients receiving DESs percutaneous coronary intervention (p < 0.05). However, no significant association was observed between AIP and MACCEs. In addition, after adjusting for possible confounding factors based on univariate analysis, multivariate analysis found a significant correlation between AIP and MACCEs.
Given the inherent complexity of the clinical efficacy of AIP, particularly considering potential gender differences, a stratified analysis was conducted to elucidate the association between AIP and MACCE, with a particular focus on gender based differences. As shown in Table , there was a significant correlation between AIP and MACCEs in female patients during the 29.8-month follow-up period, as indicated by univariate analysis (OR = 2.31, 95% CI 1.12–4.73, p = 0.023). After adjusting for potential confounding factors in multivariate analysis, a significant correlation was also observed between AIP and MACCEs (OR = 2.45, 95% CI 1.17–5.11, p = 0.018). However, regardless of whether adjustments were made, no significant correlation was observed between AIP and MACCEs in the male patients. Table illustrates the correlation between AIP and MACCEs in female patients. In the univariate logistic regression model, the OR of MACCEs was higher for every unit increase in AIP, at 2.31 (95% CI: 1.12 ~ 4.73, p = 0.023). After adjusting for several covariates, including age, heart failure, previous AMI, DBP, creatinine, UA, number of diseased vessels, and target lesion location, this relationship remained statistically significant in two multivariate logistic regression models. (Model 1: OR = 2.45, 95% CI: 1.17–5.11, p = 0.018; Model 2: OR = 2.15, 95% CI: 1.01–4.58, p = 0.047).
Furthermore, subgroup analyses were conducted to ascertain the influence of potential confounding factors, including gender, age, hypertension, and diabetes mellitus (Fig. ). Significant interactions were identified between the gender-stratified subgroups and the effect of AIP on MACCEs (p-value for interaction = 0.01). While no significant associations were observed in other subgroups, a trend was evident.
The present study sought to investigate the relationship between AIP and MACCEs among patients with ACS who were undergoing PCI with DES. Our findings revealed a significant correlation between AIP and MACCEs in female patients. An increasing number of studies have focused on cardiovascular risk in the female population, showing that the clinical features and prognosis of ACS patients differ by gender, this is evidenced by the fact that female patients are likely to have worse clinical outcomes than male patients , . Accordingly, an exploratory subgroup analysis was conducted, focusing on the variables of sex, age, hypertension and diabetes mellitus. The findings indicated that elevated AIP was linked to an elevated risk of MACCEs in female patients, but not in the overall cohort. However, the precise mechanism underlying this association remains unclear. It is established that elevated HDL-C levels are associated with a protective effect against the development of atherosclerosis, whereas elevated TG levels are associated with an increased risk of cardiovascular disease. It is possible that lower HDL-C levels may inhibit the anti-atherogenic properties of HDL. Furthermore, these changes in the lipid profile may precede the development of glycemic dysregulation – . The present study has shown that the AIP represented by the logarithm of the ratio of plasma concentrations of TG to HDL-C and is a superior predictor of plasma atherogenicity compared to isolated lipid levels , and demonstrated a strong correlation with an increased incidence of subclinical or symptomatic CAD , . In addition, AIP has been identified as a significant prognostic factor for cardiovascular events in CAD patients, whether or not they have undergone PCI , , . A recent systematic review and meta-analysis has demonstrated that elevated AIP is an independent prognostic factor in patients with CAD . Although reduction of LDL-C levels represents a key treatment objective in CAD, attainment of this target does not necessarily eliminate residual cardiovascular risk. This underscores the need for further investigation into more accurate risk factors in these patients . It should be pointed out that this study is not without limitations. Firstly, due to the absence of time variables in the data, no time event analysis was conducted, which may have led to an underestimation of the association between AIP and MACCEs. Secondly, Due to missing data and low incidence of cardiovascular events, a large number of patients were excluded, which may lead to underestimation of therapeutic efficacy. Thirdly, due to the influence of patients’ lifestyle and medication, these variables may have changed during the follow-up period. In short, the surveyed population comes from a center in China, which limits its universality. Therefore, it is recommended that future research consider incorporating these factors to further improve the accuracy and effectiveness of the results.
In summary, the research findings indicate that AIP is an independent prognostic factor for MACCE in female patients after DESs implantation. However, further investigation is needed to confirm these findings. In addition, further research is needed to elucidate the potential of targeted interventions to alter AIP levels and improve patient outcomes.
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Evaluating the effects of temperature and agitation on biofilm formation of bacterial pathogens isolated from raw cow milk | c665fa1f-4cbe-4aa1-b976-1c27f4bf514e | 11229293 | Microbiology[mh] | Raw milk refers to milk that unpasteurized or otherwise treated to kill harmful bacteria . Several studies have shown that consumers are motivated to drink raw milk by its perceived superior taste, nutritional value, and health benefits . In addition, lactic acid bacteria, which are commonly found in raw milk, have been shown to have antimicrobial activity against pathogens ; however, raw milk consumption is well established to be associated with high risk of foodborne disease . Pathogenic bacteria, such as Staphylococcus aureus , Streptococcus agalactiae , Klebsiella pneumoniae , Escherichia coli , Listeria monocytogenes , and Salmonella typhimurium have been identified as a cause of food poisoning outbreaks linked to raw milk in several countries . These bacteria can be present in the milk from the start or be introduced at any point during production and processing . Public health strategies focus on reducing the risk of harmful bacteria in raw milk throughout the food supply chain, before it reaches consumers . Though, several studies have shown that consumer food handling can counterbalance food safety practices during processing and culminate in foodborne disease . In particular, poor consumer food handling practices, such as leaving refrigerated foods like milk out at room temperature for too long, can create conditions that favor bacterial growth . During milk storage and processing, the capacity of bacteria to form clumps of cells encased in a matrix of their own making, called biofilms, might contribute to their missed detection during routine screening . Importantly, several studies have reported that bacterial biofilm formation can be increased at high temperature (~ 30 °C) ; however, unfortunately few studies have focused on the effect of vehicle movement (agitation) on biofilm formation in raw milk . In this study, we collected raw cow milk (RCM) from eight dairy farms with the aim of isolating pathogenic bacteria. We then evaluated the impact of temperature (4 °C, 15 °C, 30 °C) and agitation (0 rpm, 60 rpm, and 120 rpm) on biofilm formation by the isolated bacteria.
Sample collection Raw milk samples were taken from storage tanks (300 to 3000 L in volume) at eight dairy farms in Riyadh, Saudi Arabi. A total of 40 samples were collected: one from each of five different bulk tanks at each farm, with approximately 30 mL per sample. Sample temperature during collection and the distance from the farm to the laboratory recorded. The samples were collected and transported to Imam Mohammad Ibn Saud Islamic University within two hours, where they were kept cold and tested the same day . This study did not involve any human or animal subjects, so ethical approval was not required. Detection of presumptive pathogenic isolates To identify bacteria in RCM, 6 mL of each sample (out of 30 mL total) was used as follows: First, 3 mL was streaked on six plates (0.5 mL per plate) using 5% sheep blood agar (SBA; Watin Biolife, KSA) for the identification of bacterial species with low loads. Second, the remaining 3 mL was diluted 10 times in phosphate-buffered saline (PBS) with a pH of 7.4, and the diluted sample was then spread on SBA for the determination of bacterial species with high loads. Plates were kept for 48 h at 37 °C under aerobic and 5% CO 2 conditions. Colonies were isolated for differentiation of pathogenic and non-pathogenic bacteria from the initial cultures based on several factors including: colony morphology (size, shape, color, and texture), diameter, hemolytic properties, and basic chemistry tests including catalase and coagulase assays. Furthermore, following isolation, gram staining (BD, NJ, USA) was performed as recommended by the stain manufacturer to differentiate between Gram-positive and Gram-negative bacteria. Isolated colonies were incubated another two days at 37 °C under aerobic or in the presence of 5% CO 2 . Two colonies were picked and used to start a culture in 3 mL of sterile sodium chloride solution (0.45%), which was made to be as cloudy as a 0.5 McFarland standard. The bacterial suspensions were then put on each of two testing cards, VITEK 2 ID-GPB and VITEK 2 ID-GNB, and placed into a VITEK 2 system (bioMérieux) following the instructions from the manufacturer for species identification. In the event the VITEK 2 identification was made with less than 90% confidence. To ensure that the tests were being performed correctly, the laboratory also tested the following strains of bacteria, as recommended by the Clinical and Laboratory Standards Institute (CLSI): Enterococcus faecalis ATCC 29,212, Streptococcus equi subsp. zooepidemicus ATCC 43,079, Escherichia coli ATCC 35,218, Pseudomonas aeruginosa ATCC 27,853, Staphylococcus aureus ATCC 29,213, and Klebsiella pneumoniae ATCC 700,603 . MALDI-TOF MS identification We confirmed the identity of isolated bacteria by using MALDI-TOF MS system (bioMérieux) . We followed the manufacturer’s instructions for the VITEK MS system to perform this analysis. Briefly, a loop was used to pick individual bacterial colonies and transfer them to designated spots on a slide. A special solution (VITEK MS-CHCA matrix) was then applied and allowed to dry completely. The prepared slide was loaded into the VITEK MS system. This instrument analyzed the bacterial proteins in each sample, generating a unique fingerprint. Since these fingerprints are primarily based on ribosomal proteins, they can effectively differentiate between different bacterial species. The generated fingerprints were compared to a database containing known fingerprints of various bacteria. The closer the match between a sample’s fingerprint and a database entry, the higher the confidence score assigned by the instrument. Enterococcus faecalis ATCC 29,212, Streptococcus equi subsp. zooepidemicus ATCC 43,079, Escherichia coli ATCC 35,218, Pseudomonas aeruginosa ATCC 27,853, Staphylococcus aureus ATCC 29,213, and Klebsiella pneumoniae ATCC 700,603 were used as the quality control strain. Resolving discrepancies with gene sequencing Discrepancies between VITEK2 and MALDI-TOF MS were resolved by 16s rRNA sequencing as described by Geo et al. , which performed at King Faisal Specialist Hospital & Research Centre (Riyadh, Saudi Arabia). The results would be considered valid only if the similarity (homologous rate) between the sequenced gene and a known reference gene was above 99%. Biofilm formation By using crystal violet assay to measure biofilms, pathogenic bacteria that had been identified were streaked on plates of trypticase soy agar (TSA; Watin Biolife) and grown at 37 °C for a night. A single colony was picked and used to start a culture in 3 mL of trypticase soy broth (TSB). The culture was incubated at 37 °C with shaking at 250 rpm for 24 h. The overnight cultures were then added to five wells of a 96-well plate (Corning Inc. Corning, NY, USA) in triplicate and grown to a final concentration of ~ 10 7 CFU/mL in 180 µL (final volume) of TSB supplemented with 0.5% glucose (TSBg; Difco). To evaluate the effects of temperature and agitation on biofilm formation, the plates were then incubated for one day under the following conditions: first plate, 4 °C with no agitation (0 rpm); second plate, 15 °C with no agitation; third plate, 30 °C with no agitation; fourth plate, 30 °C with 60 rpm agitation; and fifth plate, 30 °C with 120 rpm agitation . We selected 30 °C as the temperature for agitation testing to reflect the average temperature in Riyadh, Saudi Arabia. After biofilm formation, gently washing was conducted on each well three times to remove planktonic cells with phosphate-buffered saline (PBS; pH7.4), and the biofilms were stained with 180 µL of 0.3% crystal violet (BD, MD, USA) for 30 min. The wells were then washed three times with PBS to remove any excess stain. Next, the wells were incubated with 180 µL of destaining solution (20% acetone and 80% ethanol) for 30 min to dissolve the bound stain. Samples of the stained biofilms were then transferred to a new plate and the absorbance was measured at 492 nm in a microplate reader (Apollo LB913, Bad Wildbad, Germany). The absorbance of the negative controls (TSBg alone with no bacteria added) was subtracted from the absorbance of the bacterial cultures . As a control Staphylococcus aureus ATCC 29,213 (weak biofilm former) and Klebsiella pneumoniae ATCC 700,603 (strong biofilm former), were used in this study. Statistical analysis Each experiment was repeated three times, with three replicates per repetition. The mean and standard deviation (SDs) of the measurements were calculated using Excel software. A mixed-model analysis was performed using SAS software (SAS institute, Inc., version 9.4) to adjust for multiple comparisons, and differences between groups were considered statistically significant if the p -value was less than 0.01.
Raw milk samples were taken from storage tanks (300 to 3000 L in volume) at eight dairy farms in Riyadh, Saudi Arabi. A total of 40 samples were collected: one from each of five different bulk tanks at each farm, with approximately 30 mL per sample. Sample temperature during collection and the distance from the farm to the laboratory recorded. The samples were collected and transported to Imam Mohammad Ibn Saud Islamic University within two hours, where they were kept cold and tested the same day . This study did not involve any human or animal subjects, so ethical approval was not required.
To identify bacteria in RCM, 6 mL of each sample (out of 30 mL total) was used as follows: First, 3 mL was streaked on six plates (0.5 mL per plate) using 5% sheep blood agar (SBA; Watin Biolife, KSA) for the identification of bacterial species with low loads. Second, the remaining 3 mL was diluted 10 times in phosphate-buffered saline (PBS) with a pH of 7.4, and the diluted sample was then spread on SBA for the determination of bacterial species with high loads. Plates were kept for 48 h at 37 °C under aerobic and 5% CO 2 conditions. Colonies were isolated for differentiation of pathogenic and non-pathogenic bacteria from the initial cultures based on several factors including: colony morphology (size, shape, color, and texture), diameter, hemolytic properties, and basic chemistry tests including catalase and coagulase assays. Furthermore, following isolation, gram staining (BD, NJ, USA) was performed as recommended by the stain manufacturer to differentiate between Gram-positive and Gram-negative bacteria. Isolated colonies were incubated another two days at 37 °C under aerobic or in the presence of 5% CO 2 . Two colonies were picked and used to start a culture in 3 mL of sterile sodium chloride solution (0.45%), which was made to be as cloudy as a 0.5 McFarland standard. The bacterial suspensions were then put on each of two testing cards, VITEK 2 ID-GPB and VITEK 2 ID-GNB, and placed into a VITEK 2 system (bioMérieux) following the instructions from the manufacturer for species identification. In the event the VITEK 2 identification was made with less than 90% confidence. To ensure that the tests were being performed correctly, the laboratory also tested the following strains of bacteria, as recommended by the Clinical and Laboratory Standards Institute (CLSI): Enterococcus faecalis ATCC 29,212, Streptococcus equi subsp. zooepidemicus ATCC 43,079, Escherichia coli ATCC 35,218, Pseudomonas aeruginosa ATCC 27,853, Staphylococcus aureus ATCC 29,213, and Klebsiella pneumoniae ATCC 700,603 .
We confirmed the identity of isolated bacteria by using MALDI-TOF MS system (bioMérieux) . We followed the manufacturer’s instructions for the VITEK MS system to perform this analysis. Briefly, a loop was used to pick individual bacterial colonies and transfer them to designated spots on a slide. A special solution (VITEK MS-CHCA matrix) was then applied and allowed to dry completely. The prepared slide was loaded into the VITEK MS system. This instrument analyzed the bacterial proteins in each sample, generating a unique fingerprint. Since these fingerprints are primarily based on ribosomal proteins, they can effectively differentiate between different bacterial species. The generated fingerprints were compared to a database containing known fingerprints of various bacteria. The closer the match between a sample’s fingerprint and a database entry, the higher the confidence score assigned by the instrument. Enterococcus faecalis ATCC 29,212, Streptococcus equi subsp. zooepidemicus ATCC 43,079, Escherichia coli ATCC 35,218, Pseudomonas aeruginosa ATCC 27,853, Staphylococcus aureus ATCC 29,213, and Klebsiella pneumoniae ATCC 700,603 were used as the quality control strain.
Discrepancies between VITEK2 and MALDI-TOF MS were resolved by 16s rRNA sequencing as described by Geo et al. , which performed at King Faisal Specialist Hospital & Research Centre (Riyadh, Saudi Arabia). The results would be considered valid only if the similarity (homologous rate) between the sequenced gene and a known reference gene was above 99%.
By using crystal violet assay to measure biofilms, pathogenic bacteria that had been identified were streaked on plates of trypticase soy agar (TSA; Watin Biolife) and grown at 37 °C for a night. A single colony was picked and used to start a culture in 3 mL of trypticase soy broth (TSB). The culture was incubated at 37 °C with shaking at 250 rpm for 24 h. The overnight cultures were then added to five wells of a 96-well plate (Corning Inc. Corning, NY, USA) in triplicate and grown to a final concentration of ~ 10 7 CFU/mL in 180 µL (final volume) of TSB supplemented with 0.5% glucose (TSBg; Difco). To evaluate the effects of temperature and agitation on biofilm formation, the plates were then incubated for one day under the following conditions: first plate, 4 °C with no agitation (0 rpm); second plate, 15 °C with no agitation; third plate, 30 °C with no agitation; fourth plate, 30 °C with 60 rpm agitation; and fifth plate, 30 °C with 120 rpm agitation . We selected 30 °C as the temperature for agitation testing to reflect the average temperature in Riyadh, Saudi Arabia. After biofilm formation, gently washing was conducted on each well three times to remove planktonic cells with phosphate-buffered saline (PBS; pH7.4), and the biofilms were stained with 180 µL of 0.3% crystal violet (BD, MD, USA) for 30 min. The wells were then washed three times with PBS to remove any excess stain. Next, the wells were incubated with 180 µL of destaining solution (20% acetone and 80% ethanol) for 30 min to dissolve the bound stain. Samples of the stained biofilms were then transferred to a new plate and the absorbance was measured at 492 nm in a microplate reader (Apollo LB913, Bad Wildbad, Germany). The absorbance of the negative controls (TSBg alone with no bacteria added) was subtracted from the absorbance of the bacterial cultures . As a control Staphylococcus aureus ATCC 29,213 (weak biofilm former) and Klebsiella pneumoniae ATCC 700,603 (strong biofilm former), were used in this study.
Each experiment was repeated three times, with three replicates per repetition. The mean and standard deviation (SDs) of the measurements were calculated using Excel software. A mixed-model analysis was performed using SAS software (SAS institute, Inc., version 9.4) to adjust for multiple comparisons, and differences between groups were considered statistically significant if the p -value was less than 0.01.
Prevalence of bacterial contamination in RCM Of the eight farm sites, three exhibited non-pathogenic bacterial contamination in their raw milk samples. Of the total of 40 raw milk samples, 15/40 (37.5%; from five farms) were contaminated with pathogenic bacteria. Overall, 346 bacteria were isolated from the 40 samples, with 329/346 (95.1%) considered as non-pathogenic and 17/346 (4.9%) as pathogenic. The 17 isolated pathogenic bacteria were (number): S. aureus (4), E. coli (4), K. pneumoniae (2), L. monocytogenes (3), S. agalactiae (1), and S. typhimurium (3). Interestingly, only one farm (site C) had two pathogens detected in the same sample, which occurred twice: S. aureus and E. coli in sample number 6, and S. agalactiae and S. typhimurium in sample number 8 (Table ). Regarding raw milk storage temperature, ten instances of contamination by pathogenic bacteria were found in milk stored above room temperature (more than 24 °C), five in milk at medium temperature (10 °C to 24 °C), and two in milk at low temperature (less than 10 °C). It is noteworthy that both instances of low-temperature contamination occurred at one farm (site A) and involved the same bacterial species, S. aureus (Table ). Regarding the relationship of transport distance to pathogenic bacterial contamination of milk, three instances of contamination (two with S. aureus and one E. coli ) occurred in samples from farms at less than 30 min driving distance (< 50 km), five instances (two E. coli , two K. pneumoniae , and one L. monocytogenes ) from farms 30 min to one hour away (50 km to 100 km), and nine instances (two S. aureus , one E. coli , two L. monocytogenes , one S. agalactiae , and three S. typhimurium ) from farms more than one hour away (> 100 km). All told, our data support that higher storage temperature and longer transport distance increase the chance of isolating a pathogenic bacterium from raw milk samples. Matched results by the two identification methods Among the 15 isolated pathogens tested, VITEK-2 and MALDI-TOF MS demonstrated a high level of agreement (93%). Fourteen isolates yielded the same identification at the species level using both methods. However, a single isolate (7%) showed a discrepancy. VITEK-2 identified it as S. agalactiae ( S. agalactiae 20,206), which was confirmed by gene sequencing for added confidence. Intriguingly, MALDI-TOF MS identified this same isolate as Nocardia asteroids . Effect of temperature on biofilm formation Different types of pathogenic bacteria isolated from raw milk stuck to plastic plates more or less strongly depending on the temperature at which they were grown (Fig. ). According to Hassan et al., the ability of a bacterium to form a biofilm can be classified as weak, moderate, or strong . When cultured at 4 °C, all isolated bacteria displayed weak biofilm formation, with OD 492 values of 0.02 ± 0.02 to 0.09 ± 0.01. The pathogen with the highest biofilm formation at 4 °C was E. coli 20,102, and that with the lowest was S. typhimurium 20,210. At 15 °C, all isolated bacteria displayed weak to moderate biofilm formation, with OD 492 values of 0.07 ± 0.05 to 0.52 ± 0.13. The pathogen with the highest biofilm formation was E. coli 20,105, and that with the lowest was S. typhimurium 20,213. Finally, when grown at 30 °C, all isolated bacteria displayed strong biofilms, with OD 492 values of 0.40 ± 0.05 to 1.63 ± 0.13. The pathogen with the highest biofilm formation was E. coli 20,204, and that with the lowest was S. typhimurium 20,213 (Fig. b). In general, all isolated pathogenic bacteria exhibited a significant ( p < 0.01) increase in biofilm formation when grown at 30 °C compared to 4 °C; the exceptions were S. typhimurium 20,213 ( p = 0.023) and S. typhimurium 20,207 ( p = 0.026). When comparing 30 °C to 15 °C, S. aureus 20,101, S. aureus 20,212, L. monocytogenes 20,214, all E. coli strains, and all K. pneumoniae strains showed a significant ( p < 0.01) increase in biofilm formation; other strains showed a trend towards increase, with p values between 0.046 and 0.018. Surprisingly, when comparing growth at 15 °C against 4 °C, only E. coli 20,203 and E. coli 20,204 exhibited a significant ( p < 0.01) increase in biofilm formation (Fig. b). Effect of agitation on biofilm formation Pathogenic bacteria isolated from raw milk demonstrated various degrees of adherence to polystyrene plates consistent with the degree of agitation during incubation (Fig. a). All strains grown at 30 °C with 0 rpm, 60 rpm, and 120 rpm agitation exhibited strong biofilm formation. When comparing biofilm formation under 60 rpm agitation to that at 0 rpm, only E. coli 20,102 exhibited a significant ( p < 0.01) increase. However, when comparing the 120 rpm condition to 0 rpm, all isolated pathogenic bacteria exhibited a significant ( p < 0.01) increase in biofilm formation except for S. aureus 20,101, S. aureus 20,104, S. agalactiae 20,206, and all L. monocytogenes strains. When comparing the 120 rpm condition to 60 rpm, all E. coli strains and S. typhimurium 20,207 exhibited a significant ( p < 0.01) increase in biofilm formation (Fig. b).
Of the eight farm sites, three exhibited non-pathogenic bacterial contamination in their raw milk samples. Of the total of 40 raw milk samples, 15/40 (37.5%; from five farms) were contaminated with pathogenic bacteria. Overall, 346 bacteria were isolated from the 40 samples, with 329/346 (95.1%) considered as non-pathogenic and 17/346 (4.9%) as pathogenic. The 17 isolated pathogenic bacteria were (number): S. aureus (4), E. coli (4), K. pneumoniae (2), L. monocytogenes (3), S. agalactiae (1), and S. typhimurium (3). Interestingly, only one farm (site C) had two pathogens detected in the same sample, which occurred twice: S. aureus and E. coli in sample number 6, and S. agalactiae and S. typhimurium in sample number 8 (Table ). Regarding raw milk storage temperature, ten instances of contamination by pathogenic bacteria were found in milk stored above room temperature (more than 24 °C), five in milk at medium temperature (10 °C to 24 °C), and two in milk at low temperature (less than 10 °C). It is noteworthy that both instances of low-temperature contamination occurred at one farm (site A) and involved the same bacterial species, S. aureus (Table ). Regarding the relationship of transport distance to pathogenic bacterial contamination of milk, three instances of contamination (two with S. aureus and one E. coli ) occurred in samples from farms at less than 30 min driving distance (< 50 km), five instances (two E. coli , two K. pneumoniae , and one L. monocytogenes ) from farms 30 min to one hour away (50 km to 100 km), and nine instances (two S. aureus , one E. coli , two L. monocytogenes , one S. agalactiae , and three S. typhimurium ) from farms more than one hour away (> 100 km). All told, our data support that higher storage temperature and longer transport distance increase the chance of isolating a pathogenic bacterium from raw milk samples. Matched results by the two identification methods Among the 15 isolated pathogens tested, VITEK-2 and MALDI-TOF MS demonstrated a high level of agreement (93%). Fourteen isolates yielded the same identification at the species level using both methods. However, a single isolate (7%) showed a discrepancy. VITEK-2 identified it as S. agalactiae ( S. agalactiae 20,206), which was confirmed by gene sequencing for added confidence. Intriguingly, MALDI-TOF MS identified this same isolate as Nocardia asteroids .
Different types of pathogenic bacteria isolated from raw milk stuck to plastic plates more or less strongly depending on the temperature at which they were grown (Fig. ). According to Hassan et al., the ability of a bacterium to form a biofilm can be classified as weak, moderate, or strong . When cultured at 4 °C, all isolated bacteria displayed weak biofilm formation, with OD 492 values of 0.02 ± 0.02 to 0.09 ± 0.01. The pathogen with the highest biofilm formation at 4 °C was E. coli 20,102, and that with the lowest was S. typhimurium 20,210. At 15 °C, all isolated bacteria displayed weak to moderate biofilm formation, with OD 492 values of 0.07 ± 0.05 to 0.52 ± 0.13. The pathogen with the highest biofilm formation was E. coli 20,105, and that with the lowest was S. typhimurium 20,213. Finally, when grown at 30 °C, all isolated bacteria displayed strong biofilms, with OD 492 values of 0.40 ± 0.05 to 1.63 ± 0.13. The pathogen with the highest biofilm formation was E. coli 20,204, and that with the lowest was S. typhimurium 20,213 (Fig. b). In general, all isolated pathogenic bacteria exhibited a significant ( p < 0.01) increase in biofilm formation when grown at 30 °C compared to 4 °C; the exceptions were S. typhimurium 20,213 ( p = 0.023) and S. typhimurium 20,207 ( p = 0.026). When comparing 30 °C to 15 °C, S. aureus 20,101, S. aureus 20,212, L. monocytogenes 20,214, all E. coli strains, and all K. pneumoniae strains showed a significant ( p < 0.01) increase in biofilm formation; other strains showed a trend towards increase, with p values between 0.046 and 0.018. Surprisingly, when comparing growth at 15 °C against 4 °C, only E. coli 20,203 and E. coli 20,204 exhibited a significant ( p < 0.01) increase in biofilm formation (Fig. b).
Pathogenic bacteria isolated from raw milk demonstrated various degrees of adherence to polystyrene plates consistent with the degree of agitation during incubation (Fig. a). All strains grown at 30 °C with 0 rpm, 60 rpm, and 120 rpm agitation exhibited strong biofilm formation. When comparing biofilm formation under 60 rpm agitation to that at 0 rpm, only E. coli 20,102 exhibited a significant ( p < 0.01) increase. However, when comparing the 120 rpm condition to 0 rpm, all isolated pathogenic bacteria exhibited a significant ( p < 0.01) increase in biofilm formation except for S. aureus 20,101, S. aureus 20,104, S. agalactiae 20,206, and all L. monocytogenes strains. When comparing the 120 rpm condition to 60 rpm, all E. coli strains and S. typhimurium 20,207 exhibited a significant ( p < 0.01) increase in biofilm formation (Fig. b).
Consumers store and transport food practices can affect its safety. Even though experts say to use insulated containers to keep chilled foods cold, especially in warm weather, several consumers don’t do this . Our findings highlight this as a major concern, as pathogen concentrations and biofilm formation were significantly lower when using low-temperature storage (< 10 °C) compared to high-temperature storage (> 10 °C). In addition, we demonstrated that low-temperature storage does not eliminate pathogens, but rather prevents bacterial overgrowth and biofilm formation. These results emphasize the significance of using an ice pack or other cold object to maintain a cool temperature during storage and transportation . Previous studies have shown that many refrigerators are not kept at the recommended temperature, that consumers are not aware of the temperature of their refrigerators, and that milk is often not stored in the coldest part of the refrigerator . In this study, we found that more bacteria grew and formed biofilms in milk that was stored at higher temperatures. Other studies have shown that consumers often thaw potentially hazardous foods at room temperature, even though it is recommended to thaw them overnight in the refrigerator . Biddle et al. suggest that the choice of thawing method is important for minimizing bacterial growth in milk . Elmoslemany et al. showed that bacteria that can form biofilms are a major cause of harmful bacteria in dairy production . This means that biofilm-forming bacteria can have a negative impact on the safety and quality of milk and dairy products . Contamination of dairy products is often linked to biofilms forming on the surfaces of milk pipes, cow skin flora, milking containers, and other equipment in dairy manufacturing . Herein, we demonstrated that isolated pathogenic bacteria from raw milk can exhibit increased biofilm formation when the milk is agitated, including by motor vehicle transport. That is, samples transported with more agitation from more distant farms showed more clumped milk and higher bacteria concentrations, which might indicate high biofilm formation. While our study investigated the impact of temperature and agitation on biofilm formation, a deeper understanding of the underlying mechanisms is crucial. Several studies have shown that environmental factors can influence biofilm formation through gene expression regulation. For instance, milk components have been shown to stimulate biofilm formation in S. aureus , with a corresponding upregulation of the ica operon (responsible for polysaccharide intercellular adhesin synthesis) under specific conditions . Future research should explore these mechanisms in detail. This could involve identifying and analyzing the expression patterns of genes involved in biofilm formation (e.g., adhesin production, motility, matrix synthesis) under varying temperature and agitation conditions. By elucidating the precise regulatory pathways involved, we can gain a more comprehensive understanding of how environmental factors influence biofilm formation at the molecular level. This knowledge can be vital for developing targeted strategies to prevent or disrupt biofilm formation in clinical and industrial settings. Several studies have previously isolated E. coli , L. monocytogenes , K. pneumoniae , S. aureus , S. agalactiae , and S. typhimurium from raw milk, consistent with the findings herein . However, to the author’s knowledge, this is the first study to examine the effects of temperature and agitation on pathogens in raw milk in the Middle East. A recent study from Australia by Roselyn and coworkers reported that temperature and agitation significantly impact the overgrowth of E. coli and L. monocytogenes . Our results demonstrate a similar trend for all isolated pathogens. More data are required to substantiate the impact of temperature and agitation on non-pathogenic bacteria such as Lactococcus spp. and Lactobacillus spp. Pasteurization, or short-term exposure to high temperature, aims to decrease the number of harmful bacteria to a safe level, giving it a shelf life of about ten days when refrigerated. This heat treatment process guarantees that raw milk is safe microbiologically; however, heat treatment degrades its nutritional value . Several studies have shown that heating milk can change its nutritional value and taste, since several of its molecular components are sensitive to high temperature; in particular, heating milk changes the structure of whey proteins, destroys essential amino acids like lysine, and reduces B vitamins. Therefore, there is interest in developing new strategies that do not rely on heat . New methods are being developed to prevent bacteria from growing and forming biofilm on surfaces. These methods include making surfaces smoother to prevent bacterial adhesion, adding antimicrobial coatings, and using anti-adhesive compounds to repel bacteria . In addition, consumer handling practices between sourcing and consumption are a critical element in reducing the risks associated with pathogen contamination and realizing the full benefit of these treatment processes of raw milk .Our study demonstrated a high level of agreement (93%) between VITEK-2 and MALDI-TOF MS for identifying isolated pathogens. This concordance rate aligns with previous research, such as the study by Jamal et al. (2014) which reported similar agreement (93.2%) between VITEK-2 and VITEK MS for bacterial identification . However, our findings also highlight the potential for discrepancies. Notably, one isolate (7%) displayed discordant results, with VITEK-2 identifying it as S. agalactiae and MALDI-TOF MS identifying it as Nocardia asteroids . While this finding aligns with the potential for discrepancies reported by Jamal et al. (2014), it underscores the importance of employing complementary identification methods, especially for critical pathogens like S. agalactiae . Further investigation into this specific discrepancy, potentially using gene sequencing, could provide valuable insights into the limitations of each method for specific bacterial strains. One limitation of this study is that the sample size used (30 mL) may not be representative of the larger volumes that consumers typically handle. Additionally, the small sample size may have affected how quickly the different temperature parameters were reached. Future simulations could be improved by using more representative sample sizes. In this study, we focused on two specific environmental factors, temperature and agitation, however other parameters known to effect bacterial behavior in milk, such as milk pH and vitamin availability, were not measured during this study. These factors are known as potentially impacting both bacterial contamination and biofilm formation. For future studies, we plan to include these additional parameters to increase the analysis and gain more comprehensive understanding. Our research showed that transporting raw milk at a cold temperature (4 °C) and with less agitation is important for controlling the level of pathogenic bacteria in the milk. Future studies should consider strain variation of pathogenic bacteria and the combined effects of agitation, time, and temperature misuse during the consumer phase on bacterial growth. Public health strategies could be improved by educating consumers about the importance of properly handling raw milk between sourcing and consumption.
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Dr. Valentin Grandis y Dr. Virgilio Ducceschi, Dos maestros italianos iniciadores de la Fisiología experimental en la Facultad de Ciencias Médicas - UNC | e0bff85f-b717-416b-b9c2-fc3fa145319f | 11370869 | Physiology[mh] | Valentín Grandis, nació en Boves, Provincia de Cuneo, Italia, el 14 de mayo de 1862. Estudió Medicina en la Universidad de Turín, destacándose por su interés en la Fisiología. Se graduó como Doctor en Medicina en 1886. Fue primero Interno y luego Asistente del Prof. Ángelo Mosso, famoso fisiólogo e investigador de muchos problemas, entre ellos el Mal de Altura, estableciendo un Laboratorio en el Monte Rosa, que adquirió fama mundial. En Firenze, el Dr. Grandis concurrió al Instituto de Fisiología del destacado fisiólogo Prof. Luigi Luciani, quien fue además de Maestro para el joven Grandis, su consejero y fiel amigo, durante muchos años. En 1890, Grandis continuó su perfeccionamiento en Leipzig (Alemania), en el Laboratorio del Prof. Karl Ludwing, el centro de mayor importancia en Fisiología. El dominio del idioma alemán, permitió al Dr. Grandis publicar sus trabajos en revistas alemanas, durante toda su vida. También el Prof. Guillermo Oswald, le ofreció un cargo en el Instituto de Físico-Química, pero Grandis no lo aceptó. Retornó a Turín, para trabajar nuevamente, en el Laboratorio del Prof. Mosso, efectuando varias investigaciones. Además era adscripto a la Cátedra de Clínica Psiquiátrica del Prof. Lombroso. Grandis, se relacionó en Turín con el químico Prof. Leilio Guareschi, con el que perfeccionó sus conocimientos en Química Biológica, aplicados posteriormente a sus investigaciones, altamente valoradas por los italianos que veían la marcha de la Fisiología moderna, gracias a los métodos químicos. El Dr. Valentín Grandis, ascendiendo en la carrera universitaria, fue designado profesor de Fisiología en la Universidad de Ferrara y profesor de Química Biológica en la Universidad de Génova . En resumen, el Dr. Valentín Grandis, con una preparación en Fisiología; Matemáticas, Físico-Química y Química Biológica, era un Fisiólogo completo. Ante las gestiones realizadas por el Ministro argentino de Instrucción Pública en Roma, el Prof. Ángelo Mosso, recomendó al Dr. Valentín Grandis, para desempeñarse en el Laboratorio de Fisiología Experimental de la Universidad de Buenos Aires. En 1887, era Prof. Titular de la Cátedra de Fisiología, en la Facultad de Medicina de la Capital, el Dr. Coronado y como Suplente el Dr. Horacio G. Piñero . El Dr. Valentín Grandis fue contratado como Jefe de Trabajos Prácticos de Fisiología Experimental y durante seis años desarrolló una notable actividad científica, logrando rodearse de discípulos que luego continuarían su ejemplo, entre ellos Carlos Mainini. Guillermo V. Stuckert y Manuel Payá. Con el Dr. Horacio Piñero publicaron los Apuntes del Curso de Fisiología en el año 1902. Con el Dr. Ivo Novi publicaron trabajos sobre reflejos cerebrales. Con el Dr. Pedro Vignean, se aislaron nuevas globulinas del huevo, publicado en Anales del Departamento Nacional de Higiene de Buenos Aires de 1901. Con el Dr. T. A. Moret, estudio la presión hidrostática del humor acuoso. Con el Dr. O. Copello, la composición química en las cenizas del cartílago durante la etapa de calcificación. Esta breve enumeración de trabajos, demuestra que Grandis dio nacimiento a una Escuela de Fisiología en Buenos Aires, rodeado de valiosos colaboradores. El 4 de diciembre de 1900, el Dr. Grandis fue encargado de los Trabajos Prácticos del Instituto de Anatomía Patológica (Sección Fisiología Patológica) del Prof. Susini. El conocimiento de la presencia de un fisiólogo distinguido en Buenos Aires, hizo pensar al Decano Dr. Sebastián Palacios y al Rector Dr. José Ortiz y Herrera, la posibilidad de contratar al Dr. Valentín Grandis, como Jefe de Trabajos Prácticos Experimentales, en Córdoba. Felizmente la aceptación permitió la firma del contrato el 8 de febrero de 1904, con retroactividad al 1 de Enero. (Archivo de la Universidad Nacional de Córdoba) El texto del contrato puede leerse en la obra del Dr. Garzón Maceda . El 22 de febrero de 1904, desde Buenos Aires el Dr. Grandis, comunicó su partida para Europa, a fin de comprar todo el material requerido para las investigaciones. Al mismo tiempo pedía al Sr. Rector Dr. José Ortiz y Herrera, activar la construcción del Laboratorio y el plano definitivo del mismo, ya revisado por la UNC; el Ministro de Justicia e Instrucción Pública y el Poder Ejecutivo Nacional. La construcción estuvo a cargo de la Dirección de Arquitectura de la Nación, bajo la supervisión del Ing. José Manuel Saravia y la Empresa Constructora del Ing. Carlos François. La Universidad disponía de un amplio terreno en la calle Santa Rosa esquina Mendoza, donde finalmente se fue construyendo paulatinamente, la totalidad de la denominada Escuela Práctica de Medicina . El edificio estuvo terminado el 14 de septiembre de 1905 y el Prof. Valentín Grandis pudo iniciar la instalación del material adquirido en Europa y desarrollar su labor de Investigación y docencia. Los aparatos e instrumentos llegaron embalados en 133 (ciento treinta y tres) cajones. Eran de inmejorable calidad y en cantidad tal que veinte años después permitió el funcionamiento de dos Cátedras: Fisiología y Química Biológica. Había una riqueza de material comparable con los mejores Laboratorios europeos El Laboratorio contaba con un subsuelo de una amplitud tal que comprendía toda la superficie de la edificación, bien ventilado y en comunicación con otras dependencias. En la planta baja existía una amplia sala para demostraciones experimentales, con su mesa cátedra con instalación de agua, gas y corriente eléctrica de varios voltajes. Dicho salón fue luego ocupado por la Biblioteca del Instituto. Había otro salón con grandes ventanales, con seis mesas provistas de todos los materiales requeridos por los alumnos y le seguía otro salón con mesas especiales, del Laboratorio de Química Biológica . Sumado a otras dependencias, tales como depósitos, bioterio, sanitarios, despensa. El Laboratorio de Fisiología Experimental descripto, se mantuvo inalterable hasta el año 1937, cuando el Prof. Oscar Orías consiguió la construcción de una segunda planta, aumentando la amplitud del edificio. En Febrero de 1906, durante el Decanato del Dr. Virgilio Moyano, se designó como ayudante colaborador al Sr. Adolfo Spadoni . El Dr. Valentín Grandis, organizó la enseñanza de la Fisiología Experimental, en un grado extraordinario para su época. Las clases teóricas eran dadas por el Prof. Dr. Carlos Díaz, hasta su jubilación en 1918. Todo el sistema de Trabajos Prácticos, diseñado por Grandis, se mantuvo hasta 1907 con la llegada del Dr. Virgilio Ducceschi, quien introdujo pequeñas modificaciones. Dicho programa de Trabajos Prácticos se mantuvo inalterable hasta 1935, cuando se hizo cargo el Dr. Oscar Orías. A final del año 1905, el Dr. Valentín Grandis fue designado como Profesor y Director del Real Instituto de Fisiología de Génova y fue condecorado como Caballero de la Corona de Italia, el 22 de diciembre de 1918. Debemos recordar que el Prof. Grandis, desde que aceptó su desempeño en Buenos Aires y luego en Córdoba, se mantenía con licencia en sus cargos de Profesor en las Universidades de Ferrara y Génova. gracias a que el gobierno peninsular en un acto de amistad hacia la República Argentina, lo mantuvo, aunque presionado, con licencia desde 1898 hasta 1906 . El 20 de septiembre de 1906, el Dr. Valentín Grandis comunicó a la Facultad de Ciencias Médicas de Córdoba, la imperiosa necesidad de volver a Italia. Se despidió de sus discípulos, alumnos y personal del Laboratorio, dando lugar a la toma de la fotografía histórica, que mostramos, con el grupo de 1906. El Ministro de Instrucción Pública, Dr. Joaquín V. González, posteriormente el fundador de la Universidad de La Plata, ante la situación planteada en Córdoba, dispuso la contratación del Dr. Virgilio Ducceschi, el que, durante doce años, va a continuar la obra que mantuvo en alto el nombre de la Ciencia italiana y a Córdoba con el brillo de su Laboratorio de Fisiología Experimental. El Dr. Valentín Grandis era un trabajador infatigable y estudioso de variados temas, demostrado en gran número de trabajos de investigación. El Prof. Dr. Alberto Marsal, obtuvo las fotocopias de más de ochenta trabajos que se encontraban en la Biblioteca del Instituto de Fisiología y las depositó en la Academia Nacional de Ciencias en 1973, como testimonio de la laboriosidad del primer docente investigador en la Facultad de Ciencias Médicas de Córdoba-UNC. Y dice al respecto: "El Prof. Valentín Grandis perteneció a ese grupo de científicos que a fines del Siglo pasado llevaron a tan alto lugar la Fisiología italiana. Córdoba le debe la formación de su Laboratorio de Fisiología Experimental, que fuera el fundamento del desarrollo actual de la posición científica de la Facultad de Ciencias Médicas" . Luego de un largo padecimiento, posiblemente una tuberculosis renal, el D. Grandis falleció el 12 de diciembre de 1928 , en Sturia, a los 66 años de edad. Se hizo cargo entonces de la Dirección del Real Instituto de Fisiología de Génova, el Dr. Cayetano Viale que por una verdadera casualidad, fue después, el organizador del Laboratorio de Fisiología de la Universidad Nacional de Rosario (R.A). Virgilio Ducceschi tuvo la oportunidad de desarrollar en Córdoba, el Laboratorio de Fisiología iniciado por el Dr. Valentín Grandis. Fundó también las Cátedras de Psicología Experimental y Química Biológica en la Facultad de Ciencias Médicas-UNC. Se encuentra en la Cátedra de Química Biológica. (Marsal A, p. 22) . Virgilio Ducceschi nació el 1 de septiembre de 1871, en Scansano, provincia de Grosseto (Italia). Estudió Medicina y Cirugía en el Instituto di Studi Superiori de Firenze, donde se laureó como Médico en 1885 con una Tesis Doctoral titulada: "La proteidemia en el perro tiroidectomizado". Su vocación le llevó desde estudiante a ingresar entre 1895 a 1900, en el Instituto de Fisiología de Florencia, bajo la dirección del Prof. Giulio Fano. En 1900 y hasta 1906, pasó al Instituto de Fisiología de la Universidad de Roma, dirigido por el Prof. Luigi Luciani. Después de su formación académica en Italia, se trasladó a Estrasburgo, al Instituto de Fisiología de los Prof. Goltz y J. Ewald y Química Biológica con el Prof. F. Hofmeister, figura principal de la bioquímica, en esa época. Sin duda que luego de estos estudios, Ducceschi se encontraba bien preparado para asumir la enseñanza universitaria . Profesor Titular por Concurso de la Cátedra de Fisiología en la Universidad de Palermo, posición que debió declinar ante la invitación de la Universidad Nacional de Córdoba . Antes de viajar a la República Argentina el Dr. Ducceschi contrajo matrimonio con Emilia Daugler de Estrasburgo, la que falleció en Córdoba el 4 de octubre de 1916. En 1907, era Profesor de la Cátedra de Fisiología en Córdoba, el Dr. Carlos Díaz, solo con la misión de mantener la enseñanza a un nivel puramente teórico. El Dr. Ducceschi, debía hacerse cargo entonces, del Laboratorio de Fisiología. Al tomar posesión del mismo, el Dr. Ducceschi se encontró con un Laboratorio montado por su antecesor, Dr. Valentín Grandis con una riqueza de medios de trabajo que pocos en el mundo podían presentar en aquella época. El Dr. Ducceschi tenía un interés especial en el estudio de los cinco sentidos y por el Sistema Nervioso, central y periférico. Propuso la creación de una Cátedra de Psicología Experimental, algo extraordinariamente nuevo para el comienzo del Siglo XX. Con tal motivo, el Poder Ejecutivo Nacional dictó un Decreto, el 15 de junio de 1907, firmado por el Presidente Dr. José Figueroa Alcorta y por el Ministro de Justicia e Instrucción Pública, Dr. Federico Pinedo, donde reconocían la importancia de las Cátedras, aunque sin relación con una determinada Facultad. Por este motivo la Cátedra de Psicología, fue con la Cátedra de Pedagogía, las dos primeras Cátedras Libres. El Dr. Ducceschi fue designado Profesor de la Cátedra de Psicología Experimental, desde el 15 de junio de 1907 hasta el 28 de febrero de 1919. Organizó la enseñanza y la investigación con carácter vocacional. La clase inaugural del año 1912, fue publicada en la Revista del Círculo Médico de Córdoba, Año III, febrero de 1913 Nº 2, titulada: "La Criminología moderna". En el Congreso Científico Internacional, (Buenos Aires1910, presentó un "Registrador mental", un elemento muy novedoso para la época. El prestigio alcanzado en el Laboratorio de Fisiología Experimental, hizo que Buenos Aires, ofreciera al Prof. Ducceschi, hacerse cargo de la Cátedra de Fisiología, en la Facultad de Medicina de la Capital, pero el Maestro lo declinó, por el compromiso moral que tenía con la Universidad Nacional de Córdoba. Dicha Cátedra fue ocupada después por el Prof. Bernardo A. Houssay, con el más alto grado de éxitos. nacionales e internacionales. En 1918, el Dr. Ducceschi, propuso al Interventor Dr. José Salinas, la creación de la Cátedra de Química Biológica, muy relacionada con la Fisiología, pero autónoma. El Dr. Salinas Interventor en la Facultad de Ciencias Médicas, aceptó la idea y por Decreto del 9 de octubre de 1918, nombró al Dr. Virgilio Ducceschi, Profesor de la nueva Cátedra. El Dr. Ducceschi ya había decidido volver a Italia, sin embargo presentó el primer Programa de Enseñanza de la asignatura y dio directivas para su funcionamiento. El sucesor en la Cátedra de Química Biológica fue el Dr. Guillermo V. Stuckert, uno de sus discípulos al que le había dirigido su Tesis de Doctorado en Medicina, en 1913 titulada: "Observaciones Experimentales sobre la eliminación del Ácido Úrico". Ducceschi tuvo otros discípulos, entre ellos: Juan Albarenque, Gregorio Martínez; Heriberto Walker; Guillermo V. Stukert; Manuel Salas; Daniel Barilari y otros, formando una sólida Escuela de Fisiología y realizando con ellos, una medulosa obra de investigación. Toda la producción de investigación producida por el Prof. Ducceschi fue publicada bajo el título de "Trabajos del Laboratorio de Fisiología y se dividieron en tres grandes series. Tomo 1, Serie primera; Tomo 1, serie 2 (1908-1910); Tomo 2, Serie 3 (1911-1914). En total fueron más de un centenar de trabajos científicos. Dichos trabajos, se realizaban en las seis mesas disponibles, con tres alumnos cada una. El Prof. Ducceschi guiaba y controlaba junto con el preparador Sr. Adolfo Spadoni, aportando siempre la luz en la resolución de los problemas investigados. Los experimentos eran sencillos pero muy demostrativos, estimulando en los alumnos el deseo de aprender y comprender la importancia de la Investigación. La laboriosidad del Prof. Ducceschi, dio como resultado numeroso trabajos que el HCD de la Facultad, acordó el recurso económico para realizar un tiraje de cuatrocientos ejemplares. En febrero de 1909, salió el primer Folleto titulado "Trabajos del Laboratorio de Fisiología Experimental de la Universidad Nacional de Córdoba". A fin del año 1909, se mandó a imprimir el segundo folleto, con trabajos sobre el Sistema Nervioso; Fisiología de la piel y otros. Además aparecen trabajos de los Dres. Albarenque, J. M.; Martínez, G.; Barilari, D; Salas M. y Stucker, F. . Terminada su actuación en Córdoba, el Dr. Virgilio Ducceschi, fue Profesor Titular por Concurso, de la Cátedra de Fisiología de la Universidad de Sassari, pero un año después fue llamado por la Universidad de Pavía y transferido en 1925 a la Cátedra de Fisiología en la Universidad de Padua. Sin embargo, siempre mantuvo los lazos de unión con la Universidad de Córdoba a la que representó con orgullo, con motivo del Octavo Centenario de la Universidad de Padua. El 14 de noviembre de 1934, el HCS de la Universidad Nacional de Córdoba, lo designó Profesor Honorario por su brillante actuación en el Laboratorio de Fisiología Experimental. El 30 de mayo de 1945, sintiéndose enfermo, escribió su testamento disponiendo como único heredero a la Universitá Degli Studi di Padova, incluso su casa particular, para que fuera destinada a formar la "Casa dell Assistente", como alojamiento y manutención de todos aquellos estudiantes con escasos recursos económicos. El Dr. Virgilio Ducceschi, murió el 19 de junio de 1952, a la edad de ochenta años, en Cittá di Piove di Scco (Italia). También dispuso en el testamento que su sepelio se realizara según el culto católico, pero simple, decoroso, sin discursos ni flores . En un dato del articulado del testamento, se refirió al dinero argentino que le había dejado en Córdoba, al Prof. Dr. Ferdinando Strada. Un dinero, que una vez cambiado aportó 3700 dólares que ingresaron al legado testamentario, junto con el producido por la venta de la Colección de Autógrafos de personalidades sudamericanas, que habían sido de su pertenencia. Sus obras sobre Historia de la Medicina, fueron depositadas según sus deseos. en la Biblioteca Pinali, de la Universidad. El presente trabajo pretende revivir la memoria de dos Maestros italianos: Valentín Grandis y Virgilio Ducceschi. Sus grandes condiciones científicas, docentes, morales y personales, hicieron posible el comienzo de la Investigación y Experimentación, unidas a la Fisiología y a la Química Biológica, en la Escuela Práctica de la Facultad de Ciencias Médicas. Los discípulos de los Profesores Grandis y Ducceschi, siguiendo sus ejemplos, se convirtieron después en destacados Académicos que mantuvieron la jerarquía y excelencia en la Universidad Nacional de Córdoba. |
Quaternized Molecular Brush‐Grafted Injectable Microgel with Anti‐Inflammatory and Drainage Properties for Efficient Therapy of Anal Fistula | 248ffe20-a186-42b2-a4a7-67e6672c685c | 11791944 | Surgical Procedures, Operative[mh] | Introduction Anal fistula is a common anorectal disease characterized by the presence of an abnormal tract connecting the anal canal/rectum to perianal skin, leading to persistent inflammation and an increased susceptibility to malignancy. Surgery is typically the primary treatment by removing the fistulous tract and ensuring drainage. However, complex anal fistula often requires extensive surgical dissection, which may lead to prolonged healing and catastrophic incontinence, with a recurrence rate up to 57% and incontinence rate up to 52%. Therefore, it is critical to develop an effective and meanwhile less traumatic therapeutic strategy for anal fistula. Less invasive therapeutic options for anal fistula currently include seton, bioprosthetic plug, and fibrin glue. A seton is placed in the fistula to facilitate the drainage of exudate without causing incontinence. However, the seton cannot provide an effective physical filling to exclude inflammatory stimuli from the fistula, and constant stimuli will cause abnormal epithelialization of the fistula wall and hinder the healing process ( Figure ). Meanwhile, seton therapy typically requires a prolonged period for effective drainage and gradual healing, which may lead to a lengthy treatment process and cause long‐term discomfort for patients. Alternatively, materials such as bioprosthetic plug and fibrin glue have been used to fill the fistula without extensive surgery. Bioprosthetic plug is typically made from porcine intestinal submucosa or human dermal matrix with superior biocompatibility. Similarly, fibrin glue is composed of fibrinogen and thrombin and therefore biologically safe. However, these materials will block the drainage after filling the fistula with a healing rate of less than 50%. Therefore, the balance between effective filling and adequate drainage is crucial for anal fistula treatment. Moreover, the persistent inflammation and abnormal epithelialization of fistula are featured by local accumulation of the inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β). Currently, there is no effective clinical therapy to neutralize inflammatory cytokines. Thus, a biocompatible and anti‐inflammatory material that is simultaneously filling and draining is highly desirable for the treatment of anal fistula. Herein, a new class of quaternized molecular brush‐grafted injectable microgel (denoted as GAA@CNT‐ g ‐PVBTMA) with excellent anti‐inflammatory and drainage properties is successfully developed by thermal polymerization of quaternized carbon nanotube molecular brush (CNT‐ g ‐PVBTMA) with precursor solution and mechanical fragmentation (Figure ). The precursor solution is mainly composed of gelatin (Gel), acrylic acid N‐succinimidyl ester (AAc‐NHS), and acrylated β‐cyclodextrin (Ac‐β‐CD). Due to the incorporation of CNT‐ g ‐PVBTMA, our GAA@CNT‐ g ‐PVBTMA microgel exhibits a low swell ratio of 9.9%, which could maintain stable mechanical property in the wet environment. Owing to the fragmented morphology with a porous structure, our microgel demonstrates good fluidity during injection and forms a stable seal once the shear forces are removed, which can achieve effective filling of the fistula and facilitate the drainage of exudate. More importantly, our GAA@CNT‐ g ‐PVBTMA microgel can achieve high scavenging rates of 92.6% for TNF‐α and 92.5% for IL‐1β relying on the electrostatic interactions between the positively charged CNT‐ g ‐PVBTMA and the negatively charged inflammatory cytokines. The rat inflammatory anal fistula model confirms that our microgel can reduce inflammation and epithelialization of anal fistula, and effectively promote fistula healing. Therefore, our work provides a new effective strategy for the treatment of anal fistula.
Results and Discussion The preparation of our GAA@CNT‐ g ‐PVBTMA microgel includes the synthesis of the quaternized molecular brush (i.e., CNT‐ g ‐PVBTMA), thermal polymerization of a precursor solution composed of Gel, AAc‐NHS, and Ac‐β‐CD, and mechanical fragmentation. GAA@CNT microgel was prepared following the same steps as GAA@CNT‐ g ‐PVBTMA microgel, except for replacing CNT‐ g ‐PVBTMA with carbon nanotube (CNT). Similarly, GA and GAA microgels were prepared from the given precursor solutions of Gel/AAc‐NHS and Gel/AAc‐NHS/Ac‐β‐CD, respectively. CNT‐ g ‐PVBTMA was synthesized by grafting poly[(ar‐vinylbenzyl)trimethylammonium] (PVBTMA) brushes from CNT, which was characterized by X‐ray photoelectron spectroscopy (XPS) and thermogravimetric (TGA). The presence of N 1s and Cl 2p peaks in XPS spectra (Figure , Supporting Information) and the increased weight loss in TGA curves (Figure , Supporting Information) indicate the successful graft of PVBTMA. SEM image in Figure (Supporting Information) displays inherited 1D morphology of CNT with a roughened surface after grafting CNT‐ g ‐PVBTMA. Moreover, the macroscopic particle size and microscopic morphology of the microgel were characterized. After mechanical fragmentation using silicone tubing, the digital photo and particle size of GAA@CNT‐ g ‐PVBTMA microgel are shown in Figure (Supporting Information), indicating an average particle diameter of 1.51 mm. As shown in Figure , the freeze‐dried GAA@CNT‐ g ‐PVBTMA microgel exhibits a microporous structure with an average pore diameter of 1.69 µm. In contrast, GAA microgel possesses macroporous structure (Figure , Supporting Information). These results demonstrate the successful fabrication of GAA@CNT‐ g ‐PVBTMA microgel. The anti‐swelling ability and injectability of microgels are crucial for the treatment of anal fistula. As shown in Figure , GA microgel exhibits a swelling ratio of 103.6% after soaking in PBS solution for 72 h. In comparison, GAA and GAA@CNT microgels show significantly lower swelling ratios of 26.9% and 28.5%, respectively, due to the increased cross‐linked network structure between Ac‐β‐CD and Gel/AAc‐NHS. After the incorporation of CNT‐ g ‐PVBTMA, GAA/CNT‐ g ‐PVBTMA microgel demonstrates excellent anti‐swelling property with a low swelling ratio of 9.9% (Figure ). Rheological tests were conducted to analyze the moduli changes of GAA@CNT‐ g ‐PVBTMA microgel. At room temperature (25 °C), the storage modulus (G′) of GAA@CNT‐ g ‐PVBTMA microgel is always higher than the loss modulus (G″) over time, suggesting a stable gel network of the microgel (Figure ). The strain amplitude scanning curves of GAA@CNT‐ g ‐PVBTMA microgel reveal that the intersection of G′ and G″occurs at a shear strain of 70% (Figure ). As the shear strain increases beyond this point, G' becomes lower than G″and the gel network is destroyed. The modulus changes of the microgel were further assessed through cyclic amplitude scanning. As depicted in Figure , G' exceeds G″ at low strain (1%) and indicates its gel state; G′ decreases significantly at high strain (300%), suggesting the breakdown of gel network structure. When the strain changes to low strain (1%) again, G′ can quickly return to the initial state. As shown in Figure (Supporting Information), GAA@CNT‐ g ‐PVBTMA microgel can pass smoothly through irregularly shaped silicone tubing and exhibit good injectability (Video , Supporting Information). Moreover, our GAA@CNT‐ g ‐PVBTMA microgel can be injected to form the letters “SYSU” and retain its solid gel state once the shear forces are removed (Figure ). These results demonstrate that our GAA@CNT‐ g ‐PVBTMA microgel exhibits excellent anti‐swelling ability and injectability. In clinical practice, achieving the optimal balance between effective filling and adequate drainage is essential for the successful treatment of anal fistula. Excessive filling may hinder proper drainage, while insufficient filling may expose the fistula to constant inflammatory stimuli and delay the healing process. As shown in Figure (Supporting Information), a 4‐mm‐diameter through hole was created in the porcine tissue. Our GAA@CNT‐ g ‐PVBTMA microgel can effectively fill the entire hole and exhibit firm adhesion under water flushing (Figure ; Video , Supporting Information). To further evaluate the adhesion property of the microgel to the anal fistula tissue, we established a rat inflammatory anal fistula model by inserting a 1.6‐mm‐diameter wire through the perianal and anal area of the rat. After 14 days, the anal fistula tissue was harvested for adhesion characterization. Our GAA@CNT‐ g ‐PVBTMA microgel also demonstrates strong adhesion to the anal fistula tissue under water flushing (Video , Supporting Information). When the microgels are placed in a syringe, GAA@CNT‐ g ‐PVBTMA microgel can not only effectively fill the syringe, but also promote the drainage of PBS solution, which is attributed to the fragmented morphology with a porous structure (Figure ; Video , Supporting Information). Even after soaking in PBS solution for 3 days, our microgel still maintains excellent drainage property (Video , Supporting Information). These results demonstrate that our GAA@CNT‐ g ‐PVBTMA microgel exhibits both effective filling and draining properties, making it a superior material for the treatment of anal fistula. The accumulation of inflammatory cytokines and bacterial infections in anal fistula disrupts normal tissue repair mechanisms and can severely delay the healing process. Thus, effective anti‐inflammatory and antibacterial therapies are crucial for accelerating the healing of anal fistula. To assess the anti‐inflammatory properties of GAA and GAA@CNT‐ g ‐PVBTMA microgels, their abilities to capture the negatively charged inflammatory cytokines (e.g., TNF‐α and IL‐1β) were investigated using rat anal fistula tissue homogenate to simulate inflammatory environment. GAA and GAA@CNT‐ g ‐PVBTMA microgels were incubated in the homogenate for 720 min, and immunofluorescence staining images of TNF‐α and IL‐1β captured by the microgels were taken using a fluorescence microscope. As shown in Figure , GAA@CNT‐ g ‐PVBTMA microgel shows a higher binding affinity for TNF‐α and IL‐1β compared to GAA microgel, suggesting its higher efficiency in capturing inflammatory cytokines. The binding capacities of the microgels for the inflammatory cytokines are further quantitatively investigated. GAA microgel scavenges 47.5% of TNF‐α and 45.7% of IL‐1β after 720 min of incubation. In contrast, GAA@CNT‐ g ‐PVBTMA microgel achieves significantly higher binding efficiency, scavenging 92.6% of TNF‐α and 92.5% of IL‐1β, respectively (Figure ). The incorporation of positively charged molecular brushes in the microgel may capture more negatively charged inflammatory cytokines through electrostatic interactions, thereby improving scavenging efficiency. The antibacterial properties of the microgels against E. coli and S. aureus were evaluated using the agar plate incubation method, as displayed in Figure . The GAA@CNT‐ g ‐PVBTMA group shows fewer colony‐forming unit (CFU) of E. coli and S. aureus compared to the control and GAA groups. Additionally, GAA and GAA@CNT‐ g ‐PVBTMA microgels were co‐cultured with E. coli and S. aureus for 48 h, and the optical density (OD) values of the bacterial suspensions were measured to evaluate the antibacterial efficacy. As shown in Figure , the OD values of E. coli and S. aureus suspensions in the control and GAA groups exhibit a significant increase and suggest a rapid bacterial growth phase. In contrast, the GAA@CNT‐ g ‐PVBTMA group maintains a significantly lower OD value for 48 h, demonstrating that our GAA@CNT‐ g ‐PVBTMA microgel can effectively inhibit bacterial proliferation over a long period of time. These results demonstrate that our GAA@CNT‐ g ‐PVBTMA microgel exhibits excellent anti‐inflammatory property and good bacteriostatic property by capturing inflammatory cytokines and preventing bacteria growth. Good biocompatibility is crucial for injectable implant materials. The biocompatibility of GAA and GAA@CNT‐ g ‐PVBTMA microgels was assessed using live/dead cell viability assay and CCK‐8 assay. As shown in Figure , the morphology and cell density of L929 fibroblasts in the GAA and GAA@CNT‐ g ‐PVBTMA groups are comparable to those of the control group. Meanwhile, the quantitative analysis of CCK‐8 assay reveals no significant statistical differences in proliferation among the groups, demonstrating good in vitro biocompatibility of GAA and GAA@CNT‐ g ‐PVBTMA microgels (Figure ). To further evaluate in vivo biocompatibility, subcutaneous injections of 0.9% saline (serving as control), GAA microgel, and GAA@CNT‐ g ‐PVBTMA microgel were administered into the dorsal skin of rats, respectively. Tissue samples from the injection sites after 3 days were excised and subjected to HE and immunohistochemical staining to assess inflammatory responses, as shown in Figure . The HE staining results show no statistical differences in the infiltration areas of inflammatory cells (Figure ). Moreover, immunohistochemical staining was conducted to investigate two inflammatory markers, including CD68 (a macrophage marker) and IL‐6 (an inflammatory cytokine). As shown in Figure , there are no statistical differences in the expressions of CD68 and IL‐6 among all the groups. These results demonstrate the excellent biocompatibility of GAA and GAA@CNT‐ g ‐PVBTMA microgels both in vitro and in vivo. To further evaluate in vivo healing efficacy of GAA@CNT‐g‐PVBTMA microgel, a rat inflammatory anal fistula model was induced by inserting a thicker wire through the perianal and anal area and giving fresh drinking water with dextran sodium sulfate (DSS). After leaving the wire in place for 28 days, the tissue sample from one of the rats was subjected to HE staining to evaluate the establishment of the animal model (Figure 5a). The HE staining images clearly show a fistula with complete epithelialization, indicating the successful establishment of the anal fistula model (Figure , Supporting Information). As shown in Figure 5b , the healing status of the fistula external opening in different groups is observed after 7 days. The external opening of the GAA@CNT‐ g ‐PVBTMA group is almost completely closed with a relative external opening area of 3%. In comparison, the control group exhibits significantly slower healing, with 25% of the relative area remaining unhealed (Figure ). Rats were then euthanized on day 7, and the fistula tissues were harvested for histology analysis including HE staining and immunohistochemical staining of TNF‐α and IL‐1β. The GAA@CNT‐ g ‐PVBTMA group shows dense fibroproliferative scars, while the control and GAA groups exhibit the presence of fistula with epithelialized cells (Figure ). Quantitative analysis reveals that the expressions of TNF‐α and IL‐1β are significantly lower in the GAA@CNT‐ g ‐PVBTMA group compared to the other two groups (Figures ). To further evaluate the degree of in vivo inflammation, the fresh anal fistula tissue homogenate was taken from rats after 7 days of treatment. For comparison, homogenates from healthy perianal tissue were collected as the normal group. ELISA tests were conducted to measure the amount of IL‐1β and TNF‐α in the anal fistula tissue homogenate. The GAA@CNT‐ g ‐PVBTMA group exhibits lower levels of inflammatory cytokines compared to the control group. Although cytokine levels in the GAA@CNT‐ g ‐PVBTMA group remain slightly elevated compared to the normal group, there are no statistically significant differences in the levels of inflammatory cytokines between the two groups (Figure , Supporting Information), indicating that GAA@CNT‐ g ‐PVBTMA microgel exhibits excellent anti‐inflammatory property by significantly reducing the levels of the negatively charged inflammatory cytokines. To further evaluate the role of GAA@CNT‐ g ‐PVBTMA microgel in inducing M2 macrophages polarization in vivo, we conducted immunohistochemical analysis for two markers: iNOS, a marker for pro‐inflammatory M1 macrophages, and CD206, a marker for anti‐inflammatory M2 macrophages. As shown in Figure (Supporting Information), the GAA@CNT‐ g ‐PVBTMA group shows a lower expression of iNOS and higher expression of CD206 compared to the control and GAA groups. These results show that our GAA@CNT‐ g ‐PVBTMA microgel can effectively induce the polarization of M2 macrophages. By integrating excellent anti‐inflammatory and drainage properties, our GAA@CNT‐ g ‐PVBTMA microgel can effectively reduce inflammation and epithelialization of the fistula, thereby promoting the healing of anal fistula.
Conclusion In conclusion, we have successfully developed a new class of quaternized molecular brush‐grafted injectable microgel through thermal polymerization and mechanical fragmentation. Benefitting from the incorporation of CNT‐ g ‐PVBTMA, our GAA@CNT‐ g ‐PVBTMA microgel exhibits excellent anti‐swelling property with a low value of 9.9%. Due to the fragmented morphology with a porous structure, our GAA@CNT‐ g ‐PVBTMA microgel can effectively fill the fistula and facilitate the drainage of exudate. Remarkably, our microgel can capture inflammatory cytokines of the fistula through electrostatic interactions and exhibit excellent anti‐inflammatory property, achieving remarkable scavenging rates of 92.6% for TNF‐α and 92.5% for IL‐1β. The rat inflammatory anal fistula model confirms that our microgel can effectively reduce inflammation and epithelialization of the fistula, and promote the fistula healing. Thus, our work provides a promising and effective therapy for the treatment of anal fistula.
Experimental Section The Experimental Section is available in the .
The authors declare no conflict of interest.
Supporting Information Supplementary Video 1 Supplementary Video 2 Supplementary Video 3 Supplementary Video 4 Supplementary Video 5
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Volume imaging to interrogate cancer cell-tumor microenvironment interactions in space and time | cc7a0208-ed15-4874-9abb-c8ca2abc5ccc | 10228654 | Internal Medicine[mh] | Introduction In the past decade, cancer research and clinical oncology have experienced a paradigm shift driven by the development of immunotherapies to cure patients with aggressive cancers . Ever since, cancer research around the world has begun to untangle the diverse functions of the tumor microenvironment (TME), the non-transformed cells and extracellular matrix which constitute the niche in which cancers arise and proliferate. Whilst cancer research today appreciates an underlying spatial organization of solid tumors , this organization is too complex to be unraveled by a single two-dimensional tissue section, the historic platform for cancer staging, calling for volumetric analyses that capture larger tumor regions if not the entire lesion at cellular resolution . This is particularly true for the TME due to the heterogeneity of immune and stromal infiltrates within the complex architecture of solid tumors and the existence of rare components of the TME that despite their sparsity may have important implications in tumor biology. To visualize the full complexity of the TME, high-resolution volume imaging techniques are required . Two powerful technologies for volumetric analyses are ex vivo spatial microscopy of tissue-cleared whole organs and intravital microscopy (IVM) of cancer cells in the context of a living organism. Optical clearing to visualize tissues in three dimensions was first developed at the beginning of the 20 th century and has dramatically been refined and exploited in the past decade. Originally rediscovered by neurobiology laboratories to achieve 3D maps of the central nervous system, tissue clearing has now been widely applied to study tumor complexity . The TME is a dynamic ensemble of cells and molecules that can change dramatically throughout tumor progression . By imaging live tissue of animal models, IVM meets the need to scrutinize such dynamics and visualize immune and stromal infiltration and ECM remodeling over time. This review discusses the recent advances in tissue clearing and IVM to visualize the interaction between cancer cells and cells and molecules in their microenvironment. In addition, we discuss the utilization of these two platforms to investigate the crosstalk between tumor and TME, and to advance our understanding of cancer cell biology in the context of the complex architecture and dynamic organization of cancers.
Applying tissue clearing to inspect tumors and their TME in 3D Around seventy tissue clearing techniques have been developed to perform volume fluorescence imaging of tissue resections, whole organs, and entire organisms. This ex vivo platform allows for the visualization of cells and macromolecules within the native architecture of their tissue. The first application of clearing in the context of fluorescence microscopy of whole-mount tissue was developed to study fish and Drosophila embryos and was later widely applied in neurobiology to study the architecture of the mammalian brain . To this day, tissue clearing has been applied to myriad tissues, organs, and organisms including tumors from animal models and human patients . Technically, tissue clearing consists of sample permeabilization and clarification in refractive index matching solutions to render specimens transparent and visualize their entire architecture and internal complexity. The available techniques for tissue permeabilization and refractive index (RI) matching in cancer and other samples have been reviewed extensively . Briefly, permeabilization is achieved by lipid elimination using alcohols or detergents, with zwitterionic detergents showing the highest efficacy for delipidation . Electrophoresis can be used to drive the infiltration of antibodies and dyes into the tissue . Clearing is achieved by immersing the sample in an organic solvent or aqueous solution that matches the RI of the tissue. Organic solvents include ethyl cinnamate (ECi), benzyl alcohol/benzyl benzoate (BABB), tetrahydrofuran and dichloromethane (used in DISCO-derived techniques). Aqueous solutions include fructose gradients (as in FRUIT), fructose combined with glycerol and urea (for example, for FUnGI), more complex chemical combinations as in CUBIC (antipyrine, nicotinamide and N-butyldiethanolamine, and other chemicals), as well as commercial solutions like RapiClear® . The selection of 3D imaging modalities for cleared tissue depends on the sample size and desired optical resolution. Light-sheet fluorescence microscopes provide cellular resolution and can be used to image centimeter-scale samples. Confocal microscopes offer a higher resolution in the xy plane and have been widely utilized to image whole rodent organs and cancer resections. For image rendering and analysis, the free image processing package Fiji can be used , and specific software for volume images such as Imaris and Aivia is commercially available . 2.1 Clearing cancer organotypic cultures Organoids and organotypic cultures can pose barriers to antibody labeling and microscopic analysis due to their high cellularity and embedded growth in thick culture matrices that prevent access of labeling agents and cause extensive light scattering. Consequently, permeabilization and RI matching have proven useful for complex organoid 3D cultures in which antibody access and light penetration can be limited . Tissue clearing has enabled the visualization of glioma and GBM cells in brain organoids using ECi and FRUIT clearing . Complex cocultures comprising lung cancer cell organoids with fibroblasts and endothelial cells were cleared with a mixture of polyglycerols, DMSO and urea (HyClear). Each cell line was engineered to express a different, fluorescent protein marker allowing spatial analyses by 3D fluorescent microscopy . 2.2 Clearing approaches to interrogate the interaction between cancer cells and their epithelial neighbors Carcinomas originate from epithelial cells and interact and compete with their surrounding healthy epithelium . Tissue clearing has been used to study how the epithelial architecture changes upon transformation, and how healthy epithelial cells respond to neighboring tumors. Cytokeratins have been widely used as markers to distinguish epithelia from nonepithelial tissues by 3D imaging. Keratin-8/18 (K8/18) was used to label luminal epithelial cells, and K5 to visualize myoepithelial cells in human prostate biopsies with intraductal carcinoma cleared by iDISCO . In prostate cancer samples from FFPE patient biopsies, BABB clearing showed that the K5-expressing myoepithelial cells co-localize with K8-positive tumor regions with a differentiated ductal architecture . Both keratins are also expressed in the epithelium of the mammary ductal tree and were visualized together with genetically encoded fluorescent proteins using FUnGI . Similarly, fructose glycerol clearing was shown to be applicable to mammary glands with transplanted organoids . In the MMTV-PyMT breast cancer model, K8 staining was used to detect spontaneous metastases in lungs cleared by FLASH and to demonstrate their interaction with the airway and alveolar epithelia labelled with podoplanin and surfactant protein C (SFTPC) . K7 is a marker of luminal and glandular epithelia in the endometrium, and has been used together with CUBIC clearing to investigate the architecture of occluded glands and adenomyosis in humans, associated to cervical cancer . Genetically KrasG12D transformed cells in the pancreas, liver and lung were visualized through a combination of K19 immunolabelling with antibody staining for red and green fluorescent protein (RFP, GFP) lineage tracers . 2.3 Tissue clearing techniques for brain cancer models Many clearing techniques were established to clear the brain and study its anatomy in pathophysiology . Hence, the interaction of different brain tumors with cells of the normal brain parenchyma, including neurons and glia, could be readily investigated in 3D. The astrocyte marker glial fibrillary acidic protein (GFAP) has been used to visualize the interaction of glial cells with GBM models in mouse brains cleared in tetrahydrofuran, urea, azide and nycodenz , and with CUBIC and iDISCO . Co-invasion of human glioma cell lines and astrocytes has been investigated in brain slices labelled with anti-GFAP antibody and imaged after clearing in α-thioglycerol, fructose and RapiClear . These studies also labelled laminin to image the interaction between tumor cells and the surrounding extracellular matrix, and vimentin, which marks processes of glioma cells reaching into the surrounding environment . Astrocyte interaction with breast cancer tumor metastases in the brain administered by intracarotid or intracranial injection was visualized with CUBIC and PACT clearing . The interaction between tumors and axons in 3D has been achieved through lipid staining and clearing with urea and triton , and staining for myelin basic protein (MBP) in GBM cleared with CUBIC and iDISCO . 2.4 Clearing the tumor immune microenvironment Tissue clearing constitutes a useful approach to accurately assess the infiltration of immune cells into the tumor. A general visualization of immune infiltration can be achieved with the pan-immune cell marker CD45, as demonstrated in a mouse glioblastoma (GBM) model cleared with iDISCO and CUBIC . The infiltration of fluorescently labelled tumor-associated macrophages (TAMs) has been visualized in murine lung cancer models cleared with CUBIC . Macrophage infiltration in mouse lung adenocarcinoma tumors generated by intravenous injection of A549 cells was also achieved using an Iba-1 antibody and CUBIC clearing . Primary and secondary tumors from anaplastic thyroid tumors achieved by orthotopic and intravenous injection into mice respectively were cleared with CUBIC, and TAMs visualized using a Macrin-VT680XL nanoparticle . Clearing and imaging of infiltrating T cells has also been achieved. CD8 + cells and their contribution within the heterogeneous expression landscape of immune checkpoint molecule PD-L1 was performed in mouse mammary tumor models cleared in fructose . CD3 staining was used to evaluate the infiltration of T cells in formalin fixed paraffin-embedded (FFPE) breast cancer patient biopsies cleared with CLARITY , and surgical resections of patients with head and neck squamous cell carcinoma (HNSCC) were stained with antibodies against CD3 for T cells and CD66b for neutrophils before clearing with ECi . Recent work using FLASH has revealed differences in T and B cell infiltration in whole tumour-bearing lungs, and has proved useful in the identification of tumour-adjacent immune structures such as tertiary lymphoid structures which are defined by their histology . The study of other immune cell types in different types of tumors remains to be performed, but specialized techniques such as EMOVI have been used to detect immune cells in non-cancerous samples, including antigen presenting cells using MHC-II antibodies, T cells with a CD3 antibody, and B cells and dendritic cells with antibodies against CD21 and CD35 in lungs, lymph nodes, adipose tissue, kidney, brain, liver and heart , giving a promising outlook for the future study of these infiltrates and their interaction with cancer cells. However, fluorescence imaging of large volumes is usually restricted to 4-5 markers, which does not meet the requirements for an in-depth characterization of immune infiltrates. The development of multiplex imaging technologies compatible with 3D imaging brings promise to the spatial characterization of immune infiltration within the tumor volume (See section 6). 2.5 Visualizing the tumor vasculature with tissue clearing Tissue clearing has been used extensively to map the vasculature of biological samples including tumors. In addition to antibodies that specifically label the endothelium or perivascular cells, vessels can be easily stained in animal models through intravenous injection of fluorescent dextran or lectin. Lectin dyes bind to the luminal side of endothelial cells and have been applied to image the architecture of the vascular network in mouse models of glioma using 3DISCO and FluoClearBABB , HNSCC xenografts from human cell lines using the commercial Binaree clearing kit , human hepatoma xenografts and human pancreatic ductal adenocarcinoma (PDAC) xenografts in mice cleared with CUBIC , and a genetic PDAC mouse model cleared with FLASH . Lectins have also been used to label the vasculature of entire mice with primary and metastatic orthotopic tumor models for breast cancer, PDAC, and lung cancer cleared with vDISCO . Lectins can be used in combination with antibody labelling as shown in a mouse model for GBM, in which the vasculature was stained both with lectin and a CD31 antibody and the tumors cleared in 80% glycerol . Tumor perfusion and drug penetration throughout the tumor can also be evaluated using lectin labelling. HER-2 overexpressing breast cancer xenografts have been imaged to evaluate the penetration of a fluorescent HER-2-targeting antibody (trastuzumab) in tumors cleared with BABB . Similarly, in HeLa subcutaneous xenografts, lectin was used to map the distribution of nanoparticles in tumors cleared with CUBIC . Dextran can also be used to label blood vasculature, as shown in A549 lung tumors cleared with CUBIC-plus . Dextran permeates through fenestrated vessels and can hence be used to evaluate the integrity and permeability of the vasculature, as demonstrated in GBM xenografts cleared in ECi . Widely-used epitopes to label vasculature include α-smooth muscle actin (α-SMA, labelling pericytes), used in an orthotopic model of lung cancer cleared with CUBIC , in a breast cancer model cleared with fructose , and in a renal carcinoma brain metastasis model visualized in whole mice cleared with CUBIC . CD31 to label the endothelium has also been widely applied, for example in murine cutaneous squamous cell carcinoma tumors cleared in ECi , mammary tumors cleared in fructose , melanoma xenografts cleared in BABB, CUBIC and TDE, and primary human cancer biopsies cleared with TDE , human colon adenocarcinoma biopsies cleared in FocusClear . CD31 labelling was compared to CD34 in a study that used tissue clearing on murine GBM with CUBIC and iDISCO , and concluded higher specificity of CD34 antibody for labelling endothelium. CD34 was also used to image the vasculature of mouse and human kidney , indicating that it labels endothelium specifically in different tissues. Other markers used in the study of tumor vascularization are VEGFR3, a marker of angiogenesis and CD42c, a platelet marker, used in CUBIC-cleared lung cancer xenografts , coagulation factor VIII which was detected in melanoma xenografts and MECA-79, used to visualize high endothelial venules in fibrosarcoma and 4T1 xenografts . The lymphatic system, especially tumor draining lymph nodes, is a site of particular interest in cancer research since it is a key hub for cancer cell dissemination and immune education. Lymphangiogenesis and the architecture and function of the lymphatic vasculature plays an important role in maintaining the niche of stem cells in healthy tissues and potentially the niche of cancer stem cells as well. Lymphatics can be labelled using an anti-Lyve1 antibody as demonstrated in the liver and in DCM and DBE cleared bladder cancer resections . Lymphatics of PanIN and PDAC lesions were visualized in human and mouse samples with antibodies against Lyve-1 and podoplanin and clearing in RapiClear 1.52 . Tissue clearing of lymph nodes has been achieved with techniques such as Ce3D for imaging immune cell populations and vasculature , and has enabled to track the population dynamics of cells involved in adaptive immunity such as CD8 + T cell differentiation . 2.6 Tissue clearing to image tumor innervation Cancer innervation is a timely field of research, as depending on the context, nerves can promote or inhibit cancer growth and cancer cells can invade perineurally . Neurons can readily be mapped by many clearing protocols, and have been imaged in normal tissues of the human colon , pancreas , and in a mouse model for pancreatic neoplasia where the GFAP-positive glia and TUJ1-positive neurons were found to be enriched around lesions. This study visualized both innervation and vascularization in the same samples which suggests that angiogenesis and innervation occur in parallel during neoplastic hyperproliferation and early stages of transformation in the pancreas .
Clearing cancer organotypic cultures Organoids and organotypic cultures can pose barriers to antibody labeling and microscopic analysis due to their high cellularity and embedded growth in thick culture matrices that prevent access of labeling agents and cause extensive light scattering. Consequently, permeabilization and RI matching have proven useful for complex organoid 3D cultures in which antibody access and light penetration can be limited . Tissue clearing has enabled the visualization of glioma and GBM cells in brain organoids using ECi and FRUIT clearing . Complex cocultures comprising lung cancer cell organoids with fibroblasts and endothelial cells were cleared with a mixture of polyglycerols, DMSO and urea (HyClear). Each cell line was engineered to express a different, fluorescent protein marker allowing spatial analyses by 3D fluorescent microscopy .
Clearing approaches to interrogate the interaction between cancer cells and their epithelial neighbors Carcinomas originate from epithelial cells and interact and compete with their surrounding healthy epithelium . Tissue clearing has been used to study how the epithelial architecture changes upon transformation, and how healthy epithelial cells respond to neighboring tumors. Cytokeratins have been widely used as markers to distinguish epithelia from nonepithelial tissues by 3D imaging. Keratin-8/18 (K8/18) was used to label luminal epithelial cells, and K5 to visualize myoepithelial cells in human prostate biopsies with intraductal carcinoma cleared by iDISCO . In prostate cancer samples from FFPE patient biopsies, BABB clearing showed that the K5-expressing myoepithelial cells co-localize with K8-positive tumor regions with a differentiated ductal architecture . Both keratins are also expressed in the epithelium of the mammary ductal tree and were visualized together with genetically encoded fluorescent proteins using FUnGI . Similarly, fructose glycerol clearing was shown to be applicable to mammary glands with transplanted organoids . In the MMTV-PyMT breast cancer model, K8 staining was used to detect spontaneous metastases in lungs cleared by FLASH and to demonstrate their interaction with the airway and alveolar epithelia labelled with podoplanin and surfactant protein C (SFTPC) . K7 is a marker of luminal and glandular epithelia in the endometrium, and has been used together with CUBIC clearing to investigate the architecture of occluded glands and adenomyosis in humans, associated to cervical cancer . Genetically KrasG12D transformed cells in the pancreas, liver and lung were visualized through a combination of K19 immunolabelling with antibody staining for red and green fluorescent protein (RFP, GFP) lineage tracers .
Tissue clearing techniques for brain cancer models Many clearing techniques were established to clear the brain and study its anatomy in pathophysiology . Hence, the interaction of different brain tumors with cells of the normal brain parenchyma, including neurons and glia, could be readily investigated in 3D. The astrocyte marker glial fibrillary acidic protein (GFAP) has been used to visualize the interaction of glial cells with GBM models in mouse brains cleared in tetrahydrofuran, urea, azide and nycodenz , and with CUBIC and iDISCO . Co-invasion of human glioma cell lines and astrocytes has been investigated in brain slices labelled with anti-GFAP antibody and imaged after clearing in α-thioglycerol, fructose and RapiClear . These studies also labelled laminin to image the interaction between tumor cells and the surrounding extracellular matrix, and vimentin, which marks processes of glioma cells reaching into the surrounding environment . Astrocyte interaction with breast cancer tumor metastases in the brain administered by intracarotid or intracranial injection was visualized with CUBIC and PACT clearing . The interaction between tumors and axons in 3D has been achieved through lipid staining and clearing with urea and triton , and staining for myelin basic protein (MBP) in GBM cleared with CUBIC and iDISCO .
Clearing the tumor immune microenvironment Tissue clearing constitutes a useful approach to accurately assess the infiltration of immune cells into the tumor. A general visualization of immune infiltration can be achieved with the pan-immune cell marker CD45, as demonstrated in a mouse glioblastoma (GBM) model cleared with iDISCO and CUBIC . The infiltration of fluorescently labelled tumor-associated macrophages (TAMs) has been visualized in murine lung cancer models cleared with CUBIC . Macrophage infiltration in mouse lung adenocarcinoma tumors generated by intravenous injection of A549 cells was also achieved using an Iba-1 antibody and CUBIC clearing . Primary and secondary tumors from anaplastic thyroid tumors achieved by orthotopic and intravenous injection into mice respectively were cleared with CUBIC, and TAMs visualized using a Macrin-VT680XL nanoparticle . Clearing and imaging of infiltrating T cells has also been achieved. CD8 + cells and their contribution within the heterogeneous expression landscape of immune checkpoint molecule PD-L1 was performed in mouse mammary tumor models cleared in fructose . CD3 staining was used to evaluate the infiltration of T cells in formalin fixed paraffin-embedded (FFPE) breast cancer patient biopsies cleared with CLARITY , and surgical resections of patients with head and neck squamous cell carcinoma (HNSCC) were stained with antibodies against CD3 for T cells and CD66b for neutrophils before clearing with ECi . Recent work using FLASH has revealed differences in T and B cell infiltration in whole tumour-bearing lungs, and has proved useful in the identification of tumour-adjacent immune structures such as tertiary lymphoid structures which are defined by their histology . The study of other immune cell types in different types of tumors remains to be performed, but specialized techniques such as EMOVI have been used to detect immune cells in non-cancerous samples, including antigen presenting cells using MHC-II antibodies, T cells with a CD3 antibody, and B cells and dendritic cells with antibodies against CD21 and CD35 in lungs, lymph nodes, adipose tissue, kidney, brain, liver and heart , giving a promising outlook for the future study of these infiltrates and their interaction with cancer cells. However, fluorescence imaging of large volumes is usually restricted to 4-5 markers, which does not meet the requirements for an in-depth characterization of immune infiltrates. The development of multiplex imaging technologies compatible with 3D imaging brings promise to the spatial characterization of immune infiltration within the tumor volume (See section 6).
Visualizing the tumor vasculature with tissue clearing Tissue clearing has been used extensively to map the vasculature of biological samples including tumors. In addition to antibodies that specifically label the endothelium or perivascular cells, vessels can be easily stained in animal models through intravenous injection of fluorescent dextran or lectin. Lectin dyes bind to the luminal side of endothelial cells and have been applied to image the architecture of the vascular network in mouse models of glioma using 3DISCO and FluoClearBABB , HNSCC xenografts from human cell lines using the commercial Binaree clearing kit , human hepatoma xenografts and human pancreatic ductal adenocarcinoma (PDAC) xenografts in mice cleared with CUBIC , and a genetic PDAC mouse model cleared with FLASH . Lectins have also been used to label the vasculature of entire mice with primary and metastatic orthotopic tumor models for breast cancer, PDAC, and lung cancer cleared with vDISCO . Lectins can be used in combination with antibody labelling as shown in a mouse model for GBM, in which the vasculature was stained both with lectin and a CD31 antibody and the tumors cleared in 80% glycerol . Tumor perfusion and drug penetration throughout the tumor can also be evaluated using lectin labelling. HER-2 overexpressing breast cancer xenografts have been imaged to evaluate the penetration of a fluorescent HER-2-targeting antibody (trastuzumab) in tumors cleared with BABB . Similarly, in HeLa subcutaneous xenografts, lectin was used to map the distribution of nanoparticles in tumors cleared with CUBIC . Dextran can also be used to label blood vasculature, as shown in A549 lung tumors cleared with CUBIC-plus . Dextran permeates through fenestrated vessels and can hence be used to evaluate the integrity and permeability of the vasculature, as demonstrated in GBM xenografts cleared in ECi . Widely-used epitopes to label vasculature include α-smooth muscle actin (α-SMA, labelling pericytes), used in an orthotopic model of lung cancer cleared with CUBIC , in a breast cancer model cleared with fructose , and in a renal carcinoma brain metastasis model visualized in whole mice cleared with CUBIC . CD31 to label the endothelium has also been widely applied, for example in murine cutaneous squamous cell carcinoma tumors cleared in ECi , mammary tumors cleared in fructose , melanoma xenografts cleared in BABB, CUBIC and TDE, and primary human cancer biopsies cleared with TDE , human colon adenocarcinoma biopsies cleared in FocusClear . CD31 labelling was compared to CD34 in a study that used tissue clearing on murine GBM with CUBIC and iDISCO , and concluded higher specificity of CD34 antibody for labelling endothelium. CD34 was also used to image the vasculature of mouse and human kidney , indicating that it labels endothelium specifically in different tissues. Other markers used in the study of tumor vascularization are VEGFR3, a marker of angiogenesis and CD42c, a platelet marker, used in CUBIC-cleared lung cancer xenografts , coagulation factor VIII which was detected in melanoma xenografts and MECA-79, used to visualize high endothelial venules in fibrosarcoma and 4T1 xenografts . The lymphatic system, especially tumor draining lymph nodes, is a site of particular interest in cancer research since it is a key hub for cancer cell dissemination and immune education. Lymphangiogenesis and the architecture and function of the lymphatic vasculature plays an important role in maintaining the niche of stem cells in healthy tissues and potentially the niche of cancer stem cells as well. Lymphatics can be labelled using an anti-Lyve1 antibody as demonstrated in the liver and in DCM and DBE cleared bladder cancer resections . Lymphatics of PanIN and PDAC lesions were visualized in human and mouse samples with antibodies against Lyve-1 and podoplanin and clearing in RapiClear 1.52 . Tissue clearing of lymph nodes has been achieved with techniques such as Ce3D for imaging immune cell populations and vasculature , and has enabled to track the population dynamics of cells involved in adaptive immunity such as CD8 + T cell differentiation .
Tissue clearing to image tumor innervation Cancer innervation is a timely field of research, as depending on the context, nerves can promote or inhibit cancer growth and cancer cells can invade perineurally . Neurons can readily be mapped by many clearing protocols, and have been imaged in normal tissues of the human colon , pancreas , and in a mouse model for pancreatic neoplasia where the GFAP-positive glia and TUJ1-positive neurons were found to be enriched around lesions. This study visualized both innervation and vascularization in the same samples which suggests that angiogenesis and innervation occur in parallel during neoplastic hyperproliferation and early stages of transformation in the pancreas .
IVM: a window to visualize TME-cancer cell interaction dynamics in 4D Static volume imaging allows the molecular phenotyping in the 3D cancer space but represents a snapshot in time and lacks insight into the dynamics of the same cancer cells over time. The combination of IVM with subsequent 3D molecular analysis by tissue clearing poses an attractive solution combining the strengths of both technologies . Recently, IVM has proven extremely useful to shed light on dynamic processes at play in tumor formation and progression, immune escape and metastasis . 3.1 Recent protocols and window optimizations for IVM In recent years the field of intravital microscopy has been broadened and specialized by developments that adapted the technology for different organs. Each organ casts unique demands due to its location that dictates optical accessibility, and due to its organ architecture and underlying physical (and optical) properties. Today an abundance of IVM techniques exists tailored to specific organs. For acute imaging, most organs can be accessed surgically. On the other hand, longitudinal imaging necessitates technologies to grant long-term access to the same organ and region over multiple days to weeks . This has for long been accomplished through the help of imaging windows, small sterile rings of solid inorganic material with a microscopy cover-glass inset. Such windows can be surgically implanted in the mouse skin and peritoneum, thereby rendering inner organs optically accessible. In recent years the general window design has been considerably modified to meet unique demands of different implantation sites. Most recent optimizations include windows for the pancreas and thyroid , and flexible silicon-based imaging windows for expanding tissues and tumors . Several approaches also exist to image cancerous processes in the bone marrow, ranging from the use of endoscopic devices for the femur , to the surgical exposure of tibia and calvarium . Some organs, such as those of the respiratory system and the gastrointestinal tract, are under constant movement and need to be stabilized for high resolution image acquisition. For the colon, a window with a stabilizing ferromagnetic scaffold minimizes such motion artifacts specifically during the imaging whilst granting the organ free movement in the imaging breaks to prevent obstruction and maintain intestinal function . Using a specialized lung window for longitudinal imaging, Entenberg and colleagues could visualize all stages of the metastatic process, including tumor cell arrival, extravasation, metastatic growth and progression to micrometastases . The dorsal skinfold window represents a widely used alternative to organ specific imaging windows. In this model cancer cells are engrafted subcutaneously in a window chamber implanted in the back skin of the mouse. This approach presents a robust platform to visualize cell-cell interactions in the living organism and may be seen as a straight forward alternative to test hypotheses in vivo that were gained in in vitro experiments, albeit restricted to questions where the role of the cancer-surrounding normal organ is neglected. Alternative xenograft-based approaches have visualized tumor cell behavior through cancer cell injection in the tongue , and engraftment in the eye which allow noninvasive longitudinal IVM without the need of imaging windows. Once the organ of interest can be optically accessed, different microscopy setups enable to address a multitude of questions. Optical access into deeper tissue layers is hampered by the tissue inherent light scattering and most studies employ 2-photon microscopes to visualize several hundred μm of tissue depth. The recent development of IVM protocols for 3-photon microscopy further increases the imaging depth . In addition, fast acquisition speed, for example using a spinning disk or a resonant scanner confocal microscope, can be of advantage for moving organs and allows the capture of highly dynamic events as for example the movement of leukocytes in the blood stream and tumor interstitium . The best combination of strategies for optical access and image acquisition will depend on the cancer type and biological question asked. Dedicated protocols for the imaging of primary cancers, metastases and the tumor microenvironment in different sites help identify the optimal strategy . Whilst the majority of previous IVM studies focused their attention on cellular behavior, the coming years are poised to see a broadening of IVM utilization with ongoing developments that multiplex bioluminescent and fluorescent reporters and molecular probes to film sub-cellular processes, and approaches that enable the in vivo visualization of nanoscale structures by 3D-stimulated emission depletion (3D-STED) microscopy , thereby opening the window to view molecular biology in the living organism. 3.2 Tools and strategies to visualize cell-cell interactions in vivo Given the ability to access and visualize the anatomic site of interest, a range of fluorescent labels and detection strategies enable the identification of cancer cells and structural and cellular components of the TME in the context of the organ architecture . The recent development of a photoactivatable Cre-recombinase mouse allows to target cancerous mutations to specific regions and cells in the organ landscape . Cancer cells can be labelled genetically through the expression of fluorescent proteins, and thus traced over sequential imaging sessions of several days to weeks. Proteins that can be activated, or photo-converted to change the fluorescent protein emission spectrum, allow the tracking of individual groups of cells based on their localization . With this approach, the colonization of distant organs from breast tumors was mapped by following the dissemination of cancer cells converted in the mammary gland in comparison to cancer cells converted in the lymph node . Proliferation of cancer and TME cells can be visualized indirectly by targeting individual cells with fluorescent proteins for lineage tracing, or directly through genetically encoded fluorescent cell cycle reporters . Fluorescent dyes like the recently developed apotracker-red are an option to identify apoptotic cells . Today the toolbox of IVM-ready fluorescent labels also includes a range of probes that allow to monitor the activity of the ERK, FAK, TGF-β/SMAD3 and nuclear factor kB (NF-kB) signaling pathways . A fluorescent reporter of nuclear factor of activated T cell (NFAT) activation was recently used to directly visualize how Treg cells receive TCR signals in the TME . The tumor-surrounding matrix can be readily visualized through second harmonic generations (SHG) detection of fibrillar collagen. Furthermore, recent efforts have targeted individual matrix components with fluorescent proteins. Fluorescently tagged fibronectin (FN) was used to track T cell interaction with FN in the inflamed ear dermis, where Th1 cells were found to migrate along FN fibers . The development of a laminin beta1-Dendra2 reporter mouse allows to visualize basement-membrane dynamics in vivo and can shed light on how cancer cells invade normal tissues . Tumor vasculature is commonly visualized through intravenous injection of fluorescent dextrans, lectins or quantum dots. The choice of dextran molecular weight allows to fine tune this detection to measure vascular permeability , and to label phagocytic immune cells . IVM has been instrumental to describe a wide range of fundamental immunological principles and immune cell behaviors . Genetic reporters and intravenously injected fluorophore-conjugated antibodies are commonly used to detect immune cells . Recent developments for multiplexed in vivo antibody labelling through click chemistry offer the opportunity to drastically expand the cell types that can be visualized in the same mouse . Nevertheless, it remains challenging to quantify rare cell populations in circulation. Recent developments enable real time imaging of individual fast circulating cells in the blood stream through quantum dots , a promising step in the direction of visualizing aggregation dynamics of circulating tumor cells (CTCs), or the interplay between lymphocytes, platelets, and CTCs in circulation in vivo .
Recent protocols and window optimizations for IVM In recent years the field of intravital microscopy has been broadened and specialized by developments that adapted the technology for different organs. Each organ casts unique demands due to its location that dictates optical accessibility, and due to its organ architecture and underlying physical (and optical) properties. Today an abundance of IVM techniques exists tailored to specific organs. For acute imaging, most organs can be accessed surgically. On the other hand, longitudinal imaging necessitates technologies to grant long-term access to the same organ and region over multiple days to weeks . This has for long been accomplished through the help of imaging windows, small sterile rings of solid inorganic material with a microscopy cover-glass inset. Such windows can be surgically implanted in the mouse skin and peritoneum, thereby rendering inner organs optically accessible. In recent years the general window design has been considerably modified to meet unique demands of different implantation sites. Most recent optimizations include windows for the pancreas and thyroid , and flexible silicon-based imaging windows for expanding tissues and tumors . Several approaches also exist to image cancerous processes in the bone marrow, ranging from the use of endoscopic devices for the femur , to the surgical exposure of tibia and calvarium . Some organs, such as those of the respiratory system and the gastrointestinal tract, are under constant movement and need to be stabilized for high resolution image acquisition. For the colon, a window with a stabilizing ferromagnetic scaffold minimizes such motion artifacts specifically during the imaging whilst granting the organ free movement in the imaging breaks to prevent obstruction and maintain intestinal function . Using a specialized lung window for longitudinal imaging, Entenberg and colleagues could visualize all stages of the metastatic process, including tumor cell arrival, extravasation, metastatic growth and progression to micrometastases . The dorsal skinfold window represents a widely used alternative to organ specific imaging windows. In this model cancer cells are engrafted subcutaneously in a window chamber implanted in the back skin of the mouse. This approach presents a robust platform to visualize cell-cell interactions in the living organism and may be seen as a straight forward alternative to test hypotheses in vivo that were gained in in vitro experiments, albeit restricted to questions where the role of the cancer-surrounding normal organ is neglected. Alternative xenograft-based approaches have visualized tumor cell behavior through cancer cell injection in the tongue , and engraftment in the eye which allow noninvasive longitudinal IVM without the need of imaging windows. Once the organ of interest can be optically accessed, different microscopy setups enable to address a multitude of questions. Optical access into deeper tissue layers is hampered by the tissue inherent light scattering and most studies employ 2-photon microscopes to visualize several hundred μm of tissue depth. The recent development of IVM protocols for 3-photon microscopy further increases the imaging depth . In addition, fast acquisition speed, for example using a spinning disk or a resonant scanner confocal microscope, can be of advantage for moving organs and allows the capture of highly dynamic events as for example the movement of leukocytes in the blood stream and tumor interstitium . The best combination of strategies for optical access and image acquisition will depend on the cancer type and biological question asked. Dedicated protocols for the imaging of primary cancers, metastases and the tumor microenvironment in different sites help identify the optimal strategy . Whilst the majority of previous IVM studies focused their attention on cellular behavior, the coming years are poised to see a broadening of IVM utilization with ongoing developments that multiplex bioluminescent and fluorescent reporters and molecular probes to film sub-cellular processes, and approaches that enable the in vivo visualization of nanoscale structures by 3D-stimulated emission depletion (3D-STED) microscopy , thereby opening the window to view molecular biology in the living organism.
Tools and strategies to visualize cell-cell interactions in vivo Given the ability to access and visualize the anatomic site of interest, a range of fluorescent labels and detection strategies enable the identification of cancer cells and structural and cellular components of the TME in the context of the organ architecture . The recent development of a photoactivatable Cre-recombinase mouse allows to target cancerous mutations to specific regions and cells in the organ landscape . Cancer cells can be labelled genetically through the expression of fluorescent proteins, and thus traced over sequential imaging sessions of several days to weeks. Proteins that can be activated, or photo-converted to change the fluorescent protein emission spectrum, allow the tracking of individual groups of cells based on their localization . With this approach, the colonization of distant organs from breast tumors was mapped by following the dissemination of cancer cells converted in the mammary gland in comparison to cancer cells converted in the lymph node . Proliferation of cancer and TME cells can be visualized indirectly by targeting individual cells with fluorescent proteins for lineage tracing, or directly through genetically encoded fluorescent cell cycle reporters . Fluorescent dyes like the recently developed apotracker-red are an option to identify apoptotic cells . Today the toolbox of IVM-ready fluorescent labels also includes a range of probes that allow to monitor the activity of the ERK, FAK, TGF-β/SMAD3 and nuclear factor kB (NF-kB) signaling pathways . A fluorescent reporter of nuclear factor of activated T cell (NFAT) activation was recently used to directly visualize how Treg cells receive TCR signals in the TME . The tumor-surrounding matrix can be readily visualized through second harmonic generations (SHG) detection of fibrillar collagen. Furthermore, recent efforts have targeted individual matrix components with fluorescent proteins. Fluorescently tagged fibronectin (FN) was used to track T cell interaction with FN in the inflamed ear dermis, where Th1 cells were found to migrate along FN fibers . The development of a laminin beta1-Dendra2 reporter mouse allows to visualize basement-membrane dynamics in vivo and can shed light on how cancer cells invade normal tissues . Tumor vasculature is commonly visualized through intravenous injection of fluorescent dextrans, lectins or quantum dots. The choice of dextran molecular weight allows to fine tune this detection to measure vascular permeability , and to label phagocytic immune cells . IVM has been instrumental to describe a wide range of fundamental immunological principles and immune cell behaviors . Genetic reporters and intravenously injected fluorophore-conjugated antibodies are commonly used to detect immune cells . Recent developments for multiplexed in vivo antibody labelling through click chemistry offer the opportunity to drastically expand the cell types that can be visualized in the same mouse . Nevertheless, it remains challenging to quantify rare cell populations in circulation. Recent developments enable real time imaging of individual fast circulating cells in the blood stream through quantum dots , a promising step in the direction of visualizing aggregation dynamics of circulating tumor cells (CTCs), or the interplay between lymphocytes, platelets, and CTCs in circulation in vivo .
Applications of volume imaging to visualize cell-TME interactions in cancer 4.1 Cellular relationships between epithelial cells The crosstalk between cancer cells and their neighboring normal epithelium dictates the biology of an arising lesion already at the earliest stages when only a couple of oncogenic cells start to proliferate. IVM experiments demonstrated how the crosstalk between skin cells bearing oncogenic mutations and their wildtype neighbors can correct aberrant growths . In the ear skin, hair follicle stem cells carrying an activating Hras mutation outcompeted their wildtype neighbors, yet were integrated into clinically normal skin hair follicles. In contrast, targeting the Hras mutation to the upper noncycling region of the skin epithelium led to benign outgrowths . Tissue clearing of skin, lung, liver and pancreas showed that the morphological appearance of early lesions is governed by an interplay between the architecture of the host tissue and deregulation of cancer cell actomyosin activity . Importantly, the mechanical crosstalk extends to the nuclear level where it causes dynamic changes in nuclear morphology and may threaten genetic integrity . Genomic cancer cell evolution and the dynamic TME instruct single cell biology and behavior and lead to diversification in the tumor bulk. IVM demonstrated that this heterogeneity extends to the metabolic level in ER + breast cancer. Using fluorescent biosensors, Kondo and colleagues demonstrated how actin remodeling, phosphatidylinositol 3-kinase (PI3K) and bromodomain activity modulate glycolysis resulting in metabolically distinct tumor cell populations that reside in different areas of a lesion . How this regional diversification affects the cellular interactions amongst cancer cells, as well as the interplay of cancer cells with the TME is subject to ongoing investigation. Alongside tumor expansion cancer cells actively remodel their microenvironment, and the TME is understood to be spatially patterned with differing constituents and function in different tumor areas . 4.2 Interaction of cancer cells and the tumor vasculature In the study of the TME, vascular remodeling has received particular attention as it impacts tumor nutrition, oxygenation, dissemination, and the recruitment of immune components. Quantitative assessments of the extent of tumor vascularization can be readily achieved by both tissue clearing and IVM, and have shown that several tumor types share a tendency to increase the vascular density within their mass . In some cancers the angiogenic capabilities may extend to lymphangiogenesis, as seen in pancreatic cancer . The cellular interactions between cancer cells and the tumor vasculature are multifold and reciprocal. IVM experiments on an orthotopic HNSCC model suggest that angiogenesis is specifically induced by a subpopulation of CD44 + cancer cells . On the other hand, 3D imaging of the tumor-stroma interface in cutaneous SCC showed that the blood vasculature regionally expresses TGF-β, thereby sustaining the cancer stem cell niche and contributing to cancer cell heterogeneity . In an HRAS-driven murine model of cutaneous SCC, angiogenesis was shown through tissue clearing to be triggered during the transition from papilloma to malignant carcinoma. This de novo vascularization in late stages of lesion progression facilitates leptin infiltration into the tumor, triggering Pi3k-Akt-mTor signaling, further contributing to the benign to malignant transition . Cancer progression through vascular remodeling is also seen in the bone marrow in acute myeloid leukemia (AML), where IVM experiments visualized how the remodeling of endosteal blood vessels abrogates hematopoietic stem cells (HSCs), HSC niches and osteoblasts through physical dislodgment via the damaged vasculature. Pharmacological MMP inhibition reduced vascular permeability and healthy cell loss, thereby limiting AML infiltration, proliferation, and cell migration . 4.3 Cancer cell crosstalk with the immune microenvironment The tumor vasculature is a significant route of tumor entry for immune cells that may clear cancer cells or be recruited for their tumor protective behavior. IVM successfully captured the critical steps in the cascade of lymphocyte adhesion to tumor-associated high endothelial venules (TA-HEVs), from initial lymphocyte tethering to the vessel wall to lymphocyte rolling and sticking, and extravasation . Tumor-infiltrating lymphocyte (TIL) motility changes both longitudinally during tumor progression , as well as regionally. Cytotoxic T lymphocytes (CTLs) show reduced motility and activity in avascular tumor areas but were found to be highly motile in vicinity to peripheral blood vessels and cleared tumor cells around them. Interestingly, blood flow is essential for CTL migration . Concomitantly, CTLs were shown to first accumulate in the tumor periphery and subsequently redistribute towards the invasive tumor front . Based on their migration patterns, CTLs have been understood to display a tumor cell searching behavior in the tumor periphery, which abates in velocity and changes into a “probing” behavior in tumor cell proximity indicating target engagement . IVM imaging showed that in the process of tumor cell killing, multiple CTLs undergo temporary sublethal contacts with individual tumor cells to induce killing in a process of additive cytotoxicity . On the other hand, CTL recognition of a few tumor cells can elicit sufficient IFNγ secretion to cover distances of several hundred micrometers and inhibit tumor growth even in regions not frequented by CTLs . Cancers hijack various cellular pathways to evade the immune response. T regulatory (Treg) cells are inherently poised to recognize antigens in the TME, and auto-regulate through short-lived interactions with conventional dendritic cells (cDCs) . Neutrophils stimulate T cells in a type I interferon (IFN) signaling dependent manner . However, tumor cells can elicit the production of neutrophil extracellular traps (NETs) from neutrophils and granulocytic myeloid-derived suppressor cells (MDSCs), and these NETs enwrap cancer cells, thereby reducing interfaces with CTLs and shielding against cytotoxicity . Neutrophils can promote tumor growth by activating MDSC that in turn inhibit the immune response orchestrated by effector T cells against cancer cells, as observed in cleared murine HNSCC tumors . Peritumoral neutrophils are more motile than intratumoral neutrophils , suggesting a different interactive behavior in specific tumor areas. TAMs play a multifold role in cancer progression. As for lymphocytes and neutrophils, volume imaging data suggest that TAM behavior differs drastically between tumor subtypes, stage and among different areas of the same lesion. IVM showed that the migratory behavior of TAMs is different in genetically distinct GBMs . In an orthotopic model of anaplastic thyroid cancer, tissue clearing suggested that the infiltration of tumor-associated macrophages is significantly higher in the primary tumor than in the metastases, indicating that the microenvironment in which the tumor develops conditions the infiltration of tumor-promoting TAMs . Using the dorsal skinfold chamber to track the crosstalk between macrophages and cancer grafts by IVM identified macrophage subpopulations based on their localization in the tumor core or periphery, and found that those populations differed in Arg1 gene expression and motility. This study also demonstrated the powerful combination of IVM with single cell RNA sequencing to gain molecular insight from a single cell phenotype that can only be observed in the native organ context . The cell-cell interaction between TAMs and cancer cells is a driving factor of tumor aggression. Macrophages were found to induce cancer cell stemness though Notch-Jagged signaling , and enable cancer cell dissemination through dynamic multicellular interactions . In an orthotopic breast cancer model, monocytes were shown to arrive at the tumor as motile streaming TAMs that then differentiated into sessile perivascular macrophages. This differentiation occurred through cancer cell induced TGFβ-dependent upregulation of CXCR4 in monocytes while CXCL12 expressed by perivascular fibroblasts attracted these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiated into perivascular macrophages, promoting vascular leakiness and intravasation . Together volume imaging experiments of the recent years demonstrated that tumor- supportive and tumor-inhibiting activities of immune cells crucially depend on the regional microenvironment, and are modulated in a highly dynamic way through complex multi-cellular interactions. 4.4 The basement membrane and cancer cell invasion The basement membrane constitutes a key barrier to tumor cell migration into the surrounding parenchyma. Recent IVM experiments, using a fluorescent laminin beta1 reporter mouse, show that basement membrane components are synthesized by cancer cells as well as endothelial cells in a dynamic and regionally heterogeneous manner. Interestingly, laminin beta1 turnover is higher in cancerous areas compared to normal epithelium, suggesting a local pre-conditioning of the basement membrane preceding tumor cell invasion . IVM imaging of cancer cell invasion has been instrumental to understand the cellular processes and interaction of cancer cell dissemination. Myosin 10 filopodia support the basement membrane in early lesions, but contribute to a proinvasive phenotype later in disease . Interestingly, cells that are able to breach the basement membrane, due to experimentally induced loss of E-cadherin which increases cancer cell motility, require further signals in order to disseminate into the surrounding parenchyma . Together, 3D cellular confinement, cell density and cell-cell junction stability orchestrate epithelial fluidization and single cell release , and set out the paths of migration and leader-follower cell relationships in collective movements. Cancer cells were found to migrate along collagen fibrils and blood vessels in several tumor models . Leader cells undergo significant deformations, potentially due to the confinement by the ECM, but create paths for follower cells that have more normalized cell and nuclear shapes . This synchronous migration along common paths may reflect an expansion along newly generated space in the ECM confinement, but could also be instructed directly from leader to follower cells through extracellular vesicle exchange . Thereby, the migratory behavior differs throughout the tumor landscape. GBM comprises an invasive margin with slow directed invasion and a diffuse infiltration margin with fast but less directed cell movements. Migration along blood vessels correlated with higher velocity and direction away from the tumor core whereas intraparenchymal migration along white matter tracts was measured to be slower and less directed . In glioma, IVM showed that GFAP splice variants fine‐tune invasion by modulating migration persistence. Whilst depletion of either isoform increased the migratory capacity of glioma cells, GFAPδ‐depleted cells migrated randomly through the brain tissue, whereas GFAPα‐depleted cells displayed a directionally persistent invasion into the brain parenchyma . 4.5 Cancer cell-TME interactions in transit to distant organs Tumor cell intravasation is a multicellular process in the TME which in breast cancer models was shown to require direct contacts between a proangiogenic perivascular macrophage, a tumor cell overexpressing the actin regulator protein Mena, and an endothelial cell that together create the “tumor microenvironment of metastasis” (TMEM), allowing cancer cell intravasation through transient vascular permeability . Vascular travel is a bottle neck in tumor cell dissemination as only a portion of the cells entering the blood stream are able to seed and colonize distant organs. Real-time visualization of circulating tumor cell (CTC) subpopulations through conjugated quantum dots showed that PDAC CTCs expressing the surface protein CD24 have the ability to migrate along vessel walls to distant organs . CTC packing in cell clusters can provide support for traveling cancer cells and has been debated to occur through collective intravasation or de novo clustering in the circulation. Intravital imaging showed that breast cancer CTC clusters form in circulation through CD44 protein mediated cell aggregation . Tissue clearing showed that breast cancer cells extravasate with the help of actin-rich protrusions . CTC extravasation depends on reciprocal interactions between CTCs and endothelial cells of distant organs that can prohibit or permit CTC organ entry. The bone endothelium expresses ephrin-B2 which activates ephrin-B4 in circulating melanoma cells thereby repulsing CTCs from attaching to the bone endothelial cells and barricading against metastasis formation . In the mouse lung, extravasation of breast cancer cells is dependent on physical interaction between tumor cells and macrophages in a IL4-CXCR2 dependent manner . 4.6 Cell-cell crosstalk at metastatic sites Metastasizing cancer cells can disseminate and establish tumors in organs with disparate cellular and physicochemical characteristics, or can present strong tropism and invade only one secondary organ . Tissue clearing of entire mice has shown the diverse tropism of different cancer cell lines such as mammary MDA-231 and pancreatic OS-RC-2 cells, and their colocalization with the vasculature across the mouse body . Volume imaging uncovered a widespread heterogeneity in the seeding potential, and the likelihood for successful metastatic outgrowth of different tumor cell subpopulations. These factors are now understood to be modulated by cell intrinsic properties and CSC traits on the one hand, as well as by dynamic multi-cellular contacts at the metastatic site on the other hand. Multicellular interactions influence the survival and outgrowth of metastasis seeding cancer cells. In the brain, early extravasating cells may be cleared by phagocytosis from TAM and microglia, although these effects abated in later stages of metastasis formation . As in primary tumors, the multi-cellular arrangements of cancer cells and TME components in metastatic sites show spatial and longitudinal heterogeneity. There is a highly heterogeneous cellular interplay between metastases even from the same tumor, as illustrated in breast cancer brain metastases that involve glial cells and astrocytes of the brain parenchyma to very different extents . The communication between cancer cells and the TME at different organs may amplify the heterogeneity seen in metastatic lesions. Indeed, the proliferation dynamics of cancer cells were found to depend on the organ colonized as shown with A549 lung adenocarcinoma cells after intracardiac administration into mice . One environmental mechanism may involve the access of pro-and antitumorigenic immune cells. Neutrophils are highly motile in lung capillaries and change their behavior to slow patrolling and stationary upon recruitment to lung metastasis where they were filmed to correlate with tumor cell proliferation .
Cellular relationships between epithelial cells The crosstalk between cancer cells and their neighboring normal epithelium dictates the biology of an arising lesion already at the earliest stages when only a couple of oncogenic cells start to proliferate. IVM experiments demonstrated how the crosstalk between skin cells bearing oncogenic mutations and their wildtype neighbors can correct aberrant growths . In the ear skin, hair follicle stem cells carrying an activating Hras mutation outcompeted their wildtype neighbors, yet were integrated into clinically normal skin hair follicles. In contrast, targeting the Hras mutation to the upper noncycling region of the skin epithelium led to benign outgrowths . Tissue clearing of skin, lung, liver and pancreas showed that the morphological appearance of early lesions is governed by an interplay between the architecture of the host tissue and deregulation of cancer cell actomyosin activity . Importantly, the mechanical crosstalk extends to the nuclear level where it causes dynamic changes in nuclear morphology and may threaten genetic integrity . Genomic cancer cell evolution and the dynamic TME instruct single cell biology and behavior and lead to diversification in the tumor bulk. IVM demonstrated that this heterogeneity extends to the metabolic level in ER + breast cancer. Using fluorescent biosensors, Kondo and colleagues demonstrated how actin remodeling, phosphatidylinositol 3-kinase (PI3K) and bromodomain activity modulate glycolysis resulting in metabolically distinct tumor cell populations that reside in different areas of a lesion . How this regional diversification affects the cellular interactions amongst cancer cells, as well as the interplay of cancer cells with the TME is subject to ongoing investigation. Alongside tumor expansion cancer cells actively remodel their microenvironment, and the TME is understood to be spatially patterned with differing constituents and function in different tumor areas .
Interaction of cancer cells and the tumor vasculature In the study of the TME, vascular remodeling has received particular attention as it impacts tumor nutrition, oxygenation, dissemination, and the recruitment of immune components. Quantitative assessments of the extent of tumor vascularization can be readily achieved by both tissue clearing and IVM, and have shown that several tumor types share a tendency to increase the vascular density within their mass . In some cancers the angiogenic capabilities may extend to lymphangiogenesis, as seen in pancreatic cancer . The cellular interactions between cancer cells and the tumor vasculature are multifold and reciprocal. IVM experiments on an orthotopic HNSCC model suggest that angiogenesis is specifically induced by a subpopulation of CD44 + cancer cells . On the other hand, 3D imaging of the tumor-stroma interface in cutaneous SCC showed that the blood vasculature regionally expresses TGF-β, thereby sustaining the cancer stem cell niche and contributing to cancer cell heterogeneity . In an HRAS-driven murine model of cutaneous SCC, angiogenesis was shown through tissue clearing to be triggered during the transition from papilloma to malignant carcinoma. This de novo vascularization in late stages of lesion progression facilitates leptin infiltration into the tumor, triggering Pi3k-Akt-mTor signaling, further contributing to the benign to malignant transition . Cancer progression through vascular remodeling is also seen in the bone marrow in acute myeloid leukemia (AML), where IVM experiments visualized how the remodeling of endosteal blood vessels abrogates hematopoietic stem cells (HSCs), HSC niches and osteoblasts through physical dislodgment via the damaged vasculature. Pharmacological MMP inhibition reduced vascular permeability and healthy cell loss, thereby limiting AML infiltration, proliferation, and cell migration .
Cancer cell crosstalk with the immune microenvironment The tumor vasculature is a significant route of tumor entry for immune cells that may clear cancer cells or be recruited for their tumor protective behavior. IVM successfully captured the critical steps in the cascade of lymphocyte adhesion to tumor-associated high endothelial venules (TA-HEVs), from initial lymphocyte tethering to the vessel wall to lymphocyte rolling and sticking, and extravasation . Tumor-infiltrating lymphocyte (TIL) motility changes both longitudinally during tumor progression , as well as regionally. Cytotoxic T lymphocytes (CTLs) show reduced motility and activity in avascular tumor areas but were found to be highly motile in vicinity to peripheral blood vessels and cleared tumor cells around them. Interestingly, blood flow is essential for CTL migration . Concomitantly, CTLs were shown to first accumulate in the tumor periphery and subsequently redistribute towards the invasive tumor front . Based on their migration patterns, CTLs have been understood to display a tumor cell searching behavior in the tumor periphery, which abates in velocity and changes into a “probing” behavior in tumor cell proximity indicating target engagement . IVM imaging showed that in the process of tumor cell killing, multiple CTLs undergo temporary sublethal contacts with individual tumor cells to induce killing in a process of additive cytotoxicity . On the other hand, CTL recognition of a few tumor cells can elicit sufficient IFNγ secretion to cover distances of several hundred micrometers and inhibit tumor growth even in regions not frequented by CTLs . Cancers hijack various cellular pathways to evade the immune response. T regulatory (Treg) cells are inherently poised to recognize antigens in the TME, and auto-regulate through short-lived interactions with conventional dendritic cells (cDCs) . Neutrophils stimulate T cells in a type I interferon (IFN) signaling dependent manner . However, tumor cells can elicit the production of neutrophil extracellular traps (NETs) from neutrophils and granulocytic myeloid-derived suppressor cells (MDSCs), and these NETs enwrap cancer cells, thereby reducing interfaces with CTLs and shielding against cytotoxicity . Neutrophils can promote tumor growth by activating MDSC that in turn inhibit the immune response orchestrated by effector T cells against cancer cells, as observed in cleared murine HNSCC tumors . Peritumoral neutrophils are more motile than intratumoral neutrophils , suggesting a different interactive behavior in specific tumor areas. TAMs play a multifold role in cancer progression. As for lymphocytes and neutrophils, volume imaging data suggest that TAM behavior differs drastically between tumor subtypes, stage and among different areas of the same lesion. IVM showed that the migratory behavior of TAMs is different in genetically distinct GBMs . In an orthotopic model of anaplastic thyroid cancer, tissue clearing suggested that the infiltration of tumor-associated macrophages is significantly higher in the primary tumor than in the metastases, indicating that the microenvironment in which the tumor develops conditions the infiltration of tumor-promoting TAMs . Using the dorsal skinfold chamber to track the crosstalk between macrophages and cancer grafts by IVM identified macrophage subpopulations based on their localization in the tumor core or periphery, and found that those populations differed in Arg1 gene expression and motility. This study also demonstrated the powerful combination of IVM with single cell RNA sequencing to gain molecular insight from a single cell phenotype that can only be observed in the native organ context . The cell-cell interaction between TAMs and cancer cells is a driving factor of tumor aggression. Macrophages were found to induce cancer cell stemness though Notch-Jagged signaling , and enable cancer cell dissemination through dynamic multicellular interactions . In an orthotopic breast cancer model, monocytes were shown to arrive at the tumor as motile streaming TAMs that then differentiated into sessile perivascular macrophages. This differentiation occurred through cancer cell induced TGFβ-dependent upregulation of CXCR4 in monocytes while CXCL12 expressed by perivascular fibroblasts attracted these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiated into perivascular macrophages, promoting vascular leakiness and intravasation . Together volume imaging experiments of the recent years demonstrated that tumor- supportive and tumor-inhibiting activities of immune cells crucially depend on the regional microenvironment, and are modulated in a highly dynamic way through complex multi-cellular interactions.
The basement membrane and cancer cell invasion The basement membrane constitutes a key barrier to tumor cell migration into the surrounding parenchyma. Recent IVM experiments, using a fluorescent laminin beta1 reporter mouse, show that basement membrane components are synthesized by cancer cells as well as endothelial cells in a dynamic and regionally heterogeneous manner. Interestingly, laminin beta1 turnover is higher in cancerous areas compared to normal epithelium, suggesting a local pre-conditioning of the basement membrane preceding tumor cell invasion . IVM imaging of cancer cell invasion has been instrumental to understand the cellular processes and interaction of cancer cell dissemination. Myosin 10 filopodia support the basement membrane in early lesions, but contribute to a proinvasive phenotype later in disease . Interestingly, cells that are able to breach the basement membrane, due to experimentally induced loss of E-cadherin which increases cancer cell motility, require further signals in order to disseminate into the surrounding parenchyma . Together, 3D cellular confinement, cell density and cell-cell junction stability orchestrate epithelial fluidization and single cell release , and set out the paths of migration and leader-follower cell relationships in collective movements. Cancer cells were found to migrate along collagen fibrils and blood vessels in several tumor models . Leader cells undergo significant deformations, potentially due to the confinement by the ECM, but create paths for follower cells that have more normalized cell and nuclear shapes . This synchronous migration along common paths may reflect an expansion along newly generated space in the ECM confinement, but could also be instructed directly from leader to follower cells through extracellular vesicle exchange . Thereby, the migratory behavior differs throughout the tumor landscape. GBM comprises an invasive margin with slow directed invasion and a diffuse infiltration margin with fast but less directed cell movements. Migration along blood vessels correlated with higher velocity and direction away from the tumor core whereas intraparenchymal migration along white matter tracts was measured to be slower and less directed . In glioma, IVM showed that GFAP splice variants fine‐tune invasion by modulating migration persistence. Whilst depletion of either isoform increased the migratory capacity of glioma cells, GFAPδ‐depleted cells migrated randomly through the brain tissue, whereas GFAPα‐depleted cells displayed a directionally persistent invasion into the brain parenchyma .
Cancer cell-TME interactions in transit to distant organs Tumor cell intravasation is a multicellular process in the TME which in breast cancer models was shown to require direct contacts between a proangiogenic perivascular macrophage, a tumor cell overexpressing the actin regulator protein Mena, and an endothelial cell that together create the “tumor microenvironment of metastasis” (TMEM), allowing cancer cell intravasation through transient vascular permeability . Vascular travel is a bottle neck in tumor cell dissemination as only a portion of the cells entering the blood stream are able to seed and colonize distant organs. Real-time visualization of circulating tumor cell (CTC) subpopulations through conjugated quantum dots showed that PDAC CTCs expressing the surface protein CD24 have the ability to migrate along vessel walls to distant organs . CTC packing in cell clusters can provide support for traveling cancer cells and has been debated to occur through collective intravasation or de novo clustering in the circulation. Intravital imaging showed that breast cancer CTC clusters form in circulation through CD44 protein mediated cell aggregation . Tissue clearing showed that breast cancer cells extravasate with the help of actin-rich protrusions . CTC extravasation depends on reciprocal interactions between CTCs and endothelial cells of distant organs that can prohibit or permit CTC organ entry. The bone endothelium expresses ephrin-B2 which activates ephrin-B4 in circulating melanoma cells thereby repulsing CTCs from attaching to the bone endothelial cells and barricading against metastasis formation . In the mouse lung, extravasation of breast cancer cells is dependent on physical interaction between tumor cells and macrophages in a IL4-CXCR2 dependent manner .
Cell-cell crosstalk at metastatic sites Metastasizing cancer cells can disseminate and establish tumors in organs with disparate cellular and physicochemical characteristics, or can present strong tropism and invade only one secondary organ . Tissue clearing of entire mice has shown the diverse tropism of different cancer cell lines such as mammary MDA-231 and pancreatic OS-RC-2 cells, and their colocalization with the vasculature across the mouse body . Volume imaging uncovered a widespread heterogeneity in the seeding potential, and the likelihood for successful metastatic outgrowth of different tumor cell subpopulations. These factors are now understood to be modulated by cell intrinsic properties and CSC traits on the one hand, as well as by dynamic multi-cellular contacts at the metastatic site on the other hand. Multicellular interactions influence the survival and outgrowth of metastasis seeding cancer cells. In the brain, early extravasating cells may be cleared by phagocytosis from TAM and microglia, although these effects abated in later stages of metastasis formation . As in primary tumors, the multi-cellular arrangements of cancer cells and TME components in metastatic sites show spatial and longitudinal heterogeneity. There is a highly heterogeneous cellular interplay between metastases even from the same tumor, as illustrated in breast cancer brain metastases that involve glial cells and astrocytes of the brain parenchyma to very different extents . The communication between cancer cells and the TME at different organs may amplify the heterogeneity seen in metastatic lesions. Indeed, the proliferation dynamics of cancer cells were found to depend on the organ colonized as shown with A549 lung adenocarcinoma cells after intracardiac administration into mice . One environmental mechanism may involve the access of pro-and antitumorigenic immune cells. Neutrophils are highly motile in lung capillaries and change their behavior to slow patrolling and stationary upon recruitment to lung metastasis where they were filmed to correlate with tumor cell proliferation .
Volume imaging as a platform for therapy development Whilst historically tissue culture assays have been used to carry out cancer drug screens, an ever-growing body of literature highlights limitations in the predictive value of cell culture assays to extrapolate the effect of drugs from in vitro to an entire organism. This may be attributed to a difference in signals that cells cultured in vitro lack in comparison to cancer cells in vivo . Cancer cells receive stimuli from neighboring cancer cells, non-tumor cells, architectural constraints and systemic physical factors that shape tumor biology in vivo . Recent studies using IVM showed that even cell-intrinsic molecular characteristics may not be adequately reflected in 2D/3D culture systems . In fact, when transplanted in vivo , a panel of breast cancer cell lines showed remarkable cellular heterogeneity, even in the same nodule that was not observed in vitro . Hence, preclinical models remain essential to evaluate the effect of new therapeutic agents . Combining preclinical models with fluorescence volume imaging constitutes a powerful tool to assess potential anti-cancer treatments, many of which directly targeted against components of the TME, and to study how they affect the interaction between cancer cells and the TME. 5.1 Drug delivery is dependent on tumor vascularization and perfusion Volume imaging technologies pose an attractive platform to evaluate a new drug, map off-target effects and identify the mechanisms underlying acquired therapy resistance. Several studies used tissue clearing to assess the therapeutic delivery of drugs and drug vehicles in vivo . The distribution of nanoparticles and PEGylated liposomes, and their spatial relation with the vasculature was used to evaluate efficiency of drug delivery into the tumor . Similarly, tissue clearing visualized tumor perfusion by fluorescently tagged anti-tumor antibodies, such as the HER-2-targetting antibody trastuzumab, and a fluorescent analogue of the chemotherapeutic agent docetaxel . In cases where the therapeutic agent cannot be traced directly, drug interference with fluorescent signals from the target cells may serve as proxy for measuring drug engagement. In an elegant example, doxorubicin uptake was measured in vivo on a single cell level by exploiting the changes in fluorescence lifetime of histone-GFP fusions upon doxorubicin binding to chromatin. This uncovered drastic differences in doxorubicin binding between different peritoneal metastases in the same mouse, as well as different regions and cells in the same lesion . In a murine model of non-Hodgkin lymphoma, tissue clearing revealed that the delivery of therapeutic CD20 fragment antigen-binding (FAB) antibody regions was dependent on tumor perfusion . IVM showed that preserving endosteal vessels in AML improves the efficacy of chemotherapy . IVM of melanoma and PDAC xenografts implanted under a dorsal skinfold window was used as a platform to quantify the vascular effects of acute cyclic hypoxia and antiangiogenic treatment . A key goal of treatment development has been to educate and exploit the immune system to reject cancer progression. Immune infiltration into the tumor is also dependent on its vascularization. IVM assays are an elegant tool to measure the biodistribution and efficacy of CAR T cells, as demonstrated for primary central nervous system lymphoma (PCNSL) and GBM . Volume imaging identified tumor associated high endothelial venules (TA-HEVs) as the main sites of lymphocyte arrest and extravasation into tumors that received anti-PD-1/anti-CTLA-4 immunotherapy. Increasing TA-HEV endothelial cells (TA-HEC) frequency and maturation improved CTL recruitment and therapy response . IVM experiments showed that carboplatin chemotherapy profoundly alters the TME to promote lymphocyte adhesive interactions with the tumor vasculature resulting in improveed lymphocyte trafficking . Another IVM study in a GBM model suggested that radiotherapy may aid CAR T cell extravasation and expansion . CTLs engineered to overexpress the chemokine receptor CXCR4 displayed increased homing to the bone marrow and engraftment, and acquired greater anti-tumor immunity . Cancer-derived adenosine weakens the physical interaction between activated effector CTLs and target cells, providing cancer cells with an escape way from immunotherapy. IVM showed that antagonization of adenosine A2a receptor (ADORA2a) signaling improved CTL-target engagement and cancer cell killing . 5.2 Measuring drug efficacy A key aspect of therapy development is the measure of in vivo drug efficacy. IVM of calvarium bone marrow tracked the migratory behavior of acute myeloid leukemia (AML) cells in comparison to T cell acute lymphoblastic leukemia (T-ALL), and demonstrated distinct traits of the lineages in their response to chemotherapy and CXCR4 antagonism, highlighting the importance of carefully evaluating cancer traits in vivo for each tumor type . Tissue clearing has been beneficial to evaluate therapy efficiency in various preclinical cancer models, such as HepG2 xenografts to assess the severing of the vasculature induced by antiangiogenic therapeutic siRNAs , and HER-2-expressing breast cancer xenografts treated with colony-stimulation factor 1 receptor inhibitor PLX3397, which reduced tumor burden without impairing the infiltration of macrophages . Tissue clearing for whole organ quantifications of metastatic burden showed that targeting macrophages with clodronate liposomes reduced lung colonization by intravenously injected A549 cancer cells . KPL-4 breast cancer xenografts were found to present a normalized vasculature when treating mice with anti-VEGF antiangiogenic therapy , indicating an improvement of tumor irrigation and of the penetration of nutrients and oxygen, but potentially also of other therapeutic agents that may hence more efficiently find and eliminate cancer cells. 5.3 Immunotherapy Immunotherapy is efficient in immunogenic tumors that express immune checkpoint molecules, whether in the cancer cells or in other infiltrating cells of the TME. Tissue clearing has been used to map the expression of immune checkpoint molecule PD-L1 and immune cells throughout tumors such as MMTV-HER2/Neu and NSCLC patient resections . Such 3D quantifications are more precise than 2D analyses of tissue sections and could be applied to patient biopsies as a prognostic marker that predicts the response to immunotherapy. In addition to tissue clearing, IVM has been successfully applied to evaluate the biodistribution of immunotherapies . Furthermore, such in vivo assays have proven useful to pinpoint the reasons for immunotherapy failure, and could demonstrate for example how a fluorophore-conjugated PD-1 targeting antibody shown to target PD-1 + tumor infiltrating CD8 T-cells was immediately cleared by PD-1 - tumor associated macrophages, preventing tumor regression . 5.4 Cancer therapy side effects One key advantage of tissue clearing is the power to detect rare events in large tissue volumes in a quantitative manner and at little time expenditure. These traits make tissue clearing an attractive solution to study side effects of cancer therapies. Once organ sites suffering under off-target effects and drug toxicity are identified, IVM can add to this informative value by revealing the dynamic interplay of cell populations. Liver toxicity is a common side effect of viral therapies and Naumenko and colleagues demonstrated how IVM can be used to track the interactions between hepatocytes, Kupffer cells, CD8 T-cells and neutrophils to study oncolytic adenovirus associated liver toxicity . Apart from insights that educate drug refinement, IVM may also uncover new treatment options. For example, IVM experiments uncovered evidence that bisphosphonate drugs, which were designed to target bone, readily accumulate in microcalcifications in breast cancers where they have been suggested to stimulate TAMs and improve cancer patient survival independent of the drug’s antiresorptive effects on bone tissue . 5.5 Therapy resistance A common trait of many cancers is the ability to acquire therapy resistance over the course of a drug treatment. The underlying mechanisms can be partially attributed to genomic and epigenomic evolution and molecular adaptation of tumor cell subpopulations. The process of epithelial-to-mesenchymal transition (EMT) has been under intense debate to play a key role in cancer aggression and IVM experiments proposed that cancer cells that have undergone EMT may be a source for cancer recurrence upon chemotherapy . Volume imaging suggests that the acquisition of therapy resistance, coming from subpopulations in the tumor bulk, may be linked to specific architectural regions of a tumor lesion. Indeed, cells in the invasive front were found to display a higher degree of radiotherapy resistance and survival. Combinatorial integrin targeting sensitized these cells and abrogated tumor relapse . Apart from tumor cell intrinsic molecular paths towards drug resistance, IVM experiments also demonstrated the role of direct interactions and crosstalk between tumor cells and the TME in this context. Matrix stiffness was found to dictate PDAC chemosensitivity and IVM showed that therapy priming through focal adhesion kinase (FAK) inhibition pulses could drastically increase PDAC cell sensitivity to gemcitabine/abraxane .
Drug delivery is dependent on tumor vascularization and perfusion Volume imaging technologies pose an attractive platform to evaluate a new drug, map off-target effects and identify the mechanisms underlying acquired therapy resistance. Several studies used tissue clearing to assess the therapeutic delivery of drugs and drug vehicles in vivo . The distribution of nanoparticles and PEGylated liposomes, and their spatial relation with the vasculature was used to evaluate efficiency of drug delivery into the tumor . Similarly, tissue clearing visualized tumor perfusion by fluorescently tagged anti-tumor antibodies, such as the HER-2-targetting antibody trastuzumab, and a fluorescent analogue of the chemotherapeutic agent docetaxel . In cases where the therapeutic agent cannot be traced directly, drug interference with fluorescent signals from the target cells may serve as proxy for measuring drug engagement. In an elegant example, doxorubicin uptake was measured in vivo on a single cell level by exploiting the changes in fluorescence lifetime of histone-GFP fusions upon doxorubicin binding to chromatin. This uncovered drastic differences in doxorubicin binding between different peritoneal metastases in the same mouse, as well as different regions and cells in the same lesion . In a murine model of non-Hodgkin lymphoma, tissue clearing revealed that the delivery of therapeutic CD20 fragment antigen-binding (FAB) antibody regions was dependent on tumor perfusion . IVM showed that preserving endosteal vessels in AML improves the efficacy of chemotherapy . IVM of melanoma and PDAC xenografts implanted under a dorsal skinfold window was used as a platform to quantify the vascular effects of acute cyclic hypoxia and antiangiogenic treatment . A key goal of treatment development has been to educate and exploit the immune system to reject cancer progression. Immune infiltration into the tumor is also dependent on its vascularization. IVM assays are an elegant tool to measure the biodistribution and efficacy of CAR T cells, as demonstrated for primary central nervous system lymphoma (PCNSL) and GBM . Volume imaging identified tumor associated high endothelial venules (TA-HEVs) as the main sites of lymphocyte arrest and extravasation into tumors that received anti-PD-1/anti-CTLA-4 immunotherapy. Increasing TA-HEV endothelial cells (TA-HEC) frequency and maturation improved CTL recruitment and therapy response . IVM experiments showed that carboplatin chemotherapy profoundly alters the TME to promote lymphocyte adhesive interactions with the tumor vasculature resulting in improveed lymphocyte trafficking . Another IVM study in a GBM model suggested that radiotherapy may aid CAR T cell extravasation and expansion . CTLs engineered to overexpress the chemokine receptor CXCR4 displayed increased homing to the bone marrow and engraftment, and acquired greater anti-tumor immunity . Cancer-derived adenosine weakens the physical interaction between activated effector CTLs and target cells, providing cancer cells with an escape way from immunotherapy. IVM showed that antagonization of adenosine A2a receptor (ADORA2a) signaling improved CTL-target engagement and cancer cell killing .
Measuring drug efficacy A key aspect of therapy development is the measure of in vivo drug efficacy. IVM of calvarium bone marrow tracked the migratory behavior of acute myeloid leukemia (AML) cells in comparison to T cell acute lymphoblastic leukemia (T-ALL), and demonstrated distinct traits of the lineages in their response to chemotherapy and CXCR4 antagonism, highlighting the importance of carefully evaluating cancer traits in vivo for each tumor type . Tissue clearing has been beneficial to evaluate therapy efficiency in various preclinical cancer models, such as HepG2 xenografts to assess the severing of the vasculature induced by antiangiogenic therapeutic siRNAs , and HER-2-expressing breast cancer xenografts treated with colony-stimulation factor 1 receptor inhibitor PLX3397, which reduced tumor burden without impairing the infiltration of macrophages . Tissue clearing for whole organ quantifications of metastatic burden showed that targeting macrophages with clodronate liposomes reduced lung colonization by intravenously injected A549 cancer cells . KPL-4 breast cancer xenografts were found to present a normalized vasculature when treating mice with anti-VEGF antiangiogenic therapy , indicating an improvement of tumor irrigation and of the penetration of nutrients and oxygen, but potentially also of other therapeutic agents that may hence more efficiently find and eliminate cancer cells.
Immunotherapy Immunotherapy is efficient in immunogenic tumors that express immune checkpoint molecules, whether in the cancer cells or in other infiltrating cells of the TME. Tissue clearing has been used to map the expression of immune checkpoint molecule PD-L1 and immune cells throughout tumors such as MMTV-HER2/Neu and NSCLC patient resections . Such 3D quantifications are more precise than 2D analyses of tissue sections and could be applied to patient biopsies as a prognostic marker that predicts the response to immunotherapy. In addition to tissue clearing, IVM has been successfully applied to evaluate the biodistribution of immunotherapies . Furthermore, such in vivo assays have proven useful to pinpoint the reasons for immunotherapy failure, and could demonstrate for example how a fluorophore-conjugated PD-1 targeting antibody shown to target PD-1 + tumor infiltrating CD8 T-cells was immediately cleared by PD-1 - tumor associated macrophages, preventing tumor regression .
Cancer therapy side effects One key advantage of tissue clearing is the power to detect rare events in large tissue volumes in a quantitative manner and at little time expenditure. These traits make tissue clearing an attractive solution to study side effects of cancer therapies. Once organ sites suffering under off-target effects and drug toxicity are identified, IVM can add to this informative value by revealing the dynamic interplay of cell populations. Liver toxicity is a common side effect of viral therapies and Naumenko and colleagues demonstrated how IVM can be used to track the interactions between hepatocytes, Kupffer cells, CD8 T-cells and neutrophils to study oncolytic adenovirus associated liver toxicity . Apart from insights that educate drug refinement, IVM may also uncover new treatment options. For example, IVM experiments uncovered evidence that bisphosphonate drugs, which were designed to target bone, readily accumulate in microcalcifications in breast cancers where they have been suggested to stimulate TAMs and improve cancer patient survival independent of the drug’s antiresorptive effects on bone tissue .
Therapy resistance A common trait of many cancers is the ability to acquire therapy resistance over the course of a drug treatment. The underlying mechanisms can be partially attributed to genomic and epigenomic evolution and molecular adaptation of tumor cell subpopulations. The process of epithelial-to-mesenchymal transition (EMT) has been under intense debate to play a key role in cancer aggression and IVM experiments proposed that cancer cells that have undergone EMT may be a source for cancer recurrence upon chemotherapy . Volume imaging suggests that the acquisition of therapy resistance, coming from subpopulations in the tumor bulk, may be linked to specific architectural regions of a tumor lesion. Indeed, cells in the invasive front were found to display a higher degree of radiotherapy resistance and survival. Combinatorial integrin targeting sensitized these cells and abrogated tumor relapse . Apart from tumor cell intrinsic molecular paths towards drug resistance, IVM experiments also demonstrated the role of direct interactions and crosstalk between tumor cells and the TME in this context. Matrix stiffness was found to dictate PDAC chemosensitivity and IVM showed that therapy priming through focal adhesion kinase (FAK) inhibition pulses could drastically increase PDAC cell sensitivity to gemcitabine/abraxane .
Future perspectives in volume imaging of the TME Since its rediscovery about a decade ago, tissue clearing has been drastically refined to meet the most diverse problems in cancer research, from the study of cancer cell interactions with the infiltrating TME to the mapping of tumor vascularization and innervation, and quantifications of whole-body metastatic load. A wealth of excellent techniques exists tailored to different organs, cancers, and fluorescent readouts. The recent development of accessible whole mouse clearing techniques and antibody labelling of cells spread throughout the entire organism allows to study the differential TMEs at both the primary tumor and metastatic sites in the same organism. Today most of the tissues and architectural structures can be visualized in 3D. Open opportunities remain in the detection of pathogens, such as whole organ quantifications of the distribution of microbiota in tumors . One bottleneck of tissue clearing remains in the limited detection of multiple cell types in the same sample. However, technical solutions may not be far as pioneering studies on 2D tissue sections have pushed the boundaries in multiplexing for fluorescent microscopy. Imaging mass cytometry detects up to 32 markers , and 3D mass cytometry has recently been achieved for thick sections of around 300 um depth . Whilst tissue clearing constitutes a powerful tool to examine entire tissues, organs and organisms, refractive index matching can be combined with expansion microscopy to visualize subcellular details . With this technology, super-resolution information of the molecular cancer cell-TME interactions can be unveiled. In order to connect 3D information with an understanding of cellular dynamics, IVM today offers several platforms, such as the dorsal skin fold chamber, tumor grafts in the eye or tissue-engineered bone constructs with a skin window for longitudinal imaging. Together they enable cancer research to evaluate potential therapeutics in a more relevant setting in vivo without the expenditure of developing strategies to study cancers in their native organ site . Tumor cell grafts readily remodel their surrounding vasculature and stand in intense crosstalk with the immune system, providing ample opportunity to understand and interfere with fundamental cancer traits such as tumor angiogenesis and immune evasion. However, these strategies may give little opportunity to study the crosstalk between cancer cells and their neighboring normal epithelial cells. Furthermore, the tumor graft origin from in vitro cultures may fundamentally alter cancer cell properties. Indeed, 3D imaging showed that the angiogenic capacity of xenograft models depends on the culture conditions prior to injection, suggesting that epigenetic changes in vitro condition the communication between cancer cells and the vascular microenvironment in vivo . Furthermore, intravital imaging of lung metastases showed that spontaneously disseminating tumor cells are more capable to form distant metastasis than experimentally metastasized tumor cells . Whilst the majority of volume imaging studies have used these platforms for fundamental and preclinical research, 3D imaging also poses considerable attractiveness for cancer staging. Tissue clearing could be exploited for the analysis of cancer patient resections in a diagnostic setting. 3D mapping of vasculature and lymphatics was shown to improve the prognostic evaluation of bladder cancer . The presence of high endothelial venules in models of fibrosarcoma and mammary carcinoma was found to correlate with tumor regression upon Treg cell depletion , and to predict survival and αPD-1/αCTLA-4 immunotherapy response of melanoma patients . Pathways for 3D image automation and standardization are already being laid out, as demonstrated in the successful computational segmentation of prostate cancer patient samples . A 3D high-resolution view into a clinical cancer resection holds great informational value and can uncover unexpected subtleties in the cellular composition and spatial arrangements . For example, tissue clearing showed that luminal prostate cancer, widely expressing K8 (marker of luminal epithelium) contains regions enriched in K5 + myoepithelium which acquire a more differentiated, ductal conformation . Nevertheless, clinical adoption and utilization will require careful evaluation of 3D imaging data and side by side comparisons with conventional histopathological assessment to educate data interpretation towards the best treatment decisions and to avoid patient overtreatment.
JA and HM researched literature for the review, discussed content, wrote, reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.
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The Clinical Efficacy and Safety of | b14cc1a5-953d-4fbe-b35b-65f69ad253b8 | 11743062 | Dentistry[mh] | Introduction Alveolar bone and other osseous defects can occur because of heredity, infection, trauma, tumour resection or and disuse atrophy (Jeffcoat ; Keun ; Ucer and Khan ; Zhou et al. ). Over 2 million surgical procedures are performed annually worldwide to repair such defects. The standard treatment for bone defects is bone grafting in the form of an autograft, allograft, xenografts or synthetic bone substitutes. Despite their extensive use, bone grafts have several limitations (Polo‐Corrales, Latorre‐Esteves, and Ramirez‐Vick ). With progress in tissue engineering technology, combining bone substitutes and growth factors in applications has been widely recognized as an alternative option. Recombinant human bone morphogenetic protein 2 (rhBMP‐2), one of the best documented osteoinductive growth factors, was developed and proved to induce osteoblast differentiation and stimulate new bone formation at both homotopic and ectopic sites (Halloran, Durbano, and Nohe ). It was first prepared using Chinese hamster ovary cells and then using Escherichia coli as an alternative. It has been approved for spinal fusion procedures by absorbing it on the surface of a collagen sponge, a product commercially named INFUSE, made by Medtronic. Despite the promising initial clinical efficacy, bias in the original trials seemed to underestimate the risk of complications and adverse effects caused by the high loading quantity of rhBMP‐2 (in milligram range) and burst‐release‐related side effects derived from the mode of rhBMP‐2 delivery (Jeon et al. ; Jo et al. ; Kim et al. ; Thoma et al. ) in the compound (Carragee, Hurwitz, and Weiner ; Gillman and Jayasuriya ; James et al. ). It is critical that in novel bone substitutes, rhBMP‐2 is delivered at the lowest possible dosage following a controlled and sustained release action to maximize its osteoinductivity and minimize adverse reactions. Previously, our team had developed a novel rhBMP‐2 delivery system: the low‐dosage rhBMP‐2 (in the microgram range, 1/10 of the up‐till‐now available commercial products) incorporated in biomimetic calcium phosphate coating (rhBMP‐2/BioCaP) (Liu, De Groot, and Hunziker ; Liu et al. ). It is further characterized by local, limited and sustained release of rhBMP‐2 in a cell‐mediated manner, mimicking the principles of natural bone remodelling (Liu et al. , ). Based on this innovative technology, β‐TCP, one of the most often used calcium phosphate‐based bone substitutes (Jeong et al. ), was functionalized by the E. coli ‐derived rhBMP‐2/BioCaP coating (ErhBMP‐2/BioCaP/β‐TCP). In vitro and in vivo preclinical studies have demonstrated its unique and superior properties: microporosity, biodegradability, osteoconductivity and osteoinductivity (Wei et al. ). It delivers micro‐concentrations of ErhBMP‐2 in a controlled manner, overcoming current safety concerns, and results in optimal bone regeneration with minimal side effects. This clinical trial aimed to verify the efficacy and safety of ErhBMP‐2/BioCaP/β‐TCP as a novel bone substitute using the tooth‐extraction‐socket‐healing model. Materials And Methods 2.1 Study Approval The protocol was approved by the Ethics Committee of Shanghai Ninth People's Hospital (No. SH9H‐2019‐T231‐4) and the Academic Center for Dentistry Amsterdam (No. 202061), and registered in the Chinese Clinical Trial Registry (No. ChiCTR2000035263, https://www.chictr.org.cn/ ) on 10 August 2020. This clinical trial was conducted between August 2020 and December 2021 at the Second Dental Center, Shanghai Ninth People's Hospital, Shanghai. All screened patients were required to sign an informed consent form before enrollment. 2.2 Power Analysis Two independent statisticians performed power calculations to determine the required sample size. The primary outcome, namely the volume density of new bone formation in biopsy samples taken from the tooth extraction site 6 weeks post surgery, was pivotal in determining the sample size. Drawing on insights from a previous preclinical study (Liu et al. ), the effect size was calculated using the Social Science Statistics software, and Cohen's d ‐values were inserted into G*Power 3 (Faul et al. ). An alpha of 5%, a test power of 90% and a two‐tailed independent samples t ‐test were input into the G*Power 3 software, and the minimum sample size was calculated (Table ). We included more number of patients than required by the power calculations, taking into account possible dropouts. Therefore, 40 patients were included in the trial. The enrolled patients were assigned to (i) the ErhBMP‐2/BioCaP/β‐TCP‐treated group (15 patients), (ii) the β‐TCP‐treated group (15 patients) or (iii) the natural healing sockets group (10 patients). 2.3 Patient Selection Patients who had a single root tooth that met the indications for tooth extraction and were classified as EDS‐1 or EDS‐2 according to the extraction defect sounding (EDS) classification (Caplanis, Lozada, and Kan ) and met all the other criteria were included in the trial, as previously reported (Table ) (Sun et al. ). 2.4 ErhBMP ‐2/ BioCaP /β‐ TCP Preparation E. coli ‐derived rhBMP‐2‐functionalized β‐TCP granules (Shanghai Rebone Biomaterials Co. Ltd.; particle size, 0.25–1.00 mm) coated with biomimetic calcium phosphate (Shanghai Rebone Biomaterials) (ErhBMP‐2/BioCaP/β‐TCP) were manufactured according to established protocols (Lin et al. ; Liu et al. ; Wei et al. , ) and Good Manufacturing Practice (GMP) in compliance with the ISO 13485:2016 standard for manufacturing of medical devices. Scanning electron microscopy (SEM) observation showed that ErhBMP‐2/BioCaP/β‐TCP had a plate‐like topography with crystalline coating on the surface (Figures ). The average coating thickness was 10.4 μm, as measured from the cross‐section by SEM (Figure ). The average rhBMP‐2 concentration in the synthesized material was 126.2 μg/g of BioCaP/β‐TCP, as measured by an enzyme‐linked immunosorbent assay (ELISA) kit (Neobioscience Co. Ltd) and remained stable after 3 months (Figure ). Preclinical biosafety testing of ErhBMP‐2/BioCaP/β‐TCP was performed by Weihai Desheng Technology Testing Co. Ltd. (China), in compliance with the ISO 10993 standard (Table ). 2.5 Randomization and Blinding This was a single centre, randomized, controlled, partially double‐blind clinical trial. The trial followed the principles of Good Clinical Practice (GCP) in compliance with the ISO 14155:2020 standard for the clinical investigation of medical devices and the CONSORT guidelines for human patients (Schulz, Altman, and Moher ). Randomized block design was used in this trial. The enrolled patients were divided into five blocks based on the inclusion order, with eight subjects per block. The eight subjects in each block were simply randomized by drawing lots as follows: three subjects each in both the ErhBMP‐2/BioCaP/β‐TCP group and the β‐TCP group, and two subjects in the blank control group. The subjects and surgeons were blinded to the first two groups, but the blank control group could not be blinded. The researchers who performed measurements and analyses were completely blinded to the allocation. 2.6 Clinical Trial Procedure and the Patient Analysis Sets The design and trial procedures are summarized in Figure . Five visits were planned and conducted during the 6‐week follow‐up. At visit 2, a senior dentist performed standardized tooth extraction and socket filling surgery for the patients following the randomization results. The tooth extraction sockets were filled with ErhBMP‐2/BioCaP/β‐TCP, β‐TCP or left empty and then covered with a double layer of resorbable collagen membrane (Bio‐Gide by Geistlich Biomaterials, Wolhusen, Switzerland). Gargling with 0.12% chlorhexidine solution was recommended twice daily for 7 days after surgery. Cone beam computed tomography (CBCT) and intra‐oral scan data were collected immediately and 6 weeks after surgery. At the fifth visit, biopsy samples (diameter 2.3 mm; height 6 mm) were collected, all by one senior dentist, using trephine burs with outer diameter 3 mm from the centre of the socket. To maintain consistency and meet the need of early implant placement, at least 1.5 mm bone walls were preserved in all directions. The axial direction was kept in consistent with the axis of single rooted teeth. The biopsy samples were cut out 6 mm away from the alveolar ridge crest. Dental implants were placed when the primary stability was achieved, and bone augmentation procedures were performed when the bone mass around the implant was insufficient. Forty patients were carefully selected from a cohort of 44 screened individuals and enrolled in this study. As previously mentioned, they were randomly allocated into three groups: ErhBMP‐2/BioCaP/β‐TCP‐treated (15 patients), β‐TCP treated (15 patients) and natural healing sockets (10 patients). All participants completed the clinical trial, with no instances of loss to follow‐up or intervention discontinuation. The CONSORT flow chart of this trial is shown in Figure . The number of patients in the full analysis set (FAS) and the safety set (SS) was 40. However, three patients (one each in the ErhBMP‐2/BioCaP/β‐TCP‐treated group, the β‐TCP‐treated group and the natural healing socket group) were excluded from the FAS due to protocol violations. The remaining 37 patients were included in the sensitivity analysis set (SAS). A single patient from the natural healing socket group in SAS was excluded from histomorphometric analyses because of insufficient biopsied tissue, while other data remained unaffected. This participant was retained in the SAS for hard‐ and soft‐tissue analyses but not for histomorphometric analyses. Consequently, the remaining 36 patients formed the per‐protocol set (PPS) specifically for histomorphometric analyses. In summary, the numbers of patients in the FAS, SS, SAS and PPS were 40, 40, 37 and 36, respectively. 2.7 Measurements of Efficacy and Safety Outcomes The primary outcome of the efficacy analysis was new bone volume density in the biopsy sample 6 weeks after surgery. The secondary outcomes included (1) the unmineralized tissue volume density in the biopsy sample, (2) the residual material volume density in the biopsy site, (3) the bone width and height changes measured by CBCT scans, (4) the soft‐tissue surface sectional area and width changes measured by intra‐oral scans and (5) the number of bone augmentation procedure required during dental implant placed. The safety outcomes included (1) the soft‐tissue healing score, (2) the Oral Health Impact Profile‐14 (OHIP‐14) questionnaire, (3) adverse events (application site pain, swelling, haemorrhage, dental discomfort, oral discomfort and other discomforts), (4) the BMP‐2 concentration in the serum and (5) other laboratory blood and urine tests. 2.7.1 Bone Histomorphometry Biopsy samples were collected and immersed in 10% neutral formalin solution with trephine for 24 h. Following thorough flushing, the solution was dehydrated using alcohol gradients, and the samples were embedded in polymethyl methacrylate, which was prepared by bulk polymerization using methyl methacrylate (Zhanyun ChemE, Shanghai, China). Each biopsy sample was uniformly partitioned into five sections with 1 mm distance and cut along the cross‐sectional plane using the systematic random sampling method to reduce measurement error and increase the accuracy. They were subsequently arranged on plexiglass holders in the same order, each section with a thickness initially set at 600 μm, and were polished down to a thickness ranging from 50 to 100 μm (Figure ). Following staining with McNeal's Tetrachrome, basic fuchsine and toluidine blue, the ImageJ software (National Institutes of Health, USA) was used to measure the volume density of new bone, residual material and unmineralized tissue. 2.7.2 CBCT and Intra‐Oral Scan and Analyses The CBCT scans (field‐of‐view 8 cm [D] × 8 cm [H], resolution 0.16 mm with 80 peak kilovoltage [kVp] and 10 mA; Planmeca, Finland) were made during the screening period (visit 1) for verifying whether the tooth extraction site classification met the inclusion criteria, immediately after the tooth‐extraction‐socket‐healing surgery (visit 2) for establishing the baseline of alveolar ridge and 6 weeks after the surgery (visit 5) for measuring the alveolar ridge contour changes. The CBCT scans at visits 2 and 5 were exported as DICOM (.dcm) files and then imported into the Romexis software (Planmeca Romexis, Planmeca, Finland) for matching and measurement. The bone width changes at 1, 3 and 5 mm below the alveolar ridge crest as well as the height changes of the buccal and lingual bones were measured in the sagittal plane (Figure ). Intra‐oral scanning (3Shape TRIOS intra‐oral scanner) at visits 2 and 5 were performed and matched using the Romexis software. The changes in the surface area (5 mm from the soft‐tissue crest to the root) and surface width change at 1, 3 and 5 mm in the sagittal plane below the soft‐tissue crest were measured. 2.7.3 Safety Analyses At visits 3, 4 and 5, the soft‐tissue healing score using the scale introduced by Afat et al. (Afat, Akdoğan, and Gönül ), the OHIP‐14 questionnaire and adverse events were evaluated. At visits 1 and 5, serum from the peripheral circulatory system was also collected to test the BMP‐2 concentration by ELISA. The serum and urine were also used to perform other laboratory tests. 2.8 Statistical Analyses Prior to database locking, the statistician and principal investigator validated the datasets and analytical methods. The changes of alveolar bone and surface contour were all measured directly by the matched images (CBCT and intra‐oral scan respectively) and analysed using one‐way ANOVA. Histomorphometric data, soft‐tissue healing score, OHIP‐14 score and the continuous demographic data were analysed using one‐way ANOVA. The laboratory examination data were analysed using two‐way repeated‐measures ANOVA. Multiple between‐group comparisons were performed using post hoc analyses. The Cochran–Mantel–Haenszel (CMH) test was applied to compare categorical data, and Fisher's exact test was used for post hoc analysis after the CMH tests. GraphPad Prism software version 9 (GraphPad, San Diego, CA, USA) was used for the analysis. All statistical tests were two‐sided, and p‐ values < 0.05 were deemed statistically significant. Study Approval The protocol was approved by the Ethics Committee of Shanghai Ninth People's Hospital (No. SH9H‐2019‐T231‐4) and the Academic Center for Dentistry Amsterdam (No. 202061), and registered in the Chinese Clinical Trial Registry (No. ChiCTR2000035263, https://www.chictr.org.cn/ ) on 10 August 2020. This clinical trial was conducted between August 2020 and December 2021 at the Second Dental Center, Shanghai Ninth People's Hospital, Shanghai. All screened patients were required to sign an informed consent form before enrollment. Power Analysis Two independent statisticians performed power calculations to determine the required sample size. The primary outcome, namely the volume density of new bone formation in biopsy samples taken from the tooth extraction site 6 weeks post surgery, was pivotal in determining the sample size. Drawing on insights from a previous preclinical study (Liu et al. ), the effect size was calculated using the Social Science Statistics software, and Cohen's d ‐values were inserted into G*Power 3 (Faul et al. ). An alpha of 5%, a test power of 90% and a two‐tailed independent samples t ‐test were input into the G*Power 3 software, and the minimum sample size was calculated (Table ). We included more number of patients than required by the power calculations, taking into account possible dropouts. Therefore, 40 patients were included in the trial. The enrolled patients were assigned to (i) the ErhBMP‐2/BioCaP/β‐TCP‐treated group (15 patients), (ii) the β‐TCP‐treated group (15 patients) or (iii) the natural healing sockets group (10 patients). Patient Selection Patients who had a single root tooth that met the indications for tooth extraction and were classified as EDS‐1 or EDS‐2 according to the extraction defect sounding (EDS) classification (Caplanis, Lozada, and Kan ) and met all the other criteria were included in the trial, as previously reported (Table ) (Sun et al. ). ErhBMP ‐2/ BioCaP /β‐ TCP Preparation E. coli ‐derived rhBMP‐2‐functionalized β‐TCP granules (Shanghai Rebone Biomaterials Co. Ltd.; particle size, 0.25–1.00 mm) coated with biomimetic calcium phosphate (Shanghai Rebone Biomaterials) (ErhBMP‐2/BioCaP/β‐TCP) were manufactured according to established protocols (Lin et al. ; Liu et al. ; Wei et al. , ) and Good Manufacturing Practice (GMP) in compliance with the ISO 13485:2016 standard for manufacturing of medical devices. Scanning electron microscopy (SEM) observation showed that ErhBMP‐2/BioCaP/β‐TCP had a plate‐like topography with crystalline coating on the surface (Figures ). The average coating thickness was 10.4 μm, as measured from the cross‐section by SEM (Figure ). The average rhBMP‐2 concentration in the synthesized material was 126.2 μg/g of BioCaP/β‐TCP, as measured by an enzyme‐linked immunosorbent assay (ELISA) kit (Neobioscience Co. Ltd) and remained stable after 3 months (Figure ). Preclinical biosafety testing of ErhBMP‐2/BioCaP/β‐TCP was performed by Weihai Desheng Technology Testing Co. Ltd. (China), in compliance with the ISO 10993 standard (Table ). Randomization and Blinding This was a single centre, randomized, controlled, partially double‐blind clinical trial. The trial followed the principles of Good Clinical Practice (GCP) in compliance with the ISO 14155:2020 standard for the clinical investigation of medical devices and the CONSORT guidelines for human patients (Schulz, Altman, and Moher ). Randomized block design was used in this trial. The enrolled patients were divided into five blocks based on the inclusion order, with eight subjects per block. The eight subjects in each block were simply randomized by drawing lots as follows: three subjects each in both the ErhBMP‐2/BioCaP/β‐TCP group and the β‐TCP group, and two subjects in the blank control group. The subjects and surgeons were blinded to the first two groups, but the blank control group could not be blinded. The researchers who performed measurements and analyses were completely blinded to the allocation. Clinical Trial Procedure and the Patient Analysis Sets The design and trial procedures are summarized in Figure . Five visits were planned and conducted during the 6‐week follow‐up. At visit 2, a senior dentist performed standardized tooth extraction and socket filling surgery for the patients following the randomization results. The tooth extraction sockets were filled with ErhBMP‐2/BioCaP/β‐TCP, β‐TCP or left empty and then covered with a double layer of resorbable collagen membrane (Bio‐Gide by Geistlich Biomaterials, Wolhusen, Switzerland). Gargling with 0.12% chlorhexidine solution was recommended twice daily for 7 days after surgery. Cone beam computed tomography (CBCT) and intra‐oral scan data were collected immediately and 6 weeks after surgery. At the fifth visit, biopsy samples (diameter 2.3 mm; height 6 mm) were collected, all by one senior dentist, using trephine burs with outer diameter 3 mm from the centre of the socket. To maintain consistency and meet the need of early implant placement, at least 1.5 mm bone walls were preserved in all directions. The axial direction was kept in consistent with the axis of single rooted teeth. The biopsy samples were cut out 6 mm away from the alveolar ridge crest. Dental implants were placed when the primary stability was achieved, and bone augmentation procedures were performed when the bone mass around the implant was insufficient. Forty patients were carefully selected from a cohort of 44 screened individuals and enrolled in this study. As previously mentioned, they were randomly allocated into three groups: ErhBMP‐2/BioCaP/β‐TCP‐treated (15 patients), β‐TCP treated (15 patients) and natural healing sockets (10 patients). All participants completed the clinical trial, with no instances of loss to follow‐up or intervention discontinuation. The CONSORT flow chart of this trial is shown in Figure . The number of patients in the full analysis set (FAS) and the safety set (SS) was 40. However, three patients (one each in the ErhBMP‐2/BioCaP/β‐TCP‐treated group, the β‐TCP‐treated group and the natural healing socket group) were excluded from the FAS due to protocol violations. The remaining 37 patients were included in the sensitivity analysis set (SAS). A single patient from the natural healing socket group in SAS was excluded from histomorphometric analyses because of insufficient biopsied tissue, while other data remained unaffected. This participant was retained in the SAS for hard‐ and soft‐tissue analyses but not for histomorphometric analyses. Consequently, the remaining 36 patients formed the per‐protocol set (PPS) specifically for histomorphometric analyses. In summary, the numbers of patients in the FAS, SS, SAS and PPS were 40, 40, 37 and 36, respectively. Measurements of Efficacy and Safety Outcomes The primary outcome of the efficacy analysis was new bone volume density in the biopsy sample 6 weeks after surgery. The secondary outcomes included (1) the unmineralized tissue volume density in the biopsy sample, (2) the residual material volume density in the biopsy site, (3) the bone width and height changes measured by CBCT scans, (4) the soft‐tissue surface sectional area and width changes measured by intra‐oral scans and (5) the number of bone augmentation procedure required during dental implant placed. The safety outcomes included (1) the soft‐tissue healing score, (2) the Oral Health Impact Profile‐14 (OHIP‐14) questionnaire, (3) adverse events (application site pain, swelling, haemorrhage, dental discomfort, oral discomfort and other discomforts), (4) the BMP‐2 concentration in the serum and (5) other laboratory blood and urine tests. 2.7.1 Bone Histomorphometry Biopsy samples were collected and immersed in 10% neutral formalin solution with trephine for 24 h. Following thorough flushing, the solution was dehydrated using alcohol gradients, and the samples were embedded in polymethyl methacrylate, which was prepared by bulk polymerization using methyl methacrylate (Zhanyun ChemE, Shanghai, China). Each biopsy sample was uniformly partitioned into five sections with 1 mm distance and cut along the cross‐sectional plane using the systematic random sampling method to reduce measurement error and increase the accuracy. They were subsequently arranged on plexiglass holders in the same order, each section with a thickness initially set at 600 μm, and were polished down to a thickness ranging from 50 to 100 μm (Figure ). Following staining with McNeal's Tetrachrome, basic fuchsine and toluidine blue, the ImageJ software (National Institutes of Health, USA) was used to measure the volume density of new bone, residual material and unmineralized tissue. 2.7.2 CBCT and Intra‐Oral Scan and Analyses The CBCT scans (field‐of‐view 8 cm [D] × 8 cm [H], resolution 0.16 mm with 80 peak kilovoltage [kVp] and 10 mA; Planmeca, Finland) were made during the screening period (visit 1) for verifying whether the tooth extraction site classification met the inclusion criteria, immediately after the tooth‐extraction‐socket‐healing surgery (visit 2) for establishing the baseline of alveolar ridge and 6 weeks after the surgery (visit 5) for measuring the alveolar ridge contour changes. The CBCT scans at visits 2 and 5 were exported as DICOM (.dcm) files and then imported into the Romexis software (Planmeca Romexis, Planmeca, Finland) for matching and measurement. The bone width changes at 1, 3 and 5 mm below the alveolar ridge crest as well as the height changes of the buccal and lingual bones were measured in the sagittal plane (Figure ). Intra‐oral scanning (3Shape TRIOS intra‐oral scanner) at visits 2 and 5 were performed and matched using the Romexis software. The changes in the surface area (5 mm from the soft‐tissue crest to the root) and surface width change at 1, 3 and 5 mm in the sagittal plane below the soft‐tissue crest were measured. 2.7.3 Safety Analyses At visits 3, 4 and 5, the soft‐tissue healing score using the scale introduced by Afat et al. (Afat, Akdoğan, and Gönül ), the OHIP‐14 questionnaire and adverse events were evaluated. At visits 1 and 5, serum from the peripheral circulatory system was also collected to test the BMP‐2 concentration by ELISA. The serum and urine were also used to perform other laboratory tests. Bone Histomorphometry Biopsy samples were collected and immersed in 10% neutral formalin solution with trephine for 24 h. Following thorough flushing, the solution was dehydrated using alcohol gradients, and the samples were embedded in polymethyl methacrylate, which was prepared by bulk polymerization using methyl methacrylate (Zhanyun ChemE, Shanghai, China). Each biopsy sample was uniformly partitioned into five sections with 1 mm distance and cut along the cross‐sectional plane using the systematic random sampling method to reduce measurement error and increase the accuracy. They were subsequently arranged on plexiglass holders in the same order, each section with a thickness initially set at 600 μm, and were polished down to a thickness ranging from 50 to 100 μm (Figure ). Following staining with McNeal's Tetrachrome, basic fuchsine and toluidine blue, the ImageJ software (National Institutes of Health, USA) was used to measure the volume density of new bone, residual material and unmineralized tissue. CBCT and Intra‐Oral Scan and Analyses The CBCT scans (field‐of‐view 8 cm [D] × 8 cm [H], resolution 0.16 mm with 80 peak kilovoltage [kVp] and 10 mA; Planmeca, Finland) were made during the screening period (visit 1) for verifying whether the tooth extraction site classification met the inclusion criteria, immediately after the tooth‐extraction‐socket‐healing surgery (visit 2) for establishing the baseline of alveolar ridge and 6 weeks after the surgery (visit 5) for measuring the alveolar ridge contour changes. The CBCT scans at visits 2 and 5 were exported as DICOM (.dcm) files and then imported into the Romexis software (Planmeca Romexis, Planmeca, Finland) for matching and measurement. The bone width changes at 1, 3 and 5 mm below the alveolar ridge crest as well as the height changes of the buccal and lingual bones were measured in the sagittal plane (Figure ). Intra‐oral scanning (3Shape TRIOS intra‐oral scanner) at visits 2 and 5 were performed and matched using the Romexis software. The changes in the surface area (5 mm from the soft‐tissue crest to the root) and surface width change at 1, 3 and 5 mm in the sagittal plane below the soft‐tissue crest were measured. Safety Analyses At visits 3, 4 and 5, the soft‐tissue healing score using the scale introduced by Afat et al. (Afat, Akdoğan, and Gönül ), the OHIP‐14 questionnaire and adverse events were evaluated. At visits 1 and 5, serum from the peripheral circulatory system was also collected to test the BMP‐2 concentration by ELISA. The serum and urine were also used to perform other laboratory tests. Statistical Analyses Prior to database locking, the statistician and principal investigator validated the datasets and analytical methods. The changes of alveolar bone and surface contour were all measured directly by the matched images (CBCT and intra‐oral scan respectively) and analysed using one‐way ANOVA. Histomorphometric data, soft‐tissue healing score, OHIP‐14 score and the continuous demographic data were analysed using one‐way ANOVA. The laboratory examination data were analysed using two‐way repeated‐measures ANOVA. Multiple between‐group comparisons were performed using post hoc analyses. The Cochran–Mantel–Haenszel (CMH) test was applied to compare categorical data, and Fisher's exact test was used for post hoc analysis after the CMH tests. GraphPad Prism software version 9 (GraphPad, San Diego, CA, USA) was used for the analysis. All statistical tests were two‐sided, and p‐ values < 0.05 were deemed statistically significant. Results 3.1 Study Participants and Characteristics Forty patients (24 females and 16 males) were included in the trial, and no statistically significant differences were observed among the groups in terms of age, sex or smoking status (Table ). The baseline data of tooth extraction sites in the enrolled patients showed no statistically significant differences among the groups in the number of defective bone walls, gingival phenotype, height of the bone defect, height of gingival recession or tooth extraction classification (Table ). 3.2 Efficacy Thirty‐six biopsy samples were taken from 36 patients (8 from the natural healing group, 14 from the ErhBMP‐2/BioCaP/β‐TCP group and 14 from the β‐TCP group). All biopsy samples obtained 6 weeks after tooth extraction were used to delineate the new bone, residual materials and unmineralized tissue (Figure ). Residual materials were evident in both ErhBMP‐2/BioCaP/β‐TCP‐treated (Figure ) and β‐TCP‐treated patients (Figure ). In patients with natural healing sockets, new bone trabeculae originated from the indigenous alveolar bone and regenerated in the socket periphery, whereas fibrous tissue was predominant in the central region (Figure ). Minimal new bone was observed in the periphery of the socket, with little contact with the residual material in β‐TCP‐treated patients (Figure ). In ErhBMP‐2/BioCaP/β‐TCP‐treated patients, moderate new bone formation and bone–material contact occurred in the periphery and centre of the socket (Figure ). Histomorphometric analyses were performed on biopsy specimens from 36 patients (PPS). The new bone volume densities in biopsies from ErhBMP‐2/BioCaP/β‐TCP‐treated patients, β‐TCP‐treated patients and those with natural healing sockets were 7.72 ± 6.01%, 2.96 ± 2.23% and 8.37 ± 6.31%, respectively. A statistically significant difference was observed between the β‐TCP‐treated patients and the other two groups (Figure ). The volume density of residual materials in ErhBMP‐2/BioCaP/β‐TCP‐treated patients was 10.90 ± 4.04%, which is significantly lower than that in β‐TCP‐treated patients (15.73 ± 4.52%) (Figure ). Regarding unmineralized tissue volume density , the value in patients with natural healing sockets (91.63 ± 6.31%) was significantly higher than that in the other two groups (ErhBMP‐2/BioCaP/β‐TCP‐treated patients, 81.38 ± 4.81% and β‐TCP treated patients, 81.32 ± 4.70%) (Figure ). Alterations in the height and width of the alveolar bone at 6 weeks post tooth extraction were analysed and illustrated in Figure . The difference in buccal and lingual bone height resorption values and the changes in horizontal alveolar bone width at 3 and 5 mm below the alveolar bone crest showed no statistical significance among the three groups (Figure ). Notably, the resorption in horizontal alveolar bone width at 1 mm below the alveolar bone crest in ErhBMP‐2/BioCaP/β‐TCP‐treated patients was significantly lower than that in patients with natural healing sockets (Figure ). Although alterations in the surface contour of soft tissue exhibited a reduction 6 weeks after tooth‐extraction‐socket healing, no statistically significant differences were detected among the three groups (Table ). In dental implant surgery procedures, nine patients treated with ErhBMP‐2/BioCaP/β‐TCP, seven patients treated with β‐TCP and one patient in the natural healing socket group had sufficient bone volume for implant placement and did not require bone augmentation. The need for bone augmentation in ErhBMP‐2/BioCaP/β‐TCP‐treated patients was significantly lower than that in patients with natural healing sockets (Table ). 3.3 Safety Based on the CMH test results, the number (percentages) of patients with adverse events showed no significant differences among the groups. The soft‐tissue healing score at 1, 2 and 6 weeks after tooth extraction showed no significant differences among the three groups, and the same scientific analysis results were indicated in the OHIP (Table ). All ELISA tests for the serum BMP‐2 were negative, and the other dual laboratory test results from visits 1 and 5 showed no significant differences. Study Participants and Characteristics Forty patients (24 females and 16 males) were included in the trial, and no statistically significant differences were observed among the groups in terms of age, sex or smoking status (Table ). The baseline data of tooth extraction sites in the enrolled patients showed no statistically significant differences among the groups in the number of defective bone walls, gingival phenotype, height of the bone defect, height of gingival recession or tooth extraction classification (Table ). Efficacy Thirty‐six biopsy samples were taken from 36 patients (8 from the natural healing group, 14 from the ErhBMP‐2/BioCaP/β‐TCP group and 14 from the β‐TCP group). All biopsy samples obtained 6 weeks after tooth extraction were used to delineate the new bone, residual materials and unmineralized tissue (Figure ). Residual materials were evident in both ErhBMP‐2/BioCaP/β‐TCP‐treated (Figure ) and β‐TCP‐treated patients (Figure ). In patients with natural healing sockets, new bone trabeculae originated from the indigenous alveolar bone and regenerated in the socket periphery, whereas fibrous tissue was predominant in the central region (Figure ). Minimal new bone was observed in the periphery of the socket, with little contact with the residual material in β‐TCP‐treated patients (Figure ). In ErhBMP‐2/BioCaP/β‐TCP‐treated patients, moderate new bone formation and bone–material contact occurred in the periphery and centre of the socket (Figure ). Histomorphometric analyses were performed on biopsy specimens from 36 patients (PPS). The new bone volume densities in biopsies from ErhBMP‐2/BioCaP/β‐TCP‐treated patients, β‐TCP‐treated patients and those with natural healing sockets were 7.72 ± 6.01%, 2.96 ± 2.23% and 8.37 ± 6.31%, respectively. A statistically significant difference was observed between the β‐TCP‐treated patients and the other two groups (Figure ). The volume density of residual materials in ErhBMP‐2/BioCaP/β‐TCP‐treated patients was 10.90 ± 4.04%, which is significantly lower than that in β‐TCP‐treated patients (15.73 ± 4.52%) (Figure ). Regarding unmineralized tissue volume density , the value in patients with natural healing sockets (91.63 ± 6.31%) was significantly higher than that in the other two groups (ErhBMP‐2/BioCaP/β‐TCP‐treated patients, 81.38 ± 4.81% and β‐TCP treated patients, 81.32 ± 4.70%) (Figure ). Alterations in the height and width of the alveolar bone at 6 weeks post tooth extraction were analysed and illustrated in Figure . The difference in buccal and lingual bone height resorption values and the changes in horizontal alveolar bone width at 3 and 5 mm below the alveolar bone crest showed no statistical significance among the three groups (Figure ). Notably, the resorption in horizontal alveolar bone width at 1 mm below the alveolar bone crest in ErhBMP‐2/BioCaP/β‐TCP‐treated patients was significantly lower than that in patients with natural healing sockets (Figure ). Although alterations in the surface contour of soft tissue exhibited a reduction 6 weeks after tooth‐extraction‐socket healing, no statistically significant differences were detected among the three groups (Table ). In dental implant surgery procedures, nine patients treated with ErhBMP‐2/BioCaP/β‐TCP, seven patients treated with β‐TCP and one patient in the natural healing socket group had sufficient bone volume for implant placement and did not require bone augmentation. The need for bone augmentation in ErhBMP‐2/BioCaP/β‐TCP‐treated patients was significantly lower than that in patients with natural healing sockets (Table ). Safety Based on the CMH test results, the number (percentages) of patients with adverse events showed no significant differences among the groups. The soft‐tissue healing score at 1, 2 and 6 weeks after tooth extraction showed no significant differences among the three groups, and the same scientific analysis results were indicated in the OHIP (Table ). All ELISA tests for the serum BMP‐2 were negative, and the other dual laboratory test results from visits 1 and 5 showed no significant differences. Discussion The present trial explored the efficacy and safety of ErhBMP‐2/BioCaP/β‐TCP as a novel bone substitute using the tooth‐extraction‐socket‐healing model. Regarding efficacy, faster early stage bone regeneration was observed 6 weeks after tooth extraction in sockets filled with ErhBMP‐2/BioCaP/β‐TCP compared to those filled with β‐TCP, which is a conventional synthetic bone substitute. Moreover, the ErhBMP‐2/BioCaP/β‐TCP also maintained the alveolar ridge contour and positively affected tooth‐extraction‐socket healing. Concerning safety, the results preliminarily proved that ErhBMP‐2/BioCaP/β‐TCP could be considered a biocompatible bone substitute. A higher volume density of newly formed bone was observed in ErhBMP‐2/BioCaP/β‐TCP‐treated patients than in β‐TCP‐treated patients due to the incorporation of ErhBMP‐2 (which is an osteoinductive growth factor) and similar in patients with natural healing sockets. These results are inconsistent with some previous preclinical results (Liu et al. ; Wei et al. ), which demonstrated more than 3 times new bone formation in bone‐graft‐filled defects compared with natural healing socket patients when repairing critical‐sized bone defects. The differences between the present trial and preclinical studies could have arisen from differences in bone defect classification. The tooth extraction socket can heal rapidly and spontaneously, whereas spontaneous bone healing in critically sized bone defects is permanently restricted. The inconsistency in the healing period and species variation between the clinical trial and preclinical studies also contributed to the discrepancies in bone regeneration among the three groups. In natural healing sockets, bone formation is activated as early as 2–4 weeks after tooth extraction. In β‐TCP‐treated patients, there was a prolonged healing cycle. Typically, patients undergoing implant placement after ridge preservation using bone substitutes must wait 4–6 months or longer (Tonetti et al. ). An extended healing time may be required based on the phenotypic characteristics of the extraction site, the properties of the biomaterial(s) used and patient‐specific systemic factors. This trial demonstrated that ErhBMP‐2/BioCaP/β‐TCP accelerated the early bone healing compared to the traditional synthetic bone substitutes and achieved similar bone regeneration as fast as in patients with natural healing sockets. The previous CBCT analysis also demonstrated that the grey value decrease at the central area of filled materials in the ErhBMP‐2/BioCaP/β‐TCP group was faster than in the β‐TCP group (Sun et al. ), which was consistent with the volume density of residual materials in histomorphological results in this study. CBCT analysis showed that ErhBMP‐2/BioCaP/β‐TCP significantly reduced the bone width loss at 1 mm below the alveolar ridge crest. Although the mean height changes of buccal bone in the ErhBMP‐2/BioCaP/β‐TCP‐treated patients were lower than in the natural healing socket patients, the difference was not statistically significant. Dual intra‐oral scans were also used to analyse soft‐tissue contour changes; unfortunately, no positive characteristic parameters were found. There could be two potential reasons for these results: the limited sample size in this trial, and the short observation period (only 6 weeks) compared with other trials. No serious adverse events were observed. The incidence of adverse events was similar among the three groups, and most adverse events were restricted to the application sites (e.g., pain, swelling and local discomfort). Moreover, most patients in the ErhBMP‐2/BioCaP/β‐TCP‐treated group had completely healed soft tissue. None of the patients showed abnormal results with clinical significance in vital signs, physical examinations or laboratory tests. In the presented trial, no bone regeneration was found outside the socket in the ErhBMP‐2/BioCaP/β‐TCP‐treated patients who received ErhBMP‐2 in low dosage and sustained release. Considering all the results, ErhBMP‐2/BioCaP/β‐TCP used in this clinical trial can be considered safe as a bone substitute for tooth extraction socket healing. The present study has some limitations. Firstly, biopsy samples were collected by freehand surgery rather than using the surgical guide, which can get the samples more precisely. In visit 5, CBCT and intra‐oral scan data were all collected. A surgical guide could be made for the biopsies. However, it would take some days to design and print the guide in the lab when the chairside printer was not available. That means the patients should make one more visit to the clinic. Therefore, CBCT and intra‐oral scan data were used for preoperative diagnosis and virtual design of the biopsies rather than for fabricating the surgical guide. In the future, a surgical guide should be recommended for biopsies. Secondly, the observation period of 6 weeks is relatively short for bone regeneration in humans, regardless of the location and category of the defects. The trial duration was insufficient to demonstrate the efficacy of alveolar ridge preservation and long‐term safety. A 2‐year follow‐up study was initiated to evaluate the procedure's safety. The socket healing model was used to test the safety and efficiency of the materials in general. However, the purpose of using this material is not only for tooth‐extraction‐socket healing. The present study was primarily to verify its efficacy and safety; larger multi‐centre pivotal trials will be carried out to prove the efficacy and safety fully. Conclusion Within the limitations of the current clinical trial, we found that ErhBMP‐2/BioCaP/β‐TCP could be considered as an osteo‐promotive and biocompatible bone substitute. This novel bone substitute can potentially achieve safe and effective treatment for repairing bone defects. L.W. monitored the trial and was the major contributor to writing the manuscript. Y.S. and D.Y. performed the clinical trial and were involved in the data analyses. H.P. and D.W. formulated the trial design and management. Y.L. and Y.W. supervised the research and reviewed the manuscript. All authors read and approved the final manuscript. This trial was approved by the Ethics Committee of both Shanghai Ninth People's Hospital (No. SH9H‐2019‐T231‐4) and the Academic Center for Dentistry Amsterdam (No. 202061), and registered in the Chinese Clinical Trial Registry (No. ChiCTR2000035263). All participants were informed and understood the objectives and details of the study and signed a written informed consent document. The authors declare no conflicts of interest. Data S1. Supporting Information. |
Lifetime Cost-Effectiveness of Structured Education and Exercise Therapy for Knee Osteoarthritis in Australia | 4a0a2fcf-6bf3-4a21-a0a6-b43333785404 | 11445685 | Patient Education as Topic[mh] | Knee osteoarthritis is an important public health issue, with the health and economic burden increasing through aging populations and growing reliance on surgical interventions. , Contemporary clinical guidelines recommend that nonsurgical management options, including education and exercise therapy, be exhausted before total knee replacement (TKR) is considered. , Yet, individuals with knee osteoarthritis who present to a general practitioner are 3 times more likely to be referred to an orthopedic surgeon than a physiotherapist. Future projections have estimated that 161 000 TKR procedures would be performed by 2030 at a cost of 3.4 billion Australian dollars (A$), with up to one-third of procedures regarded as inappropriate based on validated appropriateness tools (eg, for people with minimal symptoms or radiologic signs). , International programs that incorporate guideline-based education and exercise therapy, such as the Good Life With Osteoarthritis: Denmark (GLA:D) program, have shown clinically promising results in improving health outcomes and short-term TKR avoidance when implemented in the Australian health system. , , However, it is unclear whether a structured education and exercise therapy program represents value for money compared with TKR. In a randomized clinical trial (RCT), Skou et al reported that a structured education and exercise therapy program with the option of later TKR was cost-effective compared with immediate TKR at 2 years. However, longer-term evaluation is crucial given that TKR has high upfront costs but long-lasting benefits. , Furthermore, the results were sensitive to baseline characteristics (age, sex, baseline costs, and quality-adjusted life-years [QALYs]), suggesting that the cost-effectiveness of these interventions may differ in certain subgroups. Structured education and exercise therapy programs are poorly funded by the Australian public health system and private health insurers. With financial costs a recognized barrier to access, , there is a need to leverage national registry data to explore the cost-effectiveness of funding a structured education and exercise therapy program at a national level prior to TKR over a longer time horizon. The aim of this study was to evaluate whether structured education and an exercise therapy program delivered to individuals with knee osteoarthritis with the option for future TKR is cost-effective across the lifespan compared with TKR in the first year from a health system perspective.
Study Design This economic evaluation is reported according to the 2022 Consolidated Health Economic Evaluation Reporting Standards ( CHEERS ) checklist. Patient consent was obtained for the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) Patient-Reported Outcomes Program and GLA:D Australia Registry. Ethical approval was obtained through the Monash University Human Research Ethics Committee to access these deidentified data. We developed a life table model in combination with a Markov model to compare a structured education and exercise therapy program with the option for future TKR vs TKR in the first year (usual care). A Markov model uses mean participant data to replicate the clinical context where individuals can transition between different health states. The lifetime model, designed using annual cycles, comprised 4 health states: no or mild pain, moderate pain, severe or extreme pain, and dead. The 3 alive health states were solely defined by participant responses to the pain domain within the 5-level EuroQuol-5 Dimension (EQ-5D-5L) instrument. Individuals start in 1 of 3 alive health states, with dead as the absorbing health state. Half-cycle corrections were made, with an annual discount rate of 5% applied to costs and outcomes. Model inputs, their sources, and parameter distribution are summarized in and eTables 1 to 11 in . , , , , , , , The model was built using Microsoft Excel, version 16.87 (Microsoft Corporation). Model Population The model population comprised adults aged 45 to 84 years who would otherwise undergo TKR in Australia (eligible patients), excluding those undergoing partial knee replacements, as they are infrequently performed. To calculate the size of our hypothetical cohort, we obtained age- and sex-specific rates of TKR for osteoarthritis in 2020-2021 and 2022 Australian population data ( ; eTable 1 in ). We estimated that 61 394 Australians underwent primary TKR in 2022. Individuals were categorized into health states based on the EQ-5D-5L pain domain. The baseline proportion of individuals in each health state was calculated from individual-level patient-reported outcome measures from the AOANJRR. Preoperative patient-reported outcome measures have been collected since mid-2018, with data available for 73.5% of procedures. For this study, data were obtained for 9889 individuals who underwent primary TKR for osteoarthritis from August 7, 2018, to December 31, 2021 (eTable 2 in ). The baseline proportions of individuals in each health state based on age and sex strata are provided in eTable 3 in . All-cause mortality rates were derived from Australian population life tables (eTable 4 in ). Mortality risk following TKR was adjusted given evidence of fewer deaths in the first 8 to 9 years following TKR and more deaths after 12 years (eTable 5 in ). As osteoarthritis is a degenerative disease that may influence multiple joints (contralateral knee, hip, or shoulder), we took a conservative approach and assumed that the rate of contralateral TKR surgery or replacement surgery at other joints would not be influenced by the intervention. Transition Probabilities Transition probabilities following TKR were derived from AOANJRR data, stratified by age and sex (eTable 6 in ). The AOANJRR collects postoperative outcomes at 6 months as most clinical improvement occurs during this period. Transition probabilities following education and exercise therapy were obtained from the GLA:D Australia Registry at 12 months (eTable 7 in ). As this database includes all individuals with osteoarthritis who participated in the GLA:D program, we restricted our analysis to those who were (1) on a waiting list for a joint replacement or surgical opinion, (2) reported that they were in so much trouble and pain that they wanted surgery, or (3) had a Knee Injury and Osteoarthritis Outcome Score-12 summary score 54.6 or less. The Knee Injury and Osteoarthritis Outcome Score-12 summary score threshold of 54.6 aligns with the preoperative score plus 1 SD from 4010 individuals who underwent primary TKR for osteoarthritis in Australia. The likelihood of revision surgery, by age and sex, was obtained from the 2022 AOANJRR annual report (eTable 8 in ). Likelihood of revision surgery may be slightly overestimated as death was censored as a noninformative factor rather than as a competing risk. The likelihood of TKR 2 years after a structured education and exercise therapy program was obtained from an RCT of 100 participants . We assumed a consistent rate of TKR in the second and subsequent years following the structured education and exercise therapy program . We also assumed that program effectiveness in delaying or avoiding TKR was similar across health states and age and sex strata. Data on race and ethnicity were not available. Uncertainty in this model input was assessed in a 1-way sensitivity analysis. Utilities Health-related quality-of-life utilities were calculated using published Australian EQ-5D-5L value sets. Baseline utilities for each health state were calculated from preoperative EQ-5D-5L scores for individuals undergoing TKR (eTable 9 in ). Postintervention utilities for TKR were calculated using AOANJRR 6-month postoperative EQ-5D-5L scores (eTable 10 in ). To account for higher baseline utilities in GLA:D Australia Registry patients, we calculated the incremental utility and disutility based on the transition between health states for education and exercise therapy (eTable 11 in ). Based on data for 268 individuals undergoing revision TKR in the AOANJRR, a disutility of 0.28 was applied in the cycle prior to TKR revision and a disutility of 0.10 for all subsequent cycles (an SE of 25% was assumed). Costs The model estimated only direct costs from a health system perspective; all costs are presented in 2022 Australian dollars and converted into US dollars ($0.6947 for A$1 ) . Primary TKR cost in public hospitals was calculated using the weighted mean of costs for minor and major complexity procedures. Primary TKR cost in private hospitals was obtained from the websites of the 3 largest private health insurers and averaged. , , The mean cost of primary TKR, adjusting for the proportion of procedures performed in public and private hospitals, was A$24 607 ($17 095). Given no published data on revision TKR costs in private hospitals, we added 20% to the revision TKR cost in public hospitals based on expert clinician advice (I.A.H. and R.d.S.), with the mean cost of revision TKR estimated at A$43 125 ($29 959). We estimated the cost of a 12-week structured education and exercise therapy program based on earlier methods . While a 6-week program has been implemented in Australia (estimated cost of A$1000 [$695]), we chose to incorporate the costs of a 12-week program in line with the original trial. We did not incorporate any other health care costs outside the base case interventions as no difference in year 2 costs were observed in the RCT by Skou et al. This assumption was tested in the 1-way sensitivity analysis by varying these costs by A$500 ($347) annually for 5 years after primary TKR or education and exercise therapy. Statistical Analysis A cost-utility analysis was performed to calculate lifetime incremental costs and QALYs (number of life-years multiplied by utility value). As we anticipated that costs and QALYs would be lower after a structured education and exercise therapy program, we calculated the incremental net monetary benefit (INMB) with an incremental cost-effectiveness ratio (ICER) threshold of A$28 033 ($19 475) per QALY gained. This threshold is based on empirical estimates of opportunity costs for decisions to fund new health technologies. A positive INMB value indicates that a structured education and exercise therapy program is cost-effective (eg, cost savings are greater than the QALY losses), whereas a negative INMB value indicates that usual care is cost-effective (eg, cost savings do not outweigh the QALY losses). Subgroup analyses were conducted for each baseline health state, age and sex stratum, and hospital type (public vs private). A 1-way deterministic sensitivity analysis was performed on all model inputs to ascertain the outcomes of input uncertainty and the robustness of model estimates. Probabilistic sensitivity analysis was performed by running 1000 Monte Carlo simulations to obtain 95% uncertainty intervals for INMB. The results are presented on a cost-effectiveness plane and cost-effectiveness acceptability curve across a range of ICER thresholds. All analyses were performed using Microsoft Excel, version 16.87.
This economic evaluation is reported according to the 2022 Consolidated Health Economic Evaluation Reporting Standards ( CHEERS ) checklist. Patient consent was obtained for the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) Patient-Reported Outcomes Program and GLA:D Australia Registry. Ethical approval was obtained through the Monash University Human Research Ethics Committee to access these deidentified data. We developed a life table model in combination with a Markov model to compare a structured education and exercise therapy program with the option for future TKR vs TKR in the first year (usual care). A Markov model uses mean participant data to replicate the clinical context where individuals can transition between different health states. The lifetime model, designed using annual cycles, comprised 4 health states: no or mild pain, moderate pain, severe or extreme pain, and dead. The 3 alive health states were solely defined by participant responses to the pain domain within the 5-level EuroQuol-5 Dimension (EQ-5D-5L) instrument. Individuals start in 1 of 3 alive health states, with dead as the absorbing health state. Half-cycle corrections were made, with an annual discount rate of 5% applied to costs and outcomes. Model inputs, their sources, and parameter distribution are summarized in and eTables 1 to 11 in . , , , , , , , The model was built using Microsoft Excel, version 16.87 (Microsoft Corporation).
The model population comprised adults aged 45 to 84 years who would otherwise undergo TKR in Australia (eligible patients), excluding those undergoing partial knee replacements, as they are infrequently performed. To calculate the size of our hypothetical cohort, we obtained age- and sex-specific rates of TKR for osteoarthritis in 2020-2021 and 2022 Australian population data ( ; eTable 1 in ). We estimated that 61 394 Australians underwent primary TKR in 2022. Individuals were categorized into health states based on the EQ-5D-5L pain domain. The baseline proportion of individuals in each health state was calculated from individual-level patient-reported outcome measures from the AOANJRR. Preoperative patient-reported outcome measures have been collected since mid-2018, with data available for 73.5% of procedures. For this study, data were obtained for 9889 individuals who underwent primary TKR for osteoarthritis from August 7, 2018, to December 31, 2021 (eTable 2 in ). The baseline proportions of individuals in each health state based on age and sex strata are provided in eTable 3 in . All-cause mortality rates were derived from Australian population life tables (eTable 4 in ). Mortality risk following TKR was adjusted given evidence of fewer deaths in the first 8 to 9 years following TKR and more deaths after 12 years (eTable 5 in ). As osteoarthritis is a degenerative disease that may influence multiple joints (contralateral knee, hip, or shoulder), we took a conservative approach and assumed that the rate of contralateral TKR surgery or replacement surgery at other joints would not be influenced by the intervention.
Transition probabilities following TKR were derived from AOANJRR data, stratified by age and sex (eTable 6 in ). The AOANJRR collects postoperative outcomes at 6 months as most clinical improvement occurs during this period. Transition probabilities following education and exercise therapy were obtained from the GLA:D Australia Registry at 12 months (eTable 7 in ). As this database includes all individuals with osteoarthritis who participated in the GLA:D program, we restricted our analysis to those who were (1) on a waiting list for a joint replacement or surgical opinion, (2) reported that they were in so much trouble and pain that they wanted surgery, or (3) had a Knee Injury and Osteoarthritis Outcome Score-12 summary score 54.6 or less. The Knee Injury and Osteoarthritis Outcome Score-12 summary score threshold of 54.6 aligns with the preoperative score plus 1 SD from 4010 individuals who underwent primary TKR for osteoarthritis in Australia. The likelihood of revision surgery, by age and sex, was obtained from the 2022 AOANJRR annual report (eTable 8 in ). Likelihood of revision surgery may be slightly overestimated as death was censored as a noninformative factor rather than as a competing risk. The likelihood of TKR 2 years after a structured education and exercise therapy program was obtained from an RCT of 100 participants . We assumed a consistent rate of TKR in the second and subsequent years following the structured education and exercise therapy program . We also assumed that program effectiveness in delaying or avoiding TKR was similar across health states and age and sex strata. Data on race and ethnicity were not available. Uncertainty in this model input was assessed in a 1-way sensitivity analysis.
Health-related quality-of-life utilities were calculated using published Australian EQ-5D-5L value sets. Baseline utilities for each health state were calculated from preoperative EQ-5D-5L scores for individuals undergoing TKR (eTable 9 in ). Postintervention utilities for TKR were calculated using AOANJRR 6-month postoperative EQ-5D-5L scores (eTable 10 in ). To account for higher baseline utilities in GLA:D Australia Registry patients, we calculated the incremental utility and disutility based on the transition between health states for education and exercise therapy (eTable 11 in ). Based on data for 268 individuals undergoing revision TKR in the AOANJRR, a disutility of 0.28 was applied in the cycle prior to TKR revision and a disutility of 0.10 for all subsequent cycles (an SE of 25% was assumed).
The model estimated only direct costs from a health system perspective; all costs are presented in 2022 Australian dollars and converted into US dollars ($0.6947 for A$1 ) . Primary TKR cost in public hospitals was calculated using the weighted mean of costs for minor and major complexity procedures. Primary TKR cost in private hospitals was obtained from the websites of the 3 largest private health insurers and averaged. , , The mean cost of primary TKR, adjusting for the proportion of procedures performed in public and private hospitals, was A$24 607 ($17 095). Given no published data on revision TKR costs in private hospitals, we added 20% to the revision TKR cost in public hospitals based on expert clinician advice (I.A.H. and R.d.S.), with the mean cost of revision TKR estimated at A$43 125 ($29 959). We estimated the cost of a 12-week structured education and exercise therapy program based on earlier methods . While a 6-week program has been implemented in Australia (estimated cost of A$1000 [$695]), we chose to incorporate the costs of a 12-week program in line with the original trial. We did not incorporate any other health care costs outside the base case interventions as no difference in year 2 costs were observed in the RCT by Skou et al. This assumption was tested in the 1-way sensitivity analysis by varying these costs by A$500 ($347) annually for 5 years after primary TKR or education and exercise therapy.
A cost-utility analysis was performed to calculate lifetime incremental costs and QALYs (number of life-years multiplied by utility value). As we anticipated that costs and QALYs would be lower after a structured education and exercise therapy program, we calculated the incremental net monetary benefit (INMB) with an incremental cost-effectiveness ratio (ICER) threshold of A$28 033 ($19 475) per QALY gained. This threshold is based on empirical estimates of opportunity costs for decisions to fund new health technologies. A positive INMB value indicates that a structured education and exercise therapy program is cost-effective (eg, cost savings are greater than the QALY losses), whereas a negative INMB value indicates that usual care is cost-effective (eg, cost savings do not outweigh the QALY losses). Subgroup analyses were conducted for each baseline health state, age and sex stratum, and hospital type (public vs private). A 1-way deterministic sensitivity analysis was performed on all model inputs to ascertain the outcomes of input uncertainty and the robustness of model estimates. Probabilistic sensitivity analysis was performed by running 1000 Monte Carlo simulations to obtain 95% uncertainty intervals for INMB. The results are presented on a cost-effectiveness plane and cost-effectiveness acceptability curve across a range of ICER thresholds. All analyses were performed using Microsoft Excel, version 16.87.
Base Case The hypothetical cohort included 61 394 adults aged 45 to 84 years (53.9% females and 46.1% males; 93.6% aged ≥55 years). Implementation of a national education and exercise therapy program for knee osteoarthritis prior to TKR was estimated to produce a cost savings of A$489 307 942 ($339 922 227), or A$7970 ($5537) per person, over the lifetime horizon compared with usual care . This estimate was derived from 19.5% individuals avoiding primary TKR and 14 418 fewer TKRs (11 995 fewer primary TKRs and 2423 fewer revision TKRs). There was no difference in total life-years between intervention and comparator. The education and exercise therapy program resulted in 0.43 fewer QALYs per person compared with usual care over the lifetime horizon. The INMB of −A$4090 (−$2841) indicated that a structured education and exercise therapy program would not be cost-effective over the lifetime horizon . Structured education and exercise therapy was only cost-effective for the first 9 years of the time horizon ( ; eTable 12 in ). Subgroup Analyses Results from subgroup analyses are presented in . Cost savings were similar when evaluating each baseline health state separately. When restricted to individuals with no or mild pain at baseline, the structured education and exercise therapy program was cost-effective (INMB, A$11 [$8]) as between-group incremental QALY losses were lower compared with the base case (−0.29 vs −0.43). The intervention was not cost-effective over the lifetime horizon when restricted to individuals with moderate pain or severe or extreme pain at baseline (INMB, −A$4156 [−$2887] and −A$6788 [−$4716], respectively). The INMB for the intervention increased with older age categories (45-54 years, −A$17 326 [−$12 036]; 55-64 years −A$4996 [−$3471]; 65-74 years, −A$3161 [−$2196]; 75-84 years, −A$1166 [−$810]), yet usual care remained the cost-effective option for all age groups . Younger age groups accumulated greater QALY gains from usual care (TKR in first year) over more years, resulting in greater incremental QALY losses compared with the structured education and exercise therapy. The greatest cost savings were observed in the 75- to 84-year age group as individuals died before requiring TKR. The INMB was higher for males than females (−A$1938 [−$1346] vs −A$5934 [−$4122], respectively), likely driven by more males having no or mild pain at baseline (eTable 2 in ). Usual care remained the cost-effective option in both public and private hospitals. However, a larger negative INMB was evident for public hospitals vs private hospitals (−A$5414 [−$3761] vs −A$3327 [−$2311], respectively) due to smaller between-group QALY losses in private hospitals through a greater proportion of individuals who had no or slight pain at baseline. Deterministic Sensitivity Analysis One-way sensitivity analysis results are presented in the eFigure in . While uncertainty in health-related quality of life following structured education and exercise therapy greatly influenced cost-effectiveness, the model was robust to all other model inputs. Probabilistic Sensitivity Analysis The cost-effectiveness plane shows that education and exercise therapy were associated with lower costs and smaller QALY gains in 99.2% of simulations, with a mean INMB of −A$4897 (95% CI, −A$5229 to −A$4564) (−$3402; 95% CI, −$3633 to −$3171). The cost-effectiveness acceptability curve shows that the nonsurgical management program was cost-effective in 18.6% of simulations at an ICER threshold of A$28 033 per QALY gained.
The hypothetical cohort included 61 394 adults aged 45 to 84 years (53.9% females and 46.1% males; 93.6% aged ≥55 years). Implementation of a national education and exercise therapy program for knee osteoarthritis prior to TKR was estimated to produce a cost savings of A$489 307 942 ($339 922 227), or A$7970 ($5537) per person, over the lifetime horizon compared with usual care . This estimate was derived from 19.5% individuals avoiding primary TKR and 14 418 fewer TKRs (11 995 fewer primary TKRs and 2423 fewer revision TKRs). There was no difference in total life-years between intervention and comparator. The education and exercise therapy program resulted in 0.43 fewer QALYs per person compared with usual care over the lifetime horizon. The INMB of −A$4090 (−$2841) indicated that a structured education and exercise therapy program would not be cost-effective over the lifetime horizon . Structured education and exercise therapy was only cost-effective for the first 9 years of the time horizon ( ; eTable 12 in ).
Results from subgroup analyses are presented in . Cost savings were similar when evaluating each baseline health state separately. When restricted to individuals with no or mild pain at baseline, the structured education and exercise therapy program was cost-effective (INMB, A$11 [$8]) as between-group incremental QALY losses were lower compared with the base case (−0.29 vs −0.43). The intervention was not cost-effective over the lifetime horizon when restricted to individuals with moderate pain or severe or extreme pain at baseline (INMB, −A$4156 [−$2887] and −A$6788 [−$4716], respectively). The INMB for the intervention increased with older age categories (45-54 years, −A$17 326 [−$12 036]; 55-64 years −A$4996 [−$3471]; 65-74 years, −A$3161 [−$2196]; 75-84 years, −A$1166 [−$810]), yet usual care remained the cost-effective option for all age groups . Younger age groups accumulated greater QALY gains from usual care (TKR in first year) over more years, resulting in greater incremental QALY losses compared with the structured education and exercise therapy. The greatest cost savings were observed in the 75- to 84-year age group as individuals died before requiring TKR. The INMB was higher for males than females (−A$1938 [−$1346] vs −A$5934 [−$4122], respectively), likely driven by more males having no or mild pain at baseline (eTable 2 in ). Usual care remained the cost-effective option in both public and private hospitals. However, a larger negative INMB was evident for public hospitals vs private hospitals (−A$5414 [−$3761] vs −A$3327 [−$2311], respectively) due to smaller between-group QALY losses in private hospitals through a greater proportion of individuals who had no or slight pain at baseline.
One-way sensitivity analysis results are presented in the eFigure in . While uncertainty in health-related quality of life following structured education and exercise therapy greatly influenced cost-effectiveness, the model was robust to all other model inputs.
The cost-effectiveness plane shows that education and exercise therapy were associated with lower costs and smaller QALY gains in 99.2% of simulations, with a mean INMB of −A$4897 (95% CI, −A$5229 to −A$4564) (−$3402; 95% CI, −$3633 to −$3171). The cost-effectiveness acceptability curve shows that the nonsurgical management program was cost-effective in 18.6% of simulations at an ICER threshold of A$28 033 per QALY gained.
In this economic analysis, we found that a national structured education and exercise therapy program with the option for future TKR would produce substantial cost savings but would not be cost-effective over the lifetime horizon. Our results suggest that structured education and exercise therapy is cost-effective for the first 9 years and over the lifetime in individuals with low pain levels at baseline. These findings highlight the need for considered approaches that improve the sustainability of care and ensure that the right patient gets the right care at the right time. Our results are consistent with previous studies despite important methodological differences. , , , , Previous models comparing TKR and nonsurgical management did not incorporate the likelihood of individuals undergoing TKR following nonsurgical treatment. , , This limitation is important given that the 2 interventions are not directly substitutable in clinical practice. Three previously published models , , assessed the cost-effectiveness of delaying TKR with nonsurgical management but did not consider that TKR may not be needed in a subset of the population. , Our analysis, based on 2-year RCT outcomes, explicitly addresses these methodological issues. Our results contrast somewhat with those of the RCT, as the authors reported that more intensive nonsurgical care (still centered on education and exercise therapy) was cost-effective. These contrasting findings may reflect different time horizons, as our study similarly showed that education and exercise therapy may be cost-effective up to 9 years with potentially enhanced outcomes associated with more intensive nonsurgical care. Furthermore, education and exercise therapy provided in the RCT was found to be cost-effective only when adjusting for baseline participant demographics (age, sex, baseline costs, and QALYs). Our study represents an advance over earlier trial-based analyses as we constructed a cohort using national registry data that accurately represents the demographics of patients who undergo TKR in Australia. Considering the opportunity cost of freeing up financial resources that might be invested elsewhere is important. Observed QALY losses from diverting individuals with osteoarthritis to education and exercise therapy programs prior to TKR could be ameliorated by investing in prevention programs that decrease the prevalence and/or severity of knee osteoarthritis and need for future TKR (eg, weight reduction, knee injury prevention). , Examples of successful national implementation of such population-level prevention programs are lacking. Furthermore, early savings could be invested to target longer-term behavior change known to influence osteoarthritis outcomes, including quality of life. Education and exercise therapy have a limited influence on increasing physical activity participation or body weight. , Recent research has indicated that adding a specific dietary intervention to exercise therapy may result in greater improvement in quality of life, and weight reduction has been associated with a reduced incidence of TKR. Prioritizing nonsurgical management of knee osteoarthritis to patients for whom TKR is inappropriate may be a more efficient strategy as TKR is highly effective when used appropriately. Various TKR appropriateness tools, including those that consider preoperative pain, have been developed, , , , but the cost-effectiveness of implementing them as part of a care pathway has not been assessed. We were unable to identify the proportion of inappropriate TKR procedures within the current model, and the no or mild pain subgroup should not be conflated with inappropriate procedures. Future efforts are needed to test the cost-effectiveness of valid and reliable appropriateness criteria that enable the right patient to receive the right care at the right time. Public acceptance of nonsurgical management programs for osteoarthritis such as GLA:D, while growing in Australia and internationally, , is unclear at this end point of the care pathway. Previous studies have revealed that some individuals may be willing to accept smaller benefits from nonsurgical management to avoid undergoing TKR. , Similarly, Hawker et al observed that 13% to 21% of Canadians who received a TKR were not ready or willing to undergo TKR, which was associated with poor outcomes. While the generalizability of this finding is unclear, public acceptance and cost-effectiveness may be improved by targeting nonsurgical management to individuals willing to delay or avoid TKR and accept smaller quality-of-life benefits than might be gained from surgery. Limitations This study has several limitations. We recognize that the cost-effectiveness of a structured education and exercise therapy program may have been underestimated by not incorporating other benefits. Waiting for TKR may be associated with quality-of-life deterioration and the substantial health care investment needed to meet future demand. Not incorporating the 30% of individuals waiting more than 1 year for TKR in Australian public hospitals may overestimate the cost-effectiveness of TKR. Surgery is also associated with substantial work and productivity losses in the short term, which have not been incorporated into cost-effectiveness estimates. It is unclear whether education and exercise therapy programs such as GLA:D are positively associated with return to work and productivity. Further analyses from a societal perspective (eg, examining employment absenteeism, presenteeism, early retirement, and caregiver costs) may be warranted. Our cohort may not be representative of patients with knee osteoarthritis in other health care systems (eg, primary care) or other countries. Despite restricting analysis of the GLA:D registry to individuals considered eligible for TKR, it is unclear whether this population is similar (given unmeasured factors) to those who undergo surgery. We did not have access to imaging data and recognize that radiographic changes can be used for TKR decision making. , , Furthermore, individuals in the GLA:D Australia Registry reported higher quality of life at baseline compared with those in the AOANJRR. We accounted for this baseline difference as outlined in the Methods. We acknowledge current uncertainty related to the outcomes of nonsurgical management in TKR avoidance, especially over the long term. The rate of TKR avoidance was derived from 2-year follow-up of the only published RCT, and imprecision in this estimate may substantially influence the outcomes. Furthermore, the original trial excluded individuals with severe pain (>60 mm on a 100-mm visual analog pain scale), limiting the applicability of this model input for those with severe or extreme pain at baseline. We assumed a 12-week structured education and exercise therapy program based on the original trial , but it is unclear what the most efficient version of this program may be, as a 6-week program has been implemented in Australia with positive outcomes and therapeutic exercise may only have a small overall influence on pain and physical function. Finally, the rate of TKR following education and exercise therapy was applied equally across health states and age and sex strata given a lack of stratified data on progression to TKR.
This study has several limitations. We recognize that the cost-effectiveness of a structured education and exercise therapy program may have been underestimated by not incorporating other benefits. Waiting for TKR may be associated with quality-of-life deterioration and the substantial health care investment needed to meet future demand. Not incorporating the 30% of individuals waiting more than 1 year for TKR in Australian public hospitals may overestimate the cost-effectiveness of TKR. Surgery is also associated with substantial work and productivity losses in the short term, which have not been incorporated into cost-effectiveness estimates. It is unclear whether education and exercise therapy programs such as GLA:D are positively associated with return to work and productivity. Further analyses from a societal perspective (eg, examining employment absenteeism, presenteeism, early retirement, and caregiver costs) may be warranted. Our cohort may not be representative of patients with knee osteoarthritis in other health care systems (eg, primary care) or other countries. Despite restricting analysis of the GLA:D registry to individuals considered eligible for TKR, it is unclear whether this population is similar (given unmeasured factors) to those who undergo surgery. We did not have access to imaging data and recognize that radiographic changes can be used for TKR decision making. , , Furthermore, individuals in the GLA:D Australia Registry reported higher quality of life at baseline compared with those in the AOANJRR. We accounted for this baseline difference as outlined in the Methods. We acknowledge current uncertainty related to the outcomes of nonsurgical management in TKR avoidance, especially over the long term. The rate of TKR avoidance was derived from 2-year follow-up of the only published RCT, and imprecision in this estimate may substantially influence the outcomes. Furthermore, the original trial excluded individuals with severe pain (>60 mm on a 100-mm visual analog pain scale), limiting the applicability of this model input for those with severe or extreme pain at baseline. We assumed a 12-week structured education and exercise therapy program based on the original trial , but it is unclear what the most efficient version of this program may be, as a 6-week program has been implemented in Australia with positive outcomes and therapeutic exercise may only have a small overall influence on pain and physical function. Finally, the rate of TKR following education and exercise therapy was applied equally across health states and age and sex strata given a lack of stratified data on progression to TKR.
In this economic analysis, the findings suggest that structured education and exercise therapy may be cost-effective in the first 9 years but not over a lifetime horizon. The cost-effectiveness of these programs might be improved by targeting implementation to specific patient subgroups, particularly individuals with lower preoperative pain levels. These findings highlight the need to prioritize tools that identify individuals for whom nonsurgical treatment is the preferred strategy and suggest that such tools may deliver substantial cost savings and improve timely access and the quality of care for those with knee osteoarthritis.
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An IQ consortium analysis of starting dose selection for oncology small molecule first-in-patient trials suggests an alternative NOAEL-based method can be safe while reducing time to the recommended phase 2 dose | d723832b-118e-4c5b-934a-2b655666d99e | 10638197 | Internal Medicine[mh] | The first-in-patient (FIP) nonclinical safety package is designed to enable an initial risk assessment of the drug candidate, identify appropriate clinical safety monitoring, and define the recommended starting dose (RSD) for the FIP clinical trial. The FIP trial for oncology agents is generally conducted in patients with advanced malignancies, which necessitates careful selection of the RSD to be sufficiently safe, while also potentially providing therapeutic benefit to late-stage cancer patients with limited therapeutic options that enrolled in these trials. Therefore, an optimal balance between safety and clinical benefit is desired, such that the number of patients in initial and subsequent cohorts exposed to potential sub-optimal therapeutic doses of the investigational agent is minimized . Currently, the RSD approach for small molecule oncology agents is detailed in the International Council for Harmonisation (ICH) S9 guidance where the FIP start dose is based on one-tenth the severely toxic dose in 10% (STD 10 ) of rodents or one-sixth the highest non-severely toxic dose (HNSTD) in non-rodents, as appropriate or sensitive, with interspecies scaling. DeGeorge et al. suggested a slightly different method, in which one-tenth STD 10 in rodents can define the RSD as long as one-tenth STD 10 in rodents does not cause serious irreversible toxicity in a non-rodent species . If one-tenth of the rodent STD 10 causes serious irreversible toxicities in non-rodents, then one-sixth of the non-rodent HNSTD is used to define the RSD. These approaches have largely met the development needs of chemotherapeutics with cytotoxic mechanisms. However, since the approval of ICH S9, there has been a shift in development to novel molecularly targeted agents . Molecularly targeted agents (MTA) are based on different mechanisms of action, and these targeted therapies may sometimes require alternative approaches to identify appropriate dose ranges . Therefore, it is prudent to re-evaluate how the RSD is derived for small molecule oncology agents to ensure we continue to optimize oncology clinical trial starting dose justification for the appropriate balance between risk and potential therapeutic benefit. Selection of starting dose for clinical trials depends on population (healthy volunteers or patients) and mechanism of action. Prior scientific publications have reviewed the potential approaches to determine the FIP starting dose, with authors generally focusing on the previously described toxicology-based STD 10 and HNSTD methods . Use of these methods are not considered appropriate for immune activating drugs for oncology indications where a minimum anticipated biological effect level (MABEL) approach is recommended, and also do not account for the potential nonclinical toxicity profile of novel MTA, when a toxicity-based approach for starting dose selection may be too conservative . An assessment of a no-observed-adverse-effect level (NOAEL) defined in the in vivo toxicology studies to determine the clinical starting dose is typically used in non-cancer therapeutic indications only and includes the addition of safety factors . A review of 59 approved small molecule oncology MTAs showed 58 of these FIP trials had a starting dose lower than the maximum tolerated dose (MTD) in oncology patients, which indicated starting doses based on nonclinical toxicology data were safe. The majority of clinical trials used a 3 + 3 design approach, with a median of four dose escalation steps required to reach MTD or recommended Phase 2 dose (RP2D) from the starting dose. An earlier review of 81 FIP cancer trials identified from the Scopus abstract database indicated that the derivation of the starting dose was most often based on a MTD from toxicology studies and/or considering the type of nonclinical toxicity . This analysis showed that starting doses for MTAs were based on rodent and non-rodent data in similar proportions. Overall, starting doses selected using such a toxicology-based method were safe in most cases and did not exceed the human MTD in 96% ( n = 81) of Phase I clinical trials of MTAs reviewed. Since the FIP starting dose for oncology therapeutics is now based on the ICH S9 guidance (or similar guidance from ), which was generally modeled based on experience with cytotoxic chemotherapeutic agents and is not always appropriate for current oncology products in clinical development, an alternative approach was evaluated. For this exercise, blinded data on 92 small molecule oncology compounds from 12 pharmaceutical companies that had completed dose escalation in cancer patients were gathered with the goal of investigating if a different method for selecting the RSD based on a species justified NOAEL without an added safety factor could be used to select a starting dose that was both tolerated and enabled a reduction in dose escalation cohorts and, thus, reducing the number of cancer patients exposed to potentially sub-therapeutic doses. This approach is consistent with recent FDA guidance on severely debilitating life-threatening hematological diseases where starting dose selection can be justified using HNSTD, STD10, or NOAEL without defining safety factors .
An IQ DruSafe Working Group was formed to evaluate an alternative approach to setting starting doses in oncology FIP clinical studies. The scope was small molecule marketed MTA oncology drugs or those in clinical development that had reached an MTD/RP2D. A database was created capturing 50 fields of information for each of the 92 anonymized compounds provided by 12 pharmaceutical companies (AbbVie, AstraZeneca, Amgen, Boehringer Ingelheim, Bristol Meyers Squibb, Eisai, Eli Lilly, GlaxoSmithKline, Janssen, Merck, Novartis, and Pfizer). An additional 9 fields of data were collected for the alternative starting dose scenario exercise, for a total of 5428 data points. The information collected related to the compound attributes, and nonclinical and clinical safety data. These included general target class, nonclinical toxicology species, and toxicology or clinical study information. Nonclinical and clinical information included doses, exposures (AUC), dose-limiting target toxicities, NOAEL, STD 10 , HNSTD, MTD/RP2D, dose regimens, and number of clinical dose escalation cohorts needed to reach MTD/RP2D. Many database fields used drop-down menus with fixed responses to facilitate downstream analysis. To protect the anonymity of the company data, specific compound identifiers and molecular targets were not included. The additional 9 fields collected data for the alternative starting dose exercise using either actual study NOAELs or a study-justified NOAEL (toxicities not considered dose-limiting in cancer patients, e.g., if testes toxicity in rodent or non-rodent did not support a NOAEL determination) to calculate a NOAEL-based starting dose without an additional safety factor. The allometrically scaled NOAEL-based starting dose without a safety factor was compared to the reported MTD/RP2D to assess whether it exceeded that dose. If the alternative starting dose was below the MTD/RP2D, the number of dose escalation cohorts needed to reach the established MTD/RP2D was estimated (assuming a similar approach to dose escalation, e.g., dose doubling) and the difference between the number of actual and alternative escalation cohorts calculated. The database was curated for entries exhibiting obvious errors or lacking data necessary to meet basic criteria, corrections requested by IQ Secretariat to preserve data confidentiality, and the data exported to Microsoft Excel for analysis (Supplemental Data).
Overview The companies were asked to characterize the target class for the compound (Table ). Comparing the results of the alternative assessment to the target class would assist in identifying mechanistic classes that carried higher risks for this approach. With regard to the MTA nonclinical toxicology FIP-enabling packages, 74% were conducted after the implementation of ICH S9. The dosing route was oral in 86% of cases for both nonclinical and clinical studies. The majority of nonclinical dosing regimens were daily for 4 weeks (75% of rodent and 74% of non-rodent studies), corresponding to continuous daily clinical dosing (59%). The distribution of compounds that identified a NOAEL, STD10, or HNSTD is summarized in Table along with the number of compounds for which the NOAEL was the same dose or less than the STD10 or HNSTD dose. Safety of alternative NOAEL-based method and impact on clinical dose escalation Of the 92 compounds, the NOAEL-based evaluation was possible for 66 (72%) compounds resulting in higher alternative starting doses in 60 of these cases (91%), while 6 cases had a lower or similar starting dose than was reported. Of those 66 cases evaluable, 54 (82%) of the NOAEL-based starting doses would have been tolerated in the dose escalation portion of the clinical trial, while 12 (18%) would have exceeded the MTD/RP2D (Fig. a) and was independent of species used for the evaluation. For the 60 of 66 cases with higher alternative starting doses, 49 (82%) would not have exceeded the MTD/RP2D. Of the 42 evaluated compounds that originally used HNSTD or STD 10 to set the starting dose, 39 (93%) would have been tolerated if the NOAEL-based calculation was used, while 15 (63%) of the 24 that used other starting dose methods would have been tolerated using the NOAEL-based method. The ratio of the alternative NOAEL-based starting dose to the MTD/RP2D revealed that 4 of those compounds that would have exceeded the MTD/RP2D had alternative starting doses that were within twofold of the MTD/RP2D, and an additional 4 compounds were within threefold (Fig. b). Three further compounds were between 4.5 to sixfold and 1 molecule was ~ 20-fold the MTD/RP2D. The compound with the highest alternative starting dose relative to the MTD/RP2D and a compound with a 4.5-fold ratio were both immune-oncology (IO) agents delivered by the intra-tumoral (IT) route. Since the IT route is often chosen to limit systemic exposure due to severe tolerability concerns, poor ADME properties, or practical restrictions on the dose volumes from that route, these types of compounds may not be suitable for the NOAEL-based approach and often use the MABEL approach. The only other notable trends with respect to target class for programs where the alternative starting dose exceeded the MTD/RP2D were the absence of cancer mutation and hormone therapy compounds (although these represented small percentages of the total entries), the inclusion of 1 of the 3 total proteasome inhibition compounds, and the inclusion of the only metabolic drug class compound. A review of the clinical DLTs associated with the cases where the NOAEL-based starting dose exceeded the MTD/RP2D revealed that for the compound with the highest ratio of alternative starting dose to clinical MTD/RP2D, the DLT was “not determined”, indicating that clinical safety may not have been the driver for selection of the RP2D. This may be consistent with the technical restrictions associated with the IT route of administration. Other primary DLTs listed were “liver”, “bone marrow/hematological”, and “systemic toxicity (i.e., systemic inflammation)”, with 2 compounds each, and 1 “cardiovascular-hemodynamic effect.” The remaining primary DLTs were “not determined”, “not disclosed”, or “clinical-other.” Among secondary DLTs, there were 3 “gastrointestinal” and 2 “clinical-emesis.” The nonclinical toxicity of the 12 compounds that would have exceeded the MTD/RP2D showed that none had DLTs that were non-reversible and only 1 had a DLT that was not monitorable. Analysis of the entire dataset showed that the average number of clinical cohorts required to reach the MTD/RP2D was 5.3 with a median of 5 cohorts. The average reduction in cohorts using the NOAEL-based alternative starting dose was 2.3 (43%) with a median reduction of 2 cohorts (Fig. ). The principal factors affecting the time/cohort are presented in Supplemental Fig. . As a result, taking the reduced number of cohorts needed to reach the MTD/RP2D using the NOAEL-based approach of 2.3 and the average cohort duration of 9.3 weeks, illustrates a potential time savings of 21.4 weeks in the dose escalation phase. Analysis of actual starting dose methods used As shown in Table , the majority of entries in the database used the STD 10 or HNSTD method of starting dose calculations. As expected, the median safety factors in those cases, 12 × and 8.1 × (highlighted fields), respectively, were similar to the ICH S9/DeGeorge specified safety factors of 10 × and 6 ×, respectively. The calculated number of escalations was reduced using a NOAEL-based method with no safety factor was consistent across all of the groups regardless of which method the sponsor used. In cases where a standard starting dose method (STD 10 or HNSTD) was used, the application of an alternative NOAEL-based starting dose with no safety factor would have resulted in a tolerated starting dose in > 90% of cases. In contrast, the success of using the alternative NOAEL method with no safety factor was lower (55–75%) in cases where the sponsor used a less common method such as NOAEL (with a safety factor), MABEL, or another alternative. This indicates that additional judgement should be used in determining which method to use; however, in the absence of a reason to be more conservative, a NOAEL method with no safety factor is highly likely to result in a tolerated clinical starting dose and fewer dose escalations prior to reaching RP2D. Analysis of nonclinical NOAEL The determination of a NOAEL in toxicology studies conducted to support the clinical dosing of new drugs to oncology patients is not a required endpoint in ICH S9 . However, of the 92 products submitted for the survey, a NOAEL was determined for the majority of rodent (55) and non-rodent (63) toxicology studies (Table ). In addition, for the purposes of this exercise, companies were able to justify a NOAEL based on the survey criteria (see Methods) for an additional 13 rodent and 11 non-rodent studies. For 22 compounds, a NOAEL was not determined for the rodent and non-rodent studies; of these, 1 justified a rodent NOAEL and 4 justified a non-rodent NOAEL for the alternative human starting dose. When assessing criteria for justifying the starting dose in clinical studies, it is important to understand species sensitivity to the compound with the more sensitive species most often being used. In preparing to conduct the analysis for using the alternative NOAEL approach, the submitted products were evaluated for how often a NOAEL was determined or justified in both the rodent and non-rodent toxicity studies. Using the criteria outlined for assessing an alternative NOAEL-based starting dose, 66 (72%) of the products were suitable for the exercise. There were 26 products that did not meet the criteria for the NOAEL-based starting dose assessment (Table ). Of these 26 products, NOAELs were not identified in both species for 17 products; while 23 rodent studies and 20 non-rodent studies did not report a NOAEL. The target organ toxicity findings that precluded inclusion in the assessment are presented in Table . Gastrointestinal, bone marrow or hematologic, renal and skin toxicity were the most common findings for exclusion or absence of a NOAEL determination. For these compounds, the most common observed clinical DLTs were gastrointestinal, bone marrow or hematologic, and skin. It is interesting that cardiac (hemodynamic, electrophysiological, or structural) and hepatic toxicities were not prominent causes given these tissues are often reasons for attrition of non-oncology products . Translation of nonclinical to clinical dose-limiting toxicities The database collected primary and secondary DLTs from a prespecified lexicon for nonclinical toxicology species and human clinical trials. The lexicon differed slightly between the nonclinical species and clinical trials, which reflected differences in the types of evaluations performed in the studies (e.g., histopathological evaluation in nonclinical studies). The incidence of the DLTs (Table ) found the predominant rodent and non-rodent dose-limiting toxicities were GI and bone marrow/hematological; GI and bone marrow/hematological dose-limiting toxicities were also most common DLTs in patients. When secondary dose-limiting toxicities were determined, GI and bone marrow/hematological were also among the most prominent across species. Other notable dose-limiting toxicities in humans included skin, liver, and systemic toxicity (i.e., systemic inflammation). In rodent studies, reversibility of dose-limiting toxicities was demonstrated in 58% of cases, while 17% showed partial reversibility, and 10% were predicted to reverse. Only 5% had findings that did not or were predicted not to reverse. Likewise, in non-rodents, 63% of studies showed dose-limiting findings that reversed, 13% partially reversed, and 13% predicted to reverse, leaving only 1 case with irreversible findings. In addition, only 4% of cases demonstrated findings in either rodents or non-rodents deemed unmonitorable.
The companies were asked to characterize the target class for the compound (Table ). Comparing the results of the alternative assessment to the target class would assist in identifying mechanistic classes that carried higher risks for this approach. With regard to the MTA nonclinical toxicology FIP-enabling packages, 74% were conducted after the implementation of ICH S9. The dosing route was oral in 86% of cases for both nonclinical and clinical studies. The majority of nonclinical dosing regimens were daily for 4 weeks (75% of rodent and 74% of non-rodent studies), corresponding to continuous daily clinical dosing (59%). The distribution of compounds that identified a NOAEL, STD10, or HNSTD is summarized in Table along with the number of compounds for which the NOAEL was the same dose or less than the STD10 or HNSTD dose.
Of the 92 compounds, the NOAEL-based evaluation was possible for 66 (72%) compounds resulting in higher alternative starting doses in 60 of these cases (91%), while 6 cases had a lower or similar starting dose than was reported. Of those 66 cases evaluable, 54 (82%) of the NOAEL-based starting doses would have been tolerated in the dose escalation portion of the clinical trial, while 12 (18%) would have exceeded the MTD/RP2D (Fig. a) and was independent of species used for the evaluation. For the 60 of 66 cases with higher alternative starting doses, 49 (82%) would not have exceeded the MTD/RP2D. Of the 42 evaluated compounds that originally used HNSTD or STD 10 to set the starting dose, 39 (93%) would have been tolerated if the NOAEL-based calculation was used, while 15 (63%) of the 24 that used other starting dose methods would have been tolerated using the NOAEL-based method. The ratio of the alternative NOAEL-based starting dose to the MTD/RP2D revealed that 4 of those compounds that would have exceeded the MTD/RP2D had alternative starting doses that were within twofold of the MTD/RP2D, and an additional 4 compounds were within threefold (Fig. b). Three further compounds were between 4.5 to sixfold and 1 molecule was ~ 20-fold the MTD/RP2D. The compound with the highest alternative starting dose relative to the MTD/RP2D and a compound with a 4.5-fold ratio were both immune-oncology (IO) agents delivered by the intra-tumoral (IT) route. Since the IT route is often chosen to limit systemic exposure due to severe tolerability concerns, poor ADME properties, or practical restrictions on the dose volumes from that route, these types of compounds may not be suitable for the NOAEL-based approach and often use the MABEL approach. The only other notable trends with respect to target class for programs where the alternative starting dose exceeded the MTD/RP2D were the absence of cancer mutation and hormone therapy compounds (although these represented small percentages of the total entries), the inclusion of 1 of the 3 total proteasome inhibition compounds, and the inclusion of the only metabolic drug class compound. A review of the clinical DLTs associated with the cases where the NOAEL-based starting dose exceeded the MTD/RP2D revealed that for the compound with the highest ratio of alternative starting dose to clinical MTD/RP2D, the DLT was “not determined”, indicating that clinical safety may not have been the driver for selection of the RP2D. This may be consistent with the technical restrictions associated with the IT route of administration. Other primary DLTs listed were “liver”, “bone marrow/hematological”, and “systemic toxicity (i.e., systemic inflammation)”, with 2 compounds each, and 1 “cardiovascular-hemodynamic effect.” The remaining primary DLTs were “not determined”, “not disclosed”, or “clinical-other.” Among secondary DLTs, there were 3 “gastrointestinal” and 2 “clinical-emesis.” The nonclinical toxicity of the 12 compounds that would have exceeded the MTD/RP2D showed that none had DLTs that were non-reversible and only 1 had a DLT that was not monitorable. Analysis of the entire dataset showed that the average number of clinical cohorts required to reach the MTD/RP2D was 5.3 with a median of 5 cohorts. The average reduction in cohorts using the NOAEL-based alternative starting dose was 2.3 (43%) with a median reduction of 2 cohorts (Fig. ). The principal factors affecting the time/cohort are presented in Supplemental Fig. . As a result, taking the reduced number of cohorts needed to reach the MTD/RP2D using the NOAEL-based approach of 2.3 and the average cohort duration of 9.3 weeks, illustrates a potential time savings of 21.4 weeks in the dose escalation phase.
As shown in Table , the majority of entries in the database used the STD 10 or HNSTD method of starting dose calculations. As expected, the median safety factors in those cases, 12 × and 8.1 × (highlighted fields), respectively, were similar to the ICH S9/DeGeorge specified safety factors of 10 × and 6 ×, respectively. The calculated number of escalations was reduced using a NOAEL-based method with no safety factor was consistent across all of the groups regardless of which method the sponsor used. In cases where a standard starting dose method (STD 10 or HNSTD) was used, the application of an alternative NOAEL-based starting dose with no safety factor would have resulted in a tolerated starting dose in > 90% of cases. In contrast, the success of using the alternative NOAEL method with no safety factor was lower (55–75%) in cases where the sponsor used a less common method such as NOAEL (with a safety factor), MABEL, or another alternative. This indicates that additional judgement should be used in determining which method to use; however, in the absence of a reason to be more conservative, a NOAEL method with no safety factor is highly likely to result in a tolerated clinical starting dose and fewer dose escalations prior to reaching RP2D.
The determination of a NOAEL in toxicology studies conducted to support the clinical dosing of new drugs to oncology patients is not a required endpoint in ICH S9 . However, of the 92 products submitted for the survey, a NOAEL was determined for the majority of rodent (55) and non-rodent (63) toxicology studies (Table ). In addition, for the purposes of this exercise, companies were able to justify a NOAEL based on the survey criteria (see Methods) for an additional 13 rodent and 11 non-rodent studies. For 22 compounds, a NOAEL was not determined for the rodent and non-rodent studies; of these, 1 justified a rodent NOAEL and 4 justified a non-rodent NOAEL for the alternative human starting dose. When assessing criteria for justifying the starting dose in clinical studies, it is important to understand species sensitivity to the compound with the more sensitive species most often being used. In preparing to conduct the analysis for using the alternative NOAEL approach, the submitted products were evaluated for how often a NOAEL was determined or justified in both the rodent and non-rodent toxicity studies. Using the criteria outlined for assessing an alternative NOAEL-based starting dose, 66 (72%) of the products were suitable for the exercise. There were 26 products that did not meet the criteria for the NOAEL-based starting dose assessment (Table ). Of these 26 products, NOAELs were not identified in both species for 17 products; while 23 rodent studies and 20 non-rodent studies did not report a NOAEL. The target organ toxicity findings that precluded inclusion in the assessment are presented in Table . Gastrointestinal, bone marrow or hematologic, renal and skin toxicity were the most common findings for exclusion or absence of a NOAEL determination. For these compounds, the most common observed clinical DLTs were gastrointestinal, bone marrow or hematologic, and skin. It is interesting that cardiac (hemodynamic, electrophysiological, or structural) and hepatic toxicities were not prominent causes given these tissues are often reasons for attrition of non-oncology products .
The database collected primary and secondary DLTs from a prespecified lexicon for nonclinical toxicology species and human clinical trials. The lexicon differed slightly between the nonclinical species and clinical trials, which reflected differences in the types of evaluations performed in the studies (e.g., histopathological evaluation in nonclinical studies). The incidence of the DLTs (Table ) found the predominant rodent and non-rodent dose-limiting toxicities were GI and bone marrow/hematological; GI and bone marrow/hematological dose-limiting toxicities were also most common DLTs in patients. When secondary dose-limiting toxicities were determined, GI and bone marrow/hematological were also among the most prominent across species. Other notable dose-limiting toxicities in humans included skin, liver, and systemic toxicity (i.e., systemic inflammation). In rodent studies, reversibility of dose-limiting toxicities was demonstrated in 58% of cases, while 17% showed partial reversibility, and 10% were predicted to reverse. Only 5% had findings that did not or were predicted not to reverse. Likewise, in non-rodents, 63% of studies showed dose-limiting findings that reversed, 13% partially reversed, and 13% predicted to reverse, leaving only 1 case with irreversible findings. In addition, only 4% of cases demonstrated findings in either rodents or non-rodents deemed unmonitorable.
The primary goal of selecting a FIP starting dose for cancer patients should be to define a safe dose level that is sufficiently high to potentially result in clinical benefit and to enable rapid identification of the RP2D . A review of 59 approved small molecule oncology MTAs assessed the current approaches to FIP starting dose and dose escalation. While the MTD and RP2D to starting dose ratio was variable, all FIP trials except one had a starting dose lower than the MTD, which indicates that the starting doses have overwhelmingly been safe. A majority of the clinical trials used a 3 + 3 design approach, with a median range of four dose escalation steps required to reach MTD or RP2D from the starting dose. The accelerated titration design, which achieves more rapid dose escalation, was only used in 13% of trials, and the increased use of this approach with single subject cohorts may minimize the number of patients starting clinical trials at sub-therapeutic doses. Twenty nine percent of the FIP trials required ≥ 6 dose escalation steps to reach MTD or RP2D, and there was no difference in the number of dose escalation steps between first-in-class and non-first-in-class molecules. Similarly, Le Tourneau et al. reviewed the Scopus abstract database for FIP trials of MTAs in cancer patients and concluded the derivation of the starting dose was safe but was based on diverse practices using a variety of nonclinical toxicological parameters (e.g., MTD, TDL, LD10, NOAEL). This analysis showed that starting doses for MTAs were based on rodent and non-rodent data in similar proportions, and overall, starting doses selected using a toxicity-based method did not exceed the human MTD in 96% of clinical trials reviewed. A median of five dose levels was required from the starting dose to the clinical MTD, which is a similar number of cohorts reported by Mittapalli . Our data were consistent with these findings with only 1 actual starting dose exceeding the MTD (99% tolerated), and an average of 5.3 escalations to reach MTD/RP2D (data not shown). The analysis performed in this paper provides an opportunity to review the optimal starting dose algorithm(s) to enable a reduction in the number of cancer patients starting clinical trials at sub-optimal therapeutic doses and to minimize the number of dose escalation steps and time needed to reach MTD or RP2D. An analysis of 92 small molecule oncology compounds from 12 pharmaceutical companies showed that in more than half of cases the approach used was more conservative than the ICH S9 guideline dictates. In some cases, a less aggressive starting dose was justified based on the data. However, it was rare for starting doses to be significantly more aggressive than the guideline. Our analysis demonstrates that a less conservative approach to starting dose selection, such as basing the FIP dose on the nonclinical NOAEL without a safety factor, would generally be safe and would reduce the number of dose levels tested in the clinic. To encourage sponsors to be more aggressive in starting dose selection for small molecule oncology therapeutics, thereby reducing the number of patients treated at sub-efficacious dose levels and accelerating drugs to approval, we encourage sponsors to propose and discuss with regulatory agencies using this alternative NOAEL-based method to justify higher starting doses when the data supports it as outlined in Fig. . This approach is reflected in the 2019 FDA nonclinical guidance document on severely debilitating or life-threatening hematologic disorders . Although oncology products are not within the scope of the document, this guidance has the similar goal of avoiding administration of sub-therapeutic doses to patients while still protecting patients’ safety. It is worth noting that specifying the NOAEL in toxicology study reports (which may not be common practice for oncology drug candidates) may facilitate the determination of the NOAEL-based alternative starting dose. Of note, as exemplified by the exercise, it may be possible to scientifically propose a NOAEL-based starting dose in the absence of one being identified based on the nature of the finding in the GLP toxicology study and the safety implication of the finding for oncology patients. If sponsors adjust their dose selection strategies for GLP toxicology studies to ensure determination of a NOAEL, this could lead to more broadly spaced dose levels being evaluated and, thus, using the alternative starting dose approach rationale could yield a lower starting dose. In addition, some sponsors may add dose groups to ensure a NOAEL which would not be aligned a 3Rs objective of ICH S9. In the majority of cases evaluated (82%), the NOAEL-based alternative starting dose would have been safe. The number increases to 93% when evaluating those that originally used the HNSTD or STD10 approach. The reasons for not using the HNSTD or STD10 approach vary, but one common alternative is the use of MABEL. The MABEL is typically applied to immune-oncology agents, mostly those with agonist activity, for which the pharmacological activity is of great safety concern and the translation from nonclinical to clinical safety has lower confidence . Other reasons for using non-traditional approaches include cases where there is low confidence in the translation of predicted exposure or sensitivity of toxicity between animals and humans. The lack of cross-species target potency may also add to the low confidence in translation to humans for some programs. Of the compounds in the database, 60 reported the pharmacologic activity in the rodent toxicology species as unknown and 64 reported the pharmacologic activity in non-rodent toxicology species as unknown. The NOAEL-based alternative starting dose may not be appropriate for cases with increased concern like those discussed above. Of the 12 (18%) cases where the NOAEL-based alternative starting dose would have exceeded the MTD/RP2D, nonclinical toxicities would have been monitorable in all but one instance. Of these 12 cases, only 3 used HNSTD or STD10 to select the actual starting dose. Others used MABEL, NOAEL with safety factors, or pharmacokinetic/pharmacodynamic modeling. Of the 12 cases, 8 NOAEL-based alternative starting doses were within two–threefold of the clinical MTD/RP2D. Those within twofold of the RP2D would not likely have resulted in significant toxicity as this is within a typical single cohort escalation. Those within threefold might be a slightly more, but possibly acceptable, risk of causing toxicity. Of the remaining 4 cases with NOAEL-based starting doses between 4.5- and 20-fold the MTD/RP2D, 2 were IO agents using IT delivery, one of which did not determine the clinical MTD (implying RP2D was not based on tolerability). IT delivery is often limited by the maximum feasible dose determined by formulation or delivery volume restrictions. In the other 2 cases, both with a NOAEL-based alternative starting dose sixfold the MTD/RP2D, 1 used a NOAEL-based approach with a 50-fold safety factor, implying heightened safety concern, while the other used a LOAEL-based approach with a sixfold safety factor. It should be noted that in the latter case, the actual starting dose was not only the same as the NOAEL-based alternative but was not tolerated and the RP2D was 1/5 the starting dose. Therefore, these 4 cases would not be likely candidates for the NOAEL-based alternative method. Extensive analysis of translation from nonclinical toxicology studies to clinical findings have been previously published . In general, in our dataset, similar DLTs were observed across toxicology species and humans, with the gastrointestinal system and bone marrow/hematological being the most prominent. In addition, most of the DLTs were reversible or deemed reversible with only 4 cases in rodents and only 1 case in non-rodents that were irreversible (as reported by the company). Additionally, in 78 (85%) of the cases the findings in both rodent and non-rodent were reported as monitorable. In cases without DLTs reported, monitorability was reported as not applicable accounting for differences between 4% of cases demonstrated findings in either rodents or non-rodents deemed unmonitorable and those that were reported to be monitorable. The decision tree (Fig. ) outlines points to consider highlighted in this manuscript when assessing the various approaches for selecting the FIP starting dose. Companies should review all possible starting dose calculations and consider the risk–benefit when finalizing the starting dose proposal. When considering the MABEL approach, it is recommended to follow the decision tree in Leach et al. . Another factor to consider in selection of the starting dose is the projected human exposure. The projected human exposure at the clinical starting dose in general should not exceed the exposure in animal toxicity studies at the NOAEL for which potential toxicities of concern were observed. Putting the analyses performed in this paper into perspective of the risk–benefit assessment, the potential benefit of using alternative starting dose methods compared to the risk to exceed the MTD/RP2D using that approach should be taken into consideration. A minimal increase in safety risk may be justified to offer patients with advanced life-threatening disease and limited therapeutic options the potential therapeutic benefit in FIP studies. In conclusion, retrospective analysis of previously determined starting doses for oncology therapeutics clearly indicate more aggressive starting dose selection should be considered to reduce the use of sub-therapeutic dose levels in oncology patients, and accelerate patient access to effective drugs . These analyses demonstrate that a NOAEL-based alternative starting dose without an added safety factor would have been tolerated in a majority of the cases evaluated, with an anticipated mean reduction of 2.3 cohorts and 21.4 weeks in dose escalation to reach RP2D. The results also indicate a good translation of the absence of adverse effects in animals to the absence of adverse effects in patients, a key message in the Monticello et al. analysis . Alternatively, the rigorous application of rules-based starting dose methods in DeGeorge or ICH S9 could also result in reduced number of cohorts exposed to sub-optimal dose levels with low risk to clinical trial participants. It is unlikely that any method of FIP dose calculation would be appropriate for all oncology small molecule drug candidates and setting the starting dose should be done on a case-by-case approach. However, the NOAEL-based alternative without the application of a safety factor should be calculated, particularly in cases where the HNSTD/STD10 method is typically used and considered if appropriate.
Below is the link to the electronic supplementary material. Supplementary file1 (XLSX 60 KB) Supplementary file2 (XLSX 44 KB) Supplementary file3 (DOCX 54 KB)
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Integrated microbiome and metabolome analysis reveals altered gut microbial communities and metabolite profiles in dairy cows with subclinical mastitis | aea87c6d-086d-4a29-af1f-b287bef940f9 | 11877966 | Biochemistry[mh] | Mastitis of dairy cows is a common disease with high incidence and prevalence in pasture, which leads to the reduction of milk yield and quality, as well as increased culling rate and treatment expenses, causing huge economic losses to the producer worldwide . The mastitis in cows can be categorized into SM and clinical mastitis (CM) according to the presence or absence of clinical symptoms . In practice, the CM is usually well managed in pasture, however, the prevention and treatment of SM is easily to be ignored due to its invisible clinical symptoms and high concealment . Additionally, the SM cows persistently existing in herd have long incubation period and may spread the pathogens to healthy cows, causing periodic mastitis attacks that may eventually escalate into CM . Thus, the incidence of SM and economic losses caused by SM are much higher than that of CM in cows . The occurrence of mastitis is correlated with multiple factors, such as genetic characteristics, environmental hygiene, feeding management, dietary ingredients and improper milking practices, etc . It is generally accepted that the infection by various pathogenic microorganisms derived from environment is the leading cause of mastitis in cows . Whereas, a growing body of recent studies in human, mice and cows have pinpointed that the disturbance of gastrointestinal microbiota is associated with the occurrence of mastitis and may be an important endogenous factor causing mastitis, thus proposing the concept of “gastroenterogenic mastitis” . The multitudinous and diverse microbiota which habitat in the gastrointestinal tract of dairy cows are critical regulators for nutrient digestion, metabolism and immune response of host . It is well-known that gastrointestinal microbiota and their metabolites, including short chain fatty acids(SCFAs), bile acids, products of tryptophan metabolism, could regulate intestinal barrier function by modulating the expression of tight-junction protein and function of immune cell . The gut flora disorders can increase the permeability of intestinal mucosa and lead to the disruption of intestinal barrier function, which allows the pathogenic microorganisms and metabolites to migrate to the extraintestinal distal tissues or organs through endogenous pathways including blood and lymphatic circulation, contributing to the inflammatory response in various organs of host, such as pancreas, spleen, liver and mammary gland . Transplanting the fecal microflora from mastitic dairy cows to germ-free mice was proved to induce the up-regulation of expression of endotoxins and inflammatory cytokines in serum, as well as the inflammation of mammary gland, spleen and colon in mice . Moreover, fecal microbiota transplantation from cows with mastitis(M-FMT) to recipient mice promoted the transfer of pathogenic bacteria from gut to mammary gland and destroyed the blood-milk barrier, the butyric acid produced by commensal Roseburia intestinalis could restrict the translocation of bacteria to alleviate M-FMT-induced mastitis in mice . Subacute rumen acidosis (SARA), a typical model of rumen microbiota dysbiosis arose by long-term feeding of high-concentrate diet, has been proved to decrease the diversity of ruminal and intestinal flora, and increase the permeability of rumen barrier, which lead to the release of lipopolysaccharide(LPS) into blood, significantly enhancing the concentration of inflammatory factors in circulatory system, thus inducing oxidative stress, apoptosis and inflammation in the mammary gland . Hence, the gastrointestinal microbiota and their metabolites can directly or indirectly affect the occurrence and development of mastitis by regulating the intestinal barrier function and immune response of host. Recently, multiple studies have identified the ruminal or intestinal microbes associated with somatic cell count (SCC), a widely used indicator for the diagnosis of mastitis in dairy cows. The genera unclassified_f_RF16 , genera Paenibacillus and c_Deltaproteobacteria , etc., were remarkably enriched in the rumen of dairy cows with high SCC . The abundance of microflora and metabolites correlated with inflammation in the rumen of dairy cows with mastitis were markedly changed, Pseudobutyrivibrio , Gastranaerophilale s and Moraxella , etc., with high abundance were characterized in the rumen of clinical mastitic dairy cows along with the elevation of 12-oxo-20-dihy-droxy-leukotriene B4 and 10beta-hydroxy-6beta-isobu-tyrylfuranoeremophilane, Ruminiclostridium_9 and Enterorhabdus were abundant in the rumen of SM cows accompanied by the increase of methenamine and 5-HMF . In addition, the abundance of Campylobacteraceae , Campylobacter , unidentified-Christensenellaceae , Lactobacillus-reuteri and Bacteroides-vulgatus were prominently enhanced in the feces of dairy cows with CM . Since the composition and function of gastrointestinal microbiota can be shaped by many factors, such as the host genetic background, physiological condition, growth stage, geographical environment, feeding management, etc , distinct gastrointestinal microorganisms associated with dairy cow mastitis has been identified across different studies. Furthermore, the pathological metabolic mechanisms for the relationship between digestive tract microbes, metabolites and mammary health status remain not fully understood. Herein, we obtained feces and plasma samples from healthy and SM cows raised in a farm of Wuhan, and compared the differences in gut microbiome, plasma and fecal metabolome by performing 16 S rDNA sequencing and untargeted metabolomics technology, which enhance the understanding of the relationship between SM and gut microbiota and metabolic profiles, as well as laying the theoretical foundation for formulating the prevention strategies and accurate diagnosis of SM in dairy cows.
Experimental animals and sample collection The experimental cows used in this study were selected form the dairy farm owned by Institute of Animal Science and Veterinary Medicine, Wuhan Academy of Agricultural Sciences. The Holstein dairy cows which were fed ad libitum and had free access to water, received the same total mixed ration (TMR) composed of 40% roughage and 60% concentrate twice a day at 5:00 and 17:00. The composition and nutritional levels of TMR are shown in Supplementary Table . In this study, Holstein dairy cows with similar parity, lactation days and milk yield were preliminarily selected. Subsequently, the udder health status of cows was comprehensively judged according to milk SCC for nearly one month and clinical symptoms of udder based on the degree of inflammation, which were examined by veterinary surgeon from Wuhan Academy of Agricultural Sciences. Currently, it is generally accepted that 20 × 10 4 cells/mL is the optimal SCC threshold value to distinguish between healthy and mastitis cows . Ultimately, 8 dairy cows with healthy udders (SCC (9.13 ± 2.17)×10 4 cells/mL, no clinical symptoms of redness, swelling and fever in the udder, designated as CON group, n = 8) and 8 dairy cows with subclinical mastitis(SCC (75.25 ± 9.50)×10 4 cells/mL, no clinical symptoms of redness, swelling and fever in the udder, designated as SM group, n = 8) were selected for trial. The flow chart of our experimental design is presented in Fig. A. The feces and blood samples were obtained from each experimental dairy cows on the last day after a month of elevated milk SCC (above a threshold of 20 × 10 4 cells/mL). Fresh stool samples were collected from rectum of dairy cows through sterile long-arm gloves, which were aliquoted into 2 aseptic 5 mL cryovials and snap-frozen in liquid nitrogen, and then transferred to a -80 ℃ refrigerator for storage until analysis of fecal microbiota and metabolites. Approximately 10 mL of fasting blood samples were collected from the tail vein of dairy cows into vacuum tubes containing coagulant or anticoagulant EDTA, which were kept on ice, and then centrifuged to acquire serum or plasma samples at 7000 r/min for 10 min at 4 ℃. The supernatant were retained and divided into 6 sterile cryovials, which were immediately immersed in liquid nitrogen and then stored at -80 °C until subjected to untargeted metabolomic analysis, and detection of biochemical, antioxidant indices and the concentration of inflammatory cytokines. Determination of biochemical, antioxidant indices and inflammatory cytokines in serum The content of total protein (TP), albumin (ALB), globulin (GLB), uric acid (UA), triglycerides (TG), TC, HDL-C and low-density lipoprotein cholesterol (LDLC) in the serum of experimental cows were determined by using a fully automatic biochemical analyzer (TBA120FR, Canon, Japan). T-AOC, the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and CAT, as well as the content of MDA, alkline phosphatase (AKP), non-esterified fatty acid (NEFA) and lactate dehydrogenase (LDH) in the serum were detected with commercially available kits following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The concentrations of IgA, IgG, IgM, TNF-α, IL-1β, IL-2, IL-8 and IL-10 were measured by ELISA kit in accordance with the protocols provided by manufacturer (Shanghai Enzyme-linked Biotechnology Co., Ltd, Shanghai, China). DNA extraction, 16 S rDNA amplicon sequencing Total genomic DNA from stool samples were extracted using the MagBeads FastDNA Kit (116570384, MP Biomedicals, USA) following the manufacturer’s instructions, and stored at -20 °C prior to further analysis. The quantity and quality of extracted DNA samples were measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and detected by agarose gel electrophoresis, respectively. PCR amplification of the bacterial 16 S rRNA genes V3-V4 region was performed using the forward primer 338 F (5’-ACTCCTACGGGAGGCAGCA-3’) and the reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 µL of 5× reaction buffer, 5 µL of 5×High GC buffer, 2 µL of dNTP(10mM), 1 µL of each forward and reverse primer (10 µM), 2 µL of DNA template, 8.75 µL of ddH 2 O, 0.25 µL of Q5 high-fidelity DNA polymerase. Thermal cycling was comprised of initial denaturation at 98 °C for 5 min, followed by 25 cycles consisting of denaturation at 98 °C for 30 s, annealing at 53 °C for 30 s and extension at 72 °C for 45 s, with a final extension at 72 °C for 5 min. PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2 × 250 bp sequencing was carried out using the Illumina NovaSeq platform with NovaSeq 6000 SP Reagent Kit (500 cycles). Processing, bioinformatic analysis of sequencing data The sequence data were processed and analyzed using QIIME2 (2019.4) with slight modification according to the official tutorials ( https://docs.qiime2.org/2019.4/tutorials/ ). Briefly, raw sequence data were demultiplexed using the demux plugin followed by primers cutting with cutadapt plugin. Sequences were subsequently quality filtered, denoised, merged and chimera removed using the DADA2 plugin. Non-singleton amplicon sequence variants (ASVs) were aligned with mafft and employed to construct a phylogeny with fasttree2. Taxonomy was assigned to ASVs utilizing the classify-sklearn naive Bayes taxonomy classifier within the feature-classifier plugin, referencing the Greengenes 13.8 database. ASV-level alpha diversity indices, such as Chao1 richness estimator, Observed species, Shannon diversity index, Simpson index, Faith’s PD, Pielou’s evenness and Good’s coverage, were calculated using the diversity plugin with samples rarefied to 32,302 sequences per sample, and displayed as box plots. Beta diversity analysis was conducted to investigate the structural variation of microbial communities across samples using Bray-Curtis metrics and visualized via principal coordinate analysis (PCoA). The significance of difference in microbiota structure among groups was assessed by PERMANOVA (permutational multivariate analysis of variance) using QIIME2. MEGAN and GraPhlAn were engaged to visualize the taxonomic compositions and abundances, respectively. Venn diagram was generated to exhibit the number of shared and unique ASVs among groups by employing R package “VennDiagram”, based on the occurrence of ASVs across groups regardless of their relative abundance. Linear discriminant analysis effect size (LEfSe) algorithm was devoted to identify the statistically differential abundant taxa between groups, the value of linear discriminant analysis (LDA) > 2 and P < 0.05 were considered significantly different. The prediction of microbial functions was performed by Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) (Gavin M. Douglas, et al., preprint) upon MetaCyc ( https://metacyc.org/ ) databases. Metabolites extraction, preparation and LC-MS/MS analysis The plasma samples were thawed at 4 °C, and 100 µL aliquots were mixed with 400 µL of cold extraction solution (methanol: acetonitrile,1:1(v/v)) to remove the protein. The mixed solution was vortexed for 30 s, sonicated for 10 min in water bath at 4 ℃. However, for analysis using frozen stool samples, the thawed feces (50 mg) were mixed with beads and 500 µL of cold extraction solution (methanol: acetonitrile: H 2 O, 2:2:1(v/v)) to vortex for 30s. Whereafter, the mixed samples were homogenized (35 Hz,4 min) and sonicated for 5 min in water bath at 4 ℃, and repeated for three times. Both the plasma and feces samples were incubated for 1 h at -40 ℃ to precipitate proteins. Subsequently, the samples were centrifuged at 14,000 g for 20 min at 4 °C. The supernatant was transferred to a fresh glass vial, dried in a vacuum centrifuge and redissolved in 100 µL acetonitrile/water (1:1, v/v) solvent followed by centrifugation at 14,000 g for 15 min at 4 ℃, then the supernatant was injected for LC-MS/MS analysis. To supervise the stability and repeatability of instrument analysis, quality control (QC) samples were prepared by pooling 10 µL of each sample and analyzed together with the other samples. The QC samples were inserted regularly and analyzed in every 5 samples. Untargeted metabolomic analysis of fecal and plasma samples were performed using an UHPLC (Vanquish UHPLC, Thermo) coupled to a Orbitrap. Chromatography was carried out with an ACQUITY UPLC ® HSS T3 (2.1 × 100 mm, 1.8 μm) (Waters, Milford, MA, USA). The column was maintained at 40 ℃. The flow rate and injection volume were set at 0.3 mL/min and 2 µL, respectively. For LC-ESI -MS analysis, the mobile phases consisted of (B2) 0.1% formic acid in acetonitrile (v/v) and (A2) 0.1% formic acid in water (v/v). Separation was conducted under the following gradient: 0 ~ 1 min, 8% B2; 1 ~ 8 min, 8%~98% B2; 8 ~ 10 min, 98% B2; 10 ~ 10.1 min, 98%~8% B2, 10.1 ~ 12 min, 8% B2. For LC-ESI -MS analysis, the analytes were carried out with (B3) acetonitrile and (A3) ammonium formate (5 mM). Separation was conducted under the following gradient: 0 ~ 1 min, 8% B3; 1 ~ 8 min, 8%~98% B3; 8 ~ 10 min, 98% B3; 10 ~ 10.1 min, 98%~8% B3; 10.1 ~ 12 min, 8% B3. Mass spectrometric detection of metabolites was performed on a Q Exactive HFX Hybrid Quadrupole Orbitrap mass spectrometer equipped with a heated ESI ion source (Thermo Fisher Scientific, USA). Simultaneous MS1 and MS/MS (Full MS-ddMS2 mode, data-dependent MS/MS) acquisition methods were used. The parameters were set as follows: sheath gas pressure, 40 arb; aux gas flow, 10 arb; spray voltage, 3.50 kV and − 2.50 kV for ESI and ESI , respectively; capillary temperature, 325 ℃; MS1 range, m/z 100–1000; MS1 resolving power, 70,000 FWHM; number of data dependent scans per cycle, 10; MS/MS resolving power, 17,500 FWHM; normalized collision energy, 30 eV; dynamic exclusion time, automatic. Untargeted metabolomic data processing and analysis The raw MS data were converted to MzXML file format using the MSConvert tool in the Proteowizard software package (v3.0.8789) before importing into freely available XCMS software (V3.12.0). For peak picking, the following parameters were used: centWave m/z = 10 ppm, peakwidth = c (10, 60), prefilter = c (10, 100). For peak grouping, bw = 5, mzwid = 0.025, minfrac = 0.5 were used. In the extracted ion features, only the variables having more than 50% of the nonzero measurement values in at least one group were kept. The LOESS signal correction method based on QC samples is used to correct data between batches and eliminate instrument batch errors. Substances with RSD > 30% in QC samples are filtered out during data quality control. The identification of metabolites was performed by comparing accurate m/z value (< 10 ppm) and MS/MS spectra with public databases including HMDB, MassBank, LipidMaps, mzCloud, KEGG, as well as an in-house database established with available authentic standards. Quantified data were output into excel format and subjected to univariate statistical analysis using R package. Dimension reduction analyses, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA), were conducted on the sample datasets using R package “Ropls”, which generated score plots to visually depict the differences in metabolite compositions among various samples. The variable importance in projection (VIP) value of each variable in the OPLS-DA model was calculated to indicate its contribution to the classification. The metabolites with VIP > 1, P -value < 0.05 and fold change (FC) > 1.2 or < 0.83 were designated as statistically significantly upregulated or downregulated differential metabolites, respectively. Cluster analysis of the abundance values of differential metabolite was conducted by employing the Pheatmap package (v1.0.12) within R to plot a heatmap, which effectively illustrates the abundance profiles of metabolites across different samples. Functional analysis of the differential metabolites was primarily conducted through KEGG enrichment analysis using the clusterProfiler package (v 4.6.0), which was aimed at obtaining the significant enriched metabolic pathways, thus providing insights into the biological processes influenced by the differential metabolites The advanced analysis of differential metabolites was undertaken through machine learning techniques provided by the mlr3verse package (v0.2.7), which facilitated feature selection and model interpretation. Receiver operating characteristic (ROC) curves were constructed using the pROC package (v1.18.2) to assess the predictive power of the identified differential metabolites, ultimately yielding crucial information about key metabolites within the differential set that could serve as biomarkers or indicators of specific biological states. The Spearman correlation analysis between differential metabolites in feces and plasma and fecal microflora were performed by applying the correlation function in R package, and represented by a heatmap. Statistic analysis The sample size of this study was verified to be sufficient by employing SPSS27.0 software. A post hoc power calculation indicated that the sample size in this study achieved a power of 1.0 at 0.05 α level for a 2- sided test. The experimental data are presented as mean ± standard error of the mean (SEM). The statistical analyses were carried out by IBM SPSS26.0 software (IBM, Chicago, USA). The comparison of serum biochemical and antioxidant indices, inflammatory cytokines concentration and alpha diversity index between two groups were performed using unpaired two-tailed t test (Student’s t-test). The relative abundance of bacterial taxa between two groups were analyzed by Mann-Whitney U test. Results with P < 0.05 were regarded as significantly different, 0.05 < P < 0.10 were interpreted as a tendency.
The experimental cows used in this study were selected form the dairy farm owned by Institute of Animal Science and Veterinary Medicine, Wuhan Academy of Agricultural Sciences. The Holstein dairy cows which were fed ad libitum and had free access to water, received the same total mixed ration (TMR) composed of 40% roughage and 60% concentrate twice a day at 5:00 and 17:00. The composition and nutritional levels of TMR are shown in Supplementary Table . In this study, Holstein dairy cows with similar parity, lactation days and milk yield were preliminarily selected. Subsequently, the udder health status of cows was comprehensively judged according to milk SCC for nearly one month and clinical symptoms of udder based on the degree of inflammation, which were examined by veterinary surgeon from Wuhan Academy of Agricultural Sciences. Currently, it is generally accepted that 20 × 10 4 cells/mL is the optimal SCC threshold value to distinguish between healthy and mastitis cows . Ultimately, 8 dairy cows with healthy udders (SCC (9.13 ± 2.17)×10 4 cells/mL, no clinical symptoms of redness, swelling and fever in the udder, designated as CON group, n = 8) and 8 dairy cows with subclinical mastitis(SCC (75.25 ± 9.50)×10 4 cells/mL, no clinical symptoms of redness, swelling and fever in the udder, designated as SM group, n = 8) were selected for trial. The flow chart of our experimental design is presented in Fig. A. The feces and blood samples were obtained from each experimental dairy cows on the last day after a month of elevated milk SCC (above a threshold of 20 × 10 4 cells/mL). Fresh stool samples were collected from rectum of dairy cows through sterile long-arm gloves, which were aliquoted into 2 aseptic 5 mL cryovials and snap-frozen in liquid nitrogen, and then transferred to a -80 ℃ refrigerator for storage until analysis of fecal microbiota and metabolites. Approximately 10 mL of fasting blood samples were collected from the tail vein of dairy cows into vacuum tubes containing coagulant or anticoagulant EDTA, which were kept on ice, and then centrifuged to acquire serum or plasma samples at 7000 r/min for 10 min at 4 ℃. The supernatant were retained and divided into 6 sterile cryovials, which were immediately immersed in liquid nitrogen and then stored at -80 °C until subjected to untargeted metabolomic analysis, and detection of biochemical, antioxidant indices and the concentration of inflammatory cytokines.
The content of total protein (TP), albumin (ALB), globulin (GLB), uric acid (UA), triglycerides (TG), TC, HDL-C and low-density lipoprotein cholesterol (LDLC) in the serum of experimental cows were determined by using a fully automatic biochemical analyzer (TBA120FR, Canon, Japan). T-AOC, the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and CAT, as well as the content of MDA, alkline phosphatase (AKP), non-esterified fatty acid (NEFA) and lactate dehydrogenase (LDH) in the serum were detected with commercially available kits following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The concentrations of IgA, IgG, IgM, TNF-α, IL-1β, IL-2, IL-8 and IL-10 were measured by ELISA kit in accordance with the protocols provided by manufacturer (Shanghai Enzyme-linked Biotechnology Co., Ltd, Shanghai, China).
Total genomic DNA from stool samples were extracted using the MagBeads FastDNA Kit (116570384, MP Biomedicals, USA) following the manufacturer’s instructions, and stored at -20 °C prior to further analysis. The quantity and quality of extracted DNA samples were measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and detected by agarose gel electrophoresis, respectively. PCR amplification of the bacterial 16 S rRNA genes V3-V4 region was performed using the forward primer 338 F (5’-ACTCCTACGGGAGGCAGCA-3’) and the reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 µL of 5× reaction buffer, 5 µL of 5×High GC buffer, 2 µL of dNTP(10mM), 1 µL of each forward and reverse primer (10 µM), 2 µL of DNA template, 8.75 µL of ddH 2 O, 0.25 µL of Q5 high-fidelity DNA polymerase. Thermal cycling was comprised of initial denaturation at 98 °C for 5 min, followed by 25 cycles consisting of denaturation at 98 °C for 30 s, annealing at 53 °C for 30 s and extension at 72 °C for 45 s, with a final extension at 72 °C for 5 min. PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2 × 250 bp sequencing was carried out using the Illumina NovaSeq platform with NovaSeq 6000 SP Reagent Kit (500 cycles).
The sequence data were processed and analyzed using QIIME2 (2019.4) with slight modification according to the official tutorials ( https://docs.qiime2.org/2019.4/tutorials/ ). Briefly, raw sequence data were demultiplexed using the demux plugin followed by primers cutting with cutadapt plugin. Sequences were subsequently quality filtered, denoised, merged and chimera removed using the DADA2 plugin. Non-singleton amplicon sequence variants (ASVs) were aligned with mafft and employed to construct a phylogeny with fasttree2. Taxonomy was assigned to ASVs utilizing the classify-sklearn naive Bayes taxonomy classifier within the feature-classifier plugin, referencing the Greengenes 13.8 database. ASV-level alpha diversity indices, such as Chao1 richness estimator, Observed species, Shannon diversity index, Simpson index, Faith’s PD, Pielou’s evenness and Good’s coverage, were calculated using the diversity plugin with samples rarefied to 32,302 sequences per sample, and displayed as box plots. Beta diversity analysis was conducted to investigate the structural variation of microbial communities across samples using Bray-Curtis metrics and visualized via principal coordinate analysis (PCoA). The significance of difference in microbiota structure among groups was assessed by PERMANOVA (permutational multivariate analysis of variance) using QIIME2. MEGAN and GraPhlAn were engaged to visualize the taxonomic compositions and abundances, respectively. Venn diagram was generated to exhibit the number of shared and unique ASVs among groups by employing R package “VennDiagram”, based on the occurrence of ASVs across groups regardless of their relative abundance. Linear discriminant analysis effect size (LEfSe) algorithm was devoted to identify the statistically differential abundant taxa between groups, the value of linear discriminant analysis (LDA) > 2 and P < 0.05 were considered significantly different. The prediction of microbial functions was performed by Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) (Gavin M. Douglas, et al., preprint) upon MetaCyc ( https://metacyc.org/ ) databases.
The plasma samples were thawed at 4 °C, and 100 µL aliquots were mixed with 400 µL of cold extraction solution (methanol: acetonitrile,1:1(v/v)) to remove the protein. The mixed solution was vortexed for 30 s, sonicated for 10 min in water bath at 4 ℃. However, for analysis using frozen stool samples, the thawed feces (50 mg) were mixed with beads and 500 µL of cold extraction solution (methanol: acetonitrile: H 2 O, 2:2:1(v/v)) to vortex for 30s. Whereafter, the mixed samples were homogenized (35 Hz,4 min) and sonicated for 5 min in water bath at 4 ℃, and repeated for three times. Both the plasma and feces samples were incubated for 1 h at -40 ℃ to precipitate proteins. Subsequently, the samples were centrifuged at 14,000 g for 20 min at 4 °C. The supernatant was transferred to a fresh glass vial, dried in a vacuum centrifuge and redissolved in 100 µL acetonitrile/water (1:1, v/v) solvent followed by centrifugation at 14,000 g for 15 min at 4 ℃, then the supernatant was injected for LC-MS/MS analysis. To supervise the stability and repeatability of instrument analysis, quality control (QC) samples were prepared by pooling 10 µL of each sample and analyzed together with the other samples. The QC samples were inserted regularly and analyzed in every 5 samples. Untargeted metabolomic analysis of fecal and plasma samples were performed using an UHPLC (Vanquish UHPLC, Thermo) coupled to a Orbitrap. Chromatography was carried out with an ACQUITY UPLC ® HSS T3 (2.1 × 100 mm, 1.8 μm) (Waters, Milford, MA, USA). The column was maintained at 40 ℃. The flow rate and injection volume were set at 0.3 mL/min and 2 µL, respectively. For LC-ESI -MS analysis, the mobile phases consisted of (B2) 0.1% formic acid in acetonitrile (v/v) and (A2) 0.1% formic acid in water (v/v). Separation was conducted under the following gradient: 0 ~ 1 min, 8% B2; 1 ~ 8 min, 8%~98% B2; 8 ~ 10 min, 98% B2; 10 ~ 10.1 min, 98%~8% B2, 10.1 ~ 12 min, 8% B2. For LC-ESI -MS analysis, the analytes were carried out with (B3) acetonitrile and (A3) ammonium formate (5 mM). Separation was conducted under the following gradient: 0 ~ 1 min, 8% B3; 1 ~ 8 min, 8%~98% B3; 8 ~ 10 min, 98% B3; 10 ~ 10.1 min, 98%~8% B3; 10.1 ~ 12 min, 8% B3. Mass spectrometric detection of metabolites was performed on a Q Exactive HFX Hybrid Quadrupole Orbitrap mass spectrometer equipped with a heated ESI ion source (Thermo Fisher Scientific, USA). Simultaneous MS1 and MS/MS (Full MS-ddMS2 mode, data-dependent MS/MS) acquisition methods were used. The parameters were set as follows: sheath gas pressure, 40 arb; aux gas flow, 10 arb; spray voltage, 3.50 kV and − 2.50 kV for ESI and ESI , respectively; capillary temperature, 325 ℃; MS1 range, m/z 100–1000; MS1 resolving power, 70,000 FWHM; number of data dependent scans per cycle, 10; MS/MS resolving power, 17,500 FWHM; normalized collision energy, 30 eV; dynamic exclusion time, automatic.
The raw MS data were converted to MzXML file format using the MSConvert tool in the Proteowizard software package (v3.0.8789) before importing into freely available XCMS software (V3.12.0). For peak picking, the following parameters were used: centWave m/z = 10 ppm, peakwidth = c (10, 60), prefilter = c (10, 100). For peak grouping, bw = 5, mzwid = 0.025, minfrac = 0.5 were used. In the extracted ion features, only the variables having more than 50% of the nonzero measurement values in at least one group were kept. The LOESS signal correction method based on QC samples is used to correct data between batches and eliminate instrument batch errors. Substances with RSD > 30% in QC samples are filtered out during data quality control. The identification of metabolites was performed by comparing accurate m/z value (< 10 ppm) and MS/MS spectra with public databases including HMDB, MassBank, LipidMaps, mzCloud, KEGG, as well as an in-house database established with available authentic standards. Quantified data were output into excel format and subjected to univariate statistical analysis using R package. Dimension reduction analyses, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA), were conducted on the sample datasets using R package “Ropls”, which generated score plots to visually depict the differences in metabolite compositions among various samples. The variable importance in projection (VIP) value of each variable in the OPLS-DA model was calculated to indicate its contribution to the classification. The metabolites with VIP > 1, P -value < 0.05 and fold change (FC) > 1.2 or < 0.83 were designated as statistically significantly upregulated or downregulated differential metabolites, respectively. Cluster analysis of the abundance values of differential metabolite was conducted by employing the Pheatmap package (v1.0.12) within R to plot a heatmap, which effectively illustrates the abundance profiles of metabolites across different samples. Functional analysis of the differential metabolites was primarily conducted through KEGG enrichment analysis using the clusterProfiler package (v 4.6.0), which was aimed at obtaining the significant enriched metabolic pathways, thus providing insights into the biological processes influenced by the differential metabolites The advanced analysis of differential metabolites was undertaken through machine learning techniques provided by the mlr3verse package (v0.2.7), which facilitated feature selection and model interpretation. Receiver operating characteristic (ROC) curves were constructed using the pROC package (v1.18.2) to assess the predictive power of the identified differential metabolites, ultimately yielding crucial information about key metabolites within the differential set that could serve as biomarkers or indicators of specific biological states. The Spearman correlation analysis between differential metabolites in feces and plasma and fecal microflora were performed by applying the correlation function in R package, and represented by a heatmap.
The sample size of this study was verified to be sufficient by employing SPSS27.0 software. A post hoc power calculation indicated that the sample size in this study achieved a power of 1.0 at 0.05 α level for a 2- sided test. The experimental data are presented as mean ± standard error of the mean (SEM). The statistical analyses were carried out by IBM SPSS26.0 software (IBM, Chicago, USA). The comparison of serum biochemical and antioxidant indices, inflammatory cytokines concentration and alpha diversity index between two groups were performed using unpaired two-tailed t test (Student’s t-test). The relative abundance of bacterial taxa between two groups were analyzed by Mann-Whitney U test. Results with P < 0.05 were regarded as significantly different, 0.05 < P < 0.10 were interpreted as a tendency.
The serum biochemical, antioxidant indices, and inflammatory cytokines concentrations in healthy and SM cows The content of TC and HDLC in the serum of SM cows were significantly lower than those in CON group ( P < 0.05), the UA ( P = 0.063) and LDLC ( P = 0.055) concentration tended to decrease in SM group. Whereas, there were no significant differences in serum levels of TP, ALB, GLB, AKP, TG, NEFA and LDH between CON and SM groups ( P > 0.05) (Table ). T-AOC ( P < 0.05) and CAT ( P < 0.01) activities were remarkably reduced, while the concentration of MDA ( P < 0.05) was notably increased in the serum of SM cows compared with CON group. No significant differences of SOD and GSH-PX activities were observed among CON and SM groups ( P > 0.05) (Table ). In addition, the serum IgA, IgG, IgM, TNF-α, IL-1β, IL-2, IL-8 and IL-10 concentration showed no significant differences between CON and SM groups ( P > 0.05) (Table ). The gut microbial diversity and composition of healthy and SM co A total of 1,550,335 raw reads were acquired from 16 stool samples through 16 S rDNA sequencing, which were then low-quality filtered, denoised, merged and chimera removed to yield 809,370 high-quality sequences. Whereafter, 26,634 ASVs were obtained from each sample after normalization based on a 97% sequence similarity threshold. The rarefaction curves of observed species tended to flatten gradually as the depth of sequencing increased, which indicated that the sequencing in this study have covered and reflected the vast majority of microbial species in samples, increasing the number of sequencing furtherly could detect no more microorganisms (Supplementary Fig. ). Namely, the sequencing datasets were credible, that could be applied for succedent analyses. The result of Venn diagram showed that the total intestinal ASVs of cows in SM group was higher than that of CON group, the overlapped ASVs between two groups were 4242 (Fig. B). PcoA of beta diversity based on Bray-Curtis distance presented an obvious separation of fecal microbial communities between CON and SM groups, indicating that the significant difference existed in gut microbial composition profiles of healthy and SM cows (Fig. C-D). Alpha diversity analysis, a metric used to estimate the richness and evenness of species within samples, demonstrated that the chao and shannon index of intestinal microorganisms of SM cows were significantly higher than those in CON group ( P < 0.05). There were no significant differences for observed species, pielou-e, goods_coverage and simpson indexes between CON and SM groups ( P > 0.05) (Fig. E-J). The microbial composition and structure analysis was performed to compare the relative abundance of fecal microflora at different taxonomic levels. 26,634 identified ASVs were clustered and assigned to 22 phyla and 279 genera of bacteria (Supplementary Tables and ). At phyla level, the dominant bacteria in the feces of cows in CON and SM groups were Firmicutes (59.42 ± 0.86%), Bacteroidetes (36.35 ± 0.96%) and Tenericutes (1.40 ± 0.07%), the sum of their abundance accounted for 97.17% of the overall abundance (Supplementary Table ; Supplementary Fig. A). At genus level, the bacteria with relative abundance > 1% were deemed to be predominant, which occupied more than 87% of the total abundance. The unidentified_Ruminococcaceae (31.45 ± 0.68), unidentified_Bacteroidales (15.68 ± 0.46), unclassified_Clostridiales (4.23 ± 0.14), unidentified_Rikenellaceae (3.54 ± 0.16), unidentified_RF16 (3.30 ± 0.21), unidentified_Lachnospiraceae (2.24 ± 0.12), Oscillospira (2.09 ± 0.070), f__Clostridiaceae__g__Clostridium (1.92 ± 0.12), Ruminococcus (1.88 ± 0.078) and Paludibacter (1.12 ± 0.045) were the most abundant genus in the feces of cows in CON and SM groups(Supplementary Table ; Supplementary Fig. B). The differential fecal microbiota between healthy and SM cows LEfSe analysis and wilcoxon rank-sum test were further devoted to identify the representative differential microbes between CON and SM groups. The cladogram and score plot of LEfSe revealed that the bacteria of phyla Cyanobacteria ( P < 0.05) and Proteobacteria ( P < 0.05) were significantly enriched in the gut of cows in SM group (Fig. A-B). At genus level, Coprococcus ( P < 0.05), Succiniclasticum ( P < 0.05), Desulfovibrio ( P < 0.05) and Paraprevotella ( P < 0.01) were remarkably more abundant in the feces samples of CON group. Whereas, the gut of cows in SM group harbored more Succinivibrio ( P < 0.05) in comparison with CON group (Fig. A-B). Moreover, the relative abundance of Desulfovibrio_D168 ( P < 0.05) and Bifidobacterium_pseudolongum ( P < 0.05) in SM group were significantly lower than that of CON group, while Lactobacillus_iners ( P < 0.05) showed higher relative abundance in the gut of cows in SM group compared with CON group (Fig. C). Difference of gut microbial function between healthy and SM cows The predicted function of gut microbiota in healthy and SM cows were inferred by PICRUSt2, which indicated that the microflora related to fatty acid and lipid biosynthesis was significantly enhanced in SM group ( P < 0.0001), the capacity of secondary metabolite degradation was dramatically declined in SM group ( P < 0.001). Additionally, the gut of cows in CON group tended to enrich more abundant microbes correlated with nucleic acid processing ( P = 0.065) and alcohol degradation( P = 0.097). Nevertheless, the relative abundance of microflora associated with pyrimidine deoxyribonucleotides de novo biosynthesis ( P = 0.088), superpathway of L-aspartate and L-asparagine biosynthesis( P = 0.089) tended to be predominant in the gut of cows in SM group (Fig. ). Fecal metabolites profiling in healthy and SM cows Non-targeted metabolomics technique using ultra performance liquid chromatography coupled to high-resolution mass spectrometry was employed to investigate the fecal metabolomic characteristics of healthy and SM cows. In the current study, a total of 623 metabolites including 503 at positive and 120 at negative ion mode, were identified in the feces samples of dairy cows in CON and SM groups. Subsequently, the identified metabolites were statistically analyzed at subclass level, which was displayed as a donut chart (Supplementary Fiure ). The amino acids, peptides, and analogues (9.0%), fatty acids and conjugates (6.1%), carbohydrates and carbohydrate conjugates (3.7%), eicosanoids (3.0%), purines and purine derivatives (1.9%), benzoic acids and derivatives (1.9%), lineolic acids and derivatives (1.6%), triterpenoids (1.4%), bile acids, alcohols and derivatives (1.3%), hydroxycinnamic acids and derivatives (1.0%), etc. constituted the major components of metabolites in feces samples. The PLS-DA analysis, based on the abundance of identified metabolites in each sample, suggested that the contribution of PC1 and PC2 to the variation were 20.5% and 14.2%, respectively, under the positive ion mode. In the negative ion mode, PC1 and PC2 accounted for 18.7% and 12.2% of the variation, respectively (Fig. A-B). In addition, the PLS-DA score plots showed that the samples from CON and SM groups can be effectively distinguished under both positive and negative ion modes, indicating that the fecal metabolite profiles differentiated between CON and SM groups (Fig. A-B). In total, 57 significant differential metabolites comprising 27 upregulated and 30 downregulated metabolites were screened in the feces samples of CON and SM groups, that were visualized by constructing a volcano plot to exhibit the distribution and fold changes of differential metabolites (Fig. C). The detailed information of all identified significantly differential metabolites in feces are listed in Supplementary Table . A heatmap depicting the fecal differential metabolites between CON and SM groups is shown in Fig. D, which revealed that there were obvious partitions in the composition of differential metabolites between the two groups. Thereafter, we conducted KEGG pathway enrichment analysis of the differential metabolites. The fecal differential metabolites identified between two groups were mainly enriched in the pathways, such as linoleic acid metabolism, steroid biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis, mTOR and PPAR signaling pathway, arachidonic acid metabolism and amino acid metabolism (Fig. E). Identification and characterization of fecal signature differential metabolites between healthy and SM cows In order to screen for the metabolites with significant representative characteristics, the machine learning analysis was performed on the fecal differential metabolites between CON and SM groups. The results showed that the abundance of glycylleucine, L-3-cyanoalanine, 3-methyl-L-tyrosine, 2-(Methylamino)benzoic acid, N(6)-methyllysine, and Gamma-tocotrienol etc. in the feces of cows in CON group were markedly higher than those in SM group. Nevertheless, the abundance of 3-epiecdysone, 25-hydroxycholesterol, 9(S)-HPODE, 2-amino-2-deoxyisochorismate, etc. were observably increased in SM group compared with CON group (Fig. A). Additionally, the feature importance of these differential metabolites in random forest model and its contribution for classification were relatively high, that might be signature differential metabolites in the feces of cows in CON and SM groups (Fig. A). ROC analysis, an established statistical approach for assessing the predictive power and diagnostic value, also identified several differential metabolites exhibiting potential as biomarkers of SM in dairy cows. The area under curve (AUC) of 9(S)-HPODE, 25-hydroxycholesterol, deoxycytidine, dodecanedioic acid and are all greater than 0.85, which suggested that these 4 fecal differential metabolites may be valuable indicators for SM in dairy cows (Fig. B). Plasma metabolites profiling in healthy and SM cows The metabolic profile in blood can accurately and quickly reflect the physiological and pathological state of the body, and the changes in blood metabolites composition are closely correlated with the overall health status of body. Therefore, the characteristics of plasma metabolomics of healthy and SM cows were also profiled in the current study. 6753 metabolites containing 3103 at positive and 3650 at negative ion mode, were identified in the plasma samples of cows in CON and SM groups through mapping with the public and a self-established database. At subclass level, these metabolites can be classified in to several major categories, such as amino acids, peptides, and analogues (4.78%), fatty acids and conjugates (2.47%), carbohydrates and carbohydrate conjugates (1.36%), sesquiterpenoids (0.77%), benzoic acids and derivatives (0.58%), bile acids, alcohols and derivatives (0.52%), glycerophosphocholines(0.46%), eicosanoids(0.41%), lineolic acids and derivatives(0.41%), purines and purine derivatives(0.31%) (Supplementary Fiure ). The score plots of PLS-DA analysis showed that the symbols representing plasma samples were completely separated under both positive and negative mode, suggesting significant differences in the composition of plasma metabolites between CON and SM groups (Fig. A-B). A total of 905 significantly differential metabolites were identified between the two groups, among which 457 metabolites were upregulated and 448 metabolites were downregulated (Fig. C). Moreover, the volcano plot illustrating differential metabolites between groups demonstrated that the vast majority of differential metabolites had relatively concentrated FC, and some of significantly upregulated or downregulated metabolites presented a larger range of FC (Fig. C). The details of all significantly differential metabolites identified in plasma are displayed in Supplementary Table . A heatmap plotted by hierarchical clustering analysis revealed changes in the abundance of plasma differential metabolites between two groups (Fig. D). KEGG enrichment analysis indicated that the plasma differential metabolites were mainly involved in protein digestion and absorption, taurine and hypotaurine metabolism, amino acids metabolism, pyrimidine metabolism, linoleic acid metabolism, alpha-linolenic acid metabolism, etc. (Fig. E). Identification and characterization of plasma signature differential metabolites between healthy and SM cows We conducted machine learning analysis on the top100 differential metabolites in plasma to further distinguish the signature differential metabolites between healthy and SM cows. The results showed that the abundance of monolinolein, N-cyclopropyl-3-(4-fluorophenoxy)propane-1-sulfonamide, diethylthiophosphate, L-cysteine, 9-hydroxy-10,12-octadecadienoic acid in the plasma of cows in SM groups were significantly higher than those in CON group(Fig. A). While, the abundance of 2,5-dihydro-2,4,5-trimethyloxazole, 2-(2-amino-4-ethyl-6-oxopyrimidin-1-yl)-N-butylacetamide, 12,13-EODE, Glu Thr Phe Arg, Ala Ile Gln Arg Thr etc. were prominently elevated in the plasma of cows in CON group compared with SM group (Fig. A). All these differential metabolites with relatively high feature importance scores in random forest model could potentially serve as signature differential metabolites in the plasma of cows in CON and SM groups (Fig. A). Furthermore, we estimated the signature and predictive power of differential metabolites by applying ROC analysis, among which the AUC of hippuric acid, monolinolein, 9-hydroxy-10,12-octadecadienoic acid, myristic acid, 12-hydroxydodecanoic acid, 5,6-dihydroxy PGF1α, 5,6-dehydro arachidonic acid and stearoyl serotonin were all above 0.85, indicating their potential as promising biomarkers for SM in dairy cows (Fig. B). The correlation analysis of differential intestinal microbes with metabolites in feces and plasma To further explore the relationships between gut microbiome, fecal or plasma metabolome and SM in dairy cows, Spearman’s correlation analysis and significance test were performed. The heatmap of correlations between fecal differential metabolites and differential microorganisms at several taxonomic levels in feces of dairy cows in CON and SM groups was shown in Fig. A. As for the intestinal predominant bacteria in dairy cows of SM group, Cyanobacteria and Succinivibrio were negatively correlated with amino acid metabolites and analogues (L-arginine, glycylleucine, L-3-cyanoalanine, 3-methyl-L-tyrosine). Proteobacteria and Succinivibrio were positively correlated with 3-epiecdysone, alpha-tocotrienol, dodecanedioic acid, 2-amino-2-deoxyisochorismate and adenosine, but negatively correlated with glycylleucine. With regards to the microorganisms significantly enriched in the intestinal tract of dairy cows in CON group, Paraprevotella and Coprococcus were negatively associated with manumycin A and cis-1,2-dihydronaphthalene-1,2-diol, and positively associated with fatty acid metabolites (11,12-DiHETrE, 5,6-DHET, 12,13-DHOME), amino acid metabolites and analogues (glycylleucine, 3-methyl-L-tyrosine). Furthermore, Succiniclasticum and Desulfovibrio were negatively associated with Manumycin A, fatty acid metabolites (9(S)-HPODE, dodecanedioic acid), while positively associated with amino acid metabolites and analogues (L-arginine, glycylleucine, 3-methyl-L-tyrosine). Bifidobacterium_pseudolongum showed negative correlations with manumycin A and deoxycytidine in feces (Fig. A). The correlation heatmap of fecal differential microbes and plasma differential metabolites, milk SCC of dairy cows in CON and SM groups was displayed in Fig. B. Cyanobacteria and Lactobacillus_iners in feces were positively correlated with plasma differential metabolites from linoleic acid metabolism pathway, including 9-hydroxy-10,12-octadecadienoic acid, monolinolein and alpha-linolenic acid, and arachidonic acid metabolism pathway, involving 13,14-dihydro PGF1α, 5,6-dehydro arachidonic acid, as well as metabolites related to fatty acid metabolism, such as myristic acid, 12-hydroxydodecanoic acid, 12-hydroxyoctadecanoic acid, dodecanoic acid. Proteobacteria was positively correlated with hippuric acid, myristic acid, taurine, alpha-linolenic acid, 12-hydroxyheptadecanoic acid, 5,6-dehydro arachidonic acid in plasma. Succinivibrio was positively associated with plasma amino acid metabolites or analogues (tyrosyl-hydroxyproline, L-methionine), taurine and SCC of milk. Succiniclasticum and Desulfovibrio , which were dominant in the intestinal tract of dairy cows in CON group, showed negative correlations with plasma linoleic acid metabolites (9-hydroxy-10,12-octadecadienoic acid, alpha-linolenic acid), histamine, 2-hydroxymyristic acid, 12-hydroxyheptadecanoic acid, stearoyl serotonin, taurine and SCC. Paraprevotella was positively associated with plasma fatty acid metabolites (9,10,13-TriHOME, myristoleic acid) and oligopeptides (Ile Glu Val His Gly, His Glu Tyr Lys), while negatively associated with histamine, hippuric acid, tyrosyl-hydroxyproline, monolinolein and SCC. Moreover, Bifidobacterium_pseudolongum was positively associated with 9,10,13-TriHOME, N-stearoyl GABA, whereas negatively correlated with hippuric acid and SCC (Fig. B). Interestingly, L-3-cyanoalanine, 12,13-DHOME and dodecanedioic acid were overlapped differential metabolites between feces and plasma (Supplementary Fig. ). Specifically, L-3-cyanoalanine and dodecanedioic acid showed opposite expression patterns in feces and plasma of SM cows, while, the abundance of 12,13-DHOME in both feces and plasma presented the same regulatory trend.
The content of TC and HDLC in the serum of SM cows were significantly lower than those in CON group ( P < 0.05), the UA ( P = 0.063) and LDLC ( P = 0.055) concentration tended to decrease in SM group. Whereas, there were no significant differences in serum levels of TP, ALB, GLB, AKP, TG, NEFA and LDH between CON and SM groups ( P > 0.05) (Table ). T-AOC ( P < 0.05) and CAT ( P < 0.01) activities were remarkably reduced, while the concentration of MDA ( P < 0.05) was notably increased in the serum of SM cows compared with CON group. No significant differences of SOD and GSH-PX activities were observed among CON and SM groups ( P > 0.05) (Table ). In addition, the serum IgA, IgG, IgM, TNF-α, IL-1β, IL-2, IL-8 and IL-10 concentration showed no significant differences between CON and SM groups ( P > 0.05) (Table ).
A total of 1,550,335 raw reads were acquired from 16 stool samples through 16 S rDNA sequencing, which were then low-quality filtered, denoised, merged and chimera removed to yield 809,370 high-quality sequences. Whereafter, 26,634 ASVs were obtained from each sample after normalization based on a 97% sequence similarity threshold. The rarefaction curves of observed species tended to flatten gradually as the depth of sequencing increased, which indicated that the sequencing in this study have covered and reflected the vast majority of microbial species in samples, increasing the number of sequencing furtherly could detect no more microorganisms (Supplementary Fig. ). Namely, the sequencing datasets were credible, that could be applied for succedent analyses. The result of Venn diagram showed that the total intestinal ASVs of cows in SM group was higher than that of CON group, the overlapped ASVs between two groups were 4242 (Fig. B). PcoA of beta diversity based on Bray-Curtis distance presented an obvious separation of fecal microbial communities between CON and SM groups, indicating that the significant difference existed in gut microbial composition profiles of healthy and SM cows (Fig. C-D). Alpha diversity analysis, a metric used to estimate the richness and evenness of species within samples, demonstrated that the chao and shannon index of intestinal microorganisms of SM cows were significantly higher than those in CON group ( P < 0.05). There were no significant differences for observed species, pielou-e, goods_coverage and simpson indexes between CON and SM groups ( P > 0.05) (Fig. E-J). The microbial composition and structure analysis was performed to compare the relative abundance of fecal microflora at different taxonomic levels. 26,634 identified ASVs were clustered and assigned to 22 phyla and 279 genera of bacteria (Supplementary Tables and ). At phyla level, the dominant bacteria in the feces of cows in CON and SM groups were Firmicutes (59.42 ± 0.86%), Bacteroidetes (36.35 ± 0.96%) and Tenericutes (1.40 ± 0.07%), the sum of their abundance accounted for 97.17% of the overall abundance (Supplementary Table ; Supplementary Fig. A). At genus level, the bacteria with relative abundance > 1% were deemed to be predominant, which occupied more than 87% of the total abundance. The unidentified_Ruminococcaceae (31.45 ± 0.68), unidentified_Bacteroidales (15.68 ± 0.46), unclassified_Clostridiales (4.23 ± 0.14), unidentified_Rikenellaceae (3.54 ± 0.16), unidentified_RF16 (3.30 ± 0.21), unidentified_Lachnospiraceae (2.24 ± 0.12), Oscillospira (2.09 ± 0.070), f__Clostridiaceae__g__Clostridium (1.92 ± 0.12), Ruminococcus (1.88 ± 0.078) and Paludibacter (1.12 ± 0.045) were the most abundant genus in the feces of cows in CON and SM groups(Supplementary Table ; Supplementary Fig. B).
LEfSe analysis and wilcoxon rank-sum test were further devoted to identify the representative differential microbes between CON and SM groups. The cladogram and score plot of LEfSe revealed that the bacteria of phyla Cyanobacteria ( P < 0.05) and Proteobacteria ( P < 0.05) were significantly enriched in the gut of cows in SM group (Fig. A-B). At genus level, Coprococcus ( P < 0.05), Succiniclasticum ( P < 0.05), Desulfovibrio ( P < 0.05) and Paraprevotella ( P < 0.01) were remarkably more abundant in the feces samples of CON group. Whereas, the gut of cows in SM group harbored more Succinivibrio ( P < 0.05) in comparison with CON group (Fig. A-B). Moreover, the relative abundance of Desulfovibrio_D168 ( P < 0.05) and Bifidobacterium_pseudolongum ( P < 0.05) in SM group were significantly lower than that of CON group, while Lactobacillus_iners ( P < 0.05) showed higher relative abundance in the gut of cows in SM group compared with CON group (Fig. C).
The predicted function of gut microbiota in healthy and SM cows were inferred by PICRUSt2, which indicated that the microflora related to fatty acid and lipid biosynthesis was significantly enhanced in SM group ( P < 0.0001), the capacity of secondary metabolite degradation was dramatically declined in SM group ( P < 0.001). Additionally, the gut of cows in CON group tended to enrich more abundant microbes correlated with nucleic acid processing ( P = 0.065) and alcohol degradation( P = 0.097). Nevertheless, the relative abundance of microflora associated with pyrimidine deoxyribonucleotides de novo biosynthesis ( P = 0.088), superpathway of L-aspartate and L-asparagine biosynthesis( P = 0.089) tended to be predominant in the gut of cows in SM group (Fig. ).
Non-targeted metabolomics technique using ultra performance liquid chromatography coupled to high-resolution mass spectrometry was employed to investigate the fecal metabolomic characteristics of healthy and SM cows. In the current study, a total of 623 metabolites including 503 at positive and 120 at negative ion mode, were identified in the feces samples of dairy cows in CON and SM groups. Subsequently, the identified metabolites were statistically analyzed at subclass level, which was displayed as a donut chart (Supplementary Fiure ). The amino acids, peptides, and analogues (9.0%), fatty acids and conjugates (6.1%), carbohydrates and carbohydrate conjugates (3.7%), eicosanoids (3.0%), purines and purine derivatives (1.9%), benzoic acids and derivatives (1.9%), lineolic acids and derivatives (1.6%), triterpenoids (1.4%), bile acids, alcohols and derivatives (1.3%), hydroxycinnamic acids and derivatives (1.0%), etc. constituted the major components of metabolites in feces samples. The PLS-DA analysis, based on the abundance of identified metabolites in each sample, suggested that the contribution of PC1 and PC2 to the variation were 20.5% and 14.2%, respectively, under the positive ion mode. In the negative ion mode, PC1 and PC2 accounted for 18.7% and 12.2% of the variation, respectively (Fig. A-B). In addition, the PLS-DA score plots showed that the samples from CON and SM groups can be effectively distinguished under both positive and negative ion modes, indicating that the fecal metabolite profiles differentiated between CON and SM groups (Fig. A-B). In total, 57 significant differential metabolites comprising 27 upregulated and 30 downregulated metabolites were screened in the feces samples of CON and SM groups, that were visualized by constructing a volcano plot to exhibit the distribution and fold changes of differential metabolites (Fig. C). The detailed information of all identified significantly differential metabolites in feces are listed in Supplementary Table . A heatmap depicting the fecal differential metabolites between CON and SM groups is shown in Fig. D, which revealed that there were obvious partitions in the composition of differential metabolites between the two groups. Thereafter, we conducted KEGG pathway enrichment analysis of the differential metabolites. The fecal differential metabolites identified between two groups were mainly enriched in the pathways, such as linoleic acid metabolism, steroid biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis, mTOR and PPAR signaling pathway, arachidonic acid metabolism and amino acid metabolism (Fig. E).
In order to screen for the metabolites with significant representative characteristics, the machine learning analysis was performed on the fecal differential metabolites between CON and SM groups. The results showed that the abundance of glycylleucine, L-3-cyanoalanine, 3-methyl-L-tyrosine, 2-(Methylamino)benzoic acid, N(6)-methyllysine, and Gamma-tocotrienol etc. in the feces of cows in CON group were markedly higher than those in SM group. Nevertheless, the abundance of 3-epiecdysone, 25-hydroxycholesterol, 9(S)-HPODE, 2-amino-2-deoxyisochorismate, etc. were observably increased in SM group compared with CON group (Fig. A). Additionally, the feature importance of these differential metabolites in random forest model and its contribution for classification were relatively high, that might be signature differential metabolites in the feces of cows in CON and SM groups (Fig. A). ROC analysis, an established statistical approach for assessing the predictive power and diagnostic value, also identified several differential metabolites exhibiting potential as biomarkers of SM in dairy cows. The area under curve (AUC) of 9(S)-HPODE, 25-hydroxycholesterol, deoxycytidine, dodecanedioic acid and are all greater than 0.85, which suggested that these 4 fecal differential metabolites may be valuable indicators for SM in dairy cows (Fig. B).
The metabolic profile in blood can accurately and quickly reflect the physiological and pathological state of the body, and the changes in blood metabolites composition are closely correlated with the overall health status of body. Therefore, the characteristics of plasma metabolomics of healthy and SM cows were also profiled in the current study. 6753 metabolites containing 3103 at positive and 3650 at negative ion mode, were identified in the plasma samples of cows in CON and SM groups through mapping with the public and a self-established database. At subclass level, these metabolites can be classified in to several major categories, such as amino acids, peptides, and analogues (4.78%), fatty acids and conjugates (2.47%), carbohydrates and carbohydrate conjugates (1.36%), sesquiterpenoids (0.77%), benzoic acids and derivatives (0.58%), bile acids, alcohols and derivatives (0.52%), glycerophosphocholines(0.46%), eicosanoids(0.41%), lineolic acids and derivatives(0.41%), purines and purine derivatives(0.31%) (Supplementary Fiure ). The score plots of PLS-DA analysis showed that the symbols representing plasma samples were completely separated under both positive and negative mode, suggesting significant differences in the composition of plasma metabolites between CON and SM groups (Fig. A-B). A total of 905 significantly differential metabolites were identified between the two groups, among which 457 metabolites were upregulated and 448 metabolites were downregulated (Fig. C). Moreover, the volcano plot illustrating differential metabolites between groups demonstrated that the vast majority of differential metabolites had relatively concentrated FC, and some of significantly upregulated or downregulated metabolites presented a larger range of FC (Fig. C). The details of all significantly differential metabolites identified in plasma are displayed in Supplementary Table . A heatmap plotted by hierarchical clustering analysis revealed changes in the abundance of plasma differential metabolites between two groups (Fig. D). KEGG enrichment analysis indicated that the plasma differential metabolites were mainly involved in protein digestion and absorption, taurine and hypotaurine metabolism, amino acids metabolism, pyrimidine metabolism, linoleic acid metabolism, alpha-linolenic acid metabolism, etc. (Fig. E).
We conducted machine learning analysis on the top100 differential metabolites in plasma to further distinguish the signature differential metabolites between healthy and SM cows. The results showed that the abundance of monolinolein, N-cyclopropyl-3-(4-fluorophenoxy)propane-1-sulfonamide, diethylthiophosphate, L-cysteine, 9-hydroxy-10,12-octadecadienoic acid in the plasma of cows in SM groups were significantly higher than those in CON group(Fig. A). While, the abundance of 2,5-dihydro-2,4,5-trimethyloxazole, 2-(2-amino-4-ethyl-6-oxopyrimidin-1-yl)-N-butylacetamide, 12,13-EODE, Glu Thr Phe Arg, Ala Ile Gln Arg Thr etc. were prominently elevated in the plasma of cows in CON group compared with SM group (Fig. A). All these differential metabolites with relatively high feature importance scores in random forest model could potentially serve as signature differential metabolites in the plasma of cows in CON and SM groups (Fig. A). Furthermore, we estimated the signature and predictive power of differential metabolites by applying ROC analysis, among which the AUC of hippuric acid, monolinolein, 9-hydroxy-10,12-octadecadienoic acid, myristic acid, 12-hydroxydodecanoic acid, 5,6-dihydroxy PGF1α, 5,6-dehydro arachidonic acid and stearoyl serotonin were all above 0.85, indicating their potential as promising biomarkers for SM in dairy cows (Fig. B).
To further explore the relationships between gut microbiome, fecal or plasma metabolome and SM in dairy cows, Spearman’s correlation analysis and significance test were performed. The heatmap of correlations between fecal differential metabolites and differential microorganisms at several taxonomic levels in feces of dairy cows in CON and SM groups was shown in Fig. A. As for the intestinal predominant bacteria in dairy cows of SM group, Cyanobacteria and Succinivibrio were negatively correlated with amino acid metabolites and analogues (L-arginine, glycylleucine, L-3-cyanoalanine, 3-methyl-L-tyrosine). Proteobacteria and Succinivibrio were positively correlated with 3-epiecdysone, alpha-tocotrienol, dodecanedioic acid, 2-amino-2-deoxyisochorismate and adenosine, but negatively correlated with glycylleucine. With regards to the microorganisms significantly enriched in the intestinal tract of dairy cows in CON group, Paraprevotella and Coprococcus were negatively associated with manumycin A and cis-1,2-dihydronaphthalene-1,2-diol, and positively associated with fatty acid metabolites (11,12-DiHETrE, 5,6-DHET, 12,13-DHOME), amino acid metabolites and analogues (glycylleucine, 3-methyl-L-tyrosine). Furthermore, Succiniclasticum and Desulfovibrio were negatively associated with Manumycin A, fatty acid metabolites (9(S)-HPODE, dodecanedioic acid), while positively associated with amino acid metabolites and analogues (L-arginine, glycylleucine, 3-methyl-L-tyrosine). Bifidobacterium_pseudolongum showed negative correlations with manumycin A and deoxycytidine in feces (Fig. A). The correlation heatmap of fecal differential microbes and plasma differential metabolites, milk SCC of dairy cows in CON and SM groups was displayed in Fig. B. Cyanobacteria and Lactobacillus_iners in feces were positively correlated with plasma differential metabolites from linoleic acid metabolism pathway, including 9-hydroxy-10,12-octadecadienoic acid, monolinolein and alpha-linolenic acid, and arachidonic acid metabolism pathway, involving 13,14-dihydro PGF1α, 5,6-dehydro arachidonic acid, as well as metabolites related to fatty acid metabolism, such as myristic acid, 12-hydroxydodecanoic acid, 12-hydroxyoctadecanoic acid, dodecanoic acid. Proteobacteria was positively correlated with hippuric acid, myristic acid, taurine, alpha-linolenic acid, 12-hydroxyheptadecanoic acid, 5,6-dehydro arachidonic acid in plasma. Succinivibrio was positively associated with plasma amino acid metabolites or analogues (tyrosyl-hydroxyproline, L-methionine), taurine and SCC of milk. Succiniclasticum and Desulfovibrio , which were dominant in the intestinal tract of dairy cows in CON group, showed negative correlations with plasma linoleic acid metabolites (9-hydroxy-10,12-octadecadienoic acid, alpha-linolenic acid), histamine, 2-hydroxymyristic acid, 12-hydroxyheptadecanoic acid, stearoyl serotonin, taurine and SCC. Paraprevotella was positively associated with plasma fatty acid metabolites (9,10,13-TriHOME, myristoleic acid) and oligopeptides (Ile Glu Val His Gly, His Glu Tyr Lys), while negatively associated with histamine, hippuric acid, tyrosyl-hydroxyproline, monolinolein and SCC. Moreover, Bifidobacterium_pseudolongum was positively associated with 9,10,13-TriHOME, N-stearoyl GABA, whereas negatively correlated with hippuric acid and SCC (Fig. B). Interestingly, L-3-cyanoalanine, 12,13-DHOME and dodecanedioic acid were overlapped differential metabolites between feces and plasma (Supplementary Fig. ). Specifically, L-3-cyanoalanine and dodecanedioic acid showed opposite expression patterns in feces and plasma of SM cows, while, the abundance of 12,13-DHOME in both feces and plasma presented the same regulatory trend.
In the current study, feces and plasma samples from healthy and SM cows were collected to analyze the differences in gut microbiome and metabolism, thereby exploring the relationship between intestinal microorganisms and their metabolites and udder health status, as well as dissecting the potential pathological metabolic mechanisms of SM. Firstly, we compared the differences in blood parameters between healthy and SM cows. The results indicated that the content of TC and HDL-C, the activity of CAT and T-AOC in the serum of SM cows were dramatically lower than those of healthy cows, while, the content of MDA in SM cows was significantly increased, which were consistent with the previous findings from Turk et al. and Sadek et al. . All these results provided evidences for the existence of oxidative damage and redox imbalance in SM cows. The gastrointestinal microbiota are essential regulators implicated in nutrient digestion, energy metabolism and immune response of host, which have been confirmed to be associated with multiple infectious and metabolic diseases . In the present study, we compared the difference of fecal microbial composition and function between healthy and SM cows. PCoA analysis based on Bray-Curtis distance indicated significant differences in gut microbial composition and structure between the two groups. The alpha diversity analysis revealed that the chao1 index and shannon index of intestinal microorganisms of SM group were significantly higher than those in CON group, indicating the higher diversity and richness of microflora in the gut of SM cows. The investigation on rumen microbial composition in cows with different SCC demonstrated that the shannon index in cows with high SCC was remarkably higher than cows with low SCC . The chao1, sob and ace indices of ruminal microbiota were found to be prominently elevated in mastitic cows compared with healthy cows . The results of the above two studies are similar to our findings. The taxonomic profiling of intestinal microbiota at phylum level showed that Firmicutes , Bacteroidetes and Tenericutes were the most predominant bacterial phyla in the gut of cows in CON and SM groups, which are in congruency with the results from Hu and Jiang et al. . However, the relative abundance of these three bacterial phyla presented no significant difference between the two groups, which suggested that the microbes playing an essential role in the gut of cows remained relatively stable at phylum level. LEfSe analysis showed that Cyanobacteria , Proteobacteria , Succinivibrio and Lactobacillus_iners were significantly enriched in the gut of SM cows, whereas, Paraprevotella , Coprococcus , Succiniclasticum , Desulfovibrio and Bifidobacterium_pseudolongum were remarkably enhanced in the gut of cows in CON group. These fecal differential microbes might be the marker microorganisms distinguishing SM cows from healthy cows. Proteobacteria and Succinivibrio with high abundance have also been identified in the gut of cows with CM induced by SARA . Among the 16 differential microbes identified by Zhao et al., which were significantly enriched in the intestines of mastitic cows, 8 belong to Proteobacteria , including Succinivibrionacea . Succinivibrio has been confirmed to be one of the core bacterial taxa in the rumen of dairy cows and negatively correlated with milk yield . Cyanobacteria was known as a photosynthetic gram-negative bacteria that were presumed to originate from freshwater or terrestrial environment, has also been proved to be significantly enriched in the rumen of cows with CM . Nevertheless, the investigation on the regulatory role of Cyanobacteria as intestinal microbe in host metabolism has been limited, and its correlation with mastitis remains unclear. Lactobacillus_iners , an obligate L-lactate-producing bacterium, was discovered to have a markedly higher relative abundance in the gut of mice with diabetic nephropathy . Additionally, it was associated with bacterial vaginitis in females and considered to be an opportunistic pathogen . In this study, the relative abundance of Paraprevotella , Coprococcus , Succiniclasticum , Desulfovibrio and Bifidobacterium_pseudolongum in the gut of SM cows were prominently reduced. Another previous research has shown that the abundance of Desulfovibrio and Bifidobacterium_pseudolongum were markedly decreased in the rumen of cows with CM, besides, the administration of Bifidobacterium_pseudolongum could restore the weight loss of germ-free mice caused by FMT from mastitic cows, and significantly reduce their mortality . Paraprevotella is an intestinal symbiotic bacterium isolated from feces of healthy human dornors recently, which could recruit trypsin to the bacterial surface to protect sIgA from degradation by trypsin, playing a crucial role in anti-infection and intestinal immunity of host . Succiniclasticum , a core bacterial community in the rumen of ruminants, which is specialized in fermenting and converting succinate into propionate, as well as enhancing the bioavailability of butyrate and correlating positively with feed efficiency in host . Coprococcus , being the primary butyrate producing bacteria, which could not only enhance the intestinal barrier function by upregulating the expression of claudin-1 and ZO-1 , but also increase the secretion of IL-18 by activating GRP109A in intestinal epithelia cells through its metabolite butyrate, thus inhibiting the occurrence of inflammation . Furthermore, the report from Zhao et al. also proved that Coprococcus was notably enriched in the gut of healthy cows , which agree with our results. Recently, an investigation on the intestinal microbial characteristics in 45 cohorts involving 2518 patients with inflammatory bowel disease (IBD), indicated that the abundance of Paraprevotella , Coprococcus and Desulfovibrio were memorably decreased in the gut of patients with Crohn’s disease or ulcerative colitis . Correlation analysis showed that the genus Paraprevotella , Succiniclasticum and Desulfovibrio were significantly negatively associated with SCC, Succinivibrio showed signally positive correlation with SCC. Moreover, species Bifidobacterium_pseudolongum was notably negatively associated with SCC, that was in accordance with the results from Hsieh . Collectively, the reduction in the abundance of these symbiotic bacteria or potential probiotics accompanied by the enrichment of potential pathogenic bacteria in the gut of cows might contribute to the development of SM. The fecal and blood metabolome are important tools for exploring the linkage between gut microbiome and host health, which were considered to possess important application value in the early prediction of diseases and research on pathological mechanisms . The fecal differential metabolites between healthy and SM cows are mainly enriched in lipid metabolism-related pathways, such as linoleic acid metabolism and steroid synthesis, which mutually corroborates with the results from PICRUSt in predicting the significant enhancement of lipid metabolism ability of gut microbiota in SM cows. The relative abundance of fecal differential metabolites related with lipid metabolism in SM cows were dramatically increased, including 9(S)-HPODE, 25-hydroxycholesterol, dodecanedioic acid and prostaglandin B1. The lipid peroxidation process of linoleic acid is correlated with oxidative stress and many inflammatory diseases, such as atherosclerosis and rheumatoid arthritis . 9(S)-HPODE, one of the lipid peroxidation products of linoleic acid, is rapidly reduced into stable 9-HODE in the body . The content of 9-HODE was memorably elevated in tissues of cows with mastitis caused by intramammary Streptococcus uberis infection . The correlation analysis showed that fecal 9(S)-HPODE was positively associated with Cyanobacteria , whereas, negatively correlated with Succiniclasticum and Desulfovibrio . 25-hydroxycholesterol is an oxidation product of cholesterol mediated by 25-hydroxylase, and its secretion is closely related to inflammation and immune status of tissues and organs . 25-hydroxycholesterol secreted by macrophages in response to toll-like receptor activation has been certified to modulate the production of IgA . The secretion of IL-6 and IL-8 can be upregulated by 25-hydroxycholesterol via enhancing NF-κB signaling, thus amplifying the inflammatory response . Dodecanedioic acid, a dicarboxylic acid involved in a metabolic pathway intermediate between lipids and carbohydrates, can be used as an alternate fuel substrate and promptly oxidized to produce succinic acid for gluconeogenesis in type 2 diabetes . Nevertheless, few studies have been reported on the role of dodecanedioic acid in inflammatory diseases. Prostaglandin B1, a derivative of arachidonic acid, is a metabolite of prostaglandin E1. Prostaglandins (PGs) not only mediate the acute inflammatory response, but also implicate in a variety of chronic inflammatory diseases through synergistically interacting with cytokines to activate NF-κB and induce the expression of inflammation-related genes . The fecal Desulfovibrio was negatively associated with Prostaglandin B1. Mandelic acid is an alpha hydroxy-carboxylic acid with antioxidant and anti-inflammatory activity that reduces the production of pro-inflammatory mediators and oxygen free radicals . The markedly reduced abundance of mandelic acid in SM cows was positively associated with Succiniclasticum . Gamma-tocotrienol has been proved to not only inhibit the activation of MAPK, NF-κB signaling pathway and polarization of M1 macrophage, reduce systemic inflammatory response to improve insulin resistance induced by high fat diet , but also ameliorate inflammation in liver via suppressing endoplasmic reticulum stress and the expression of IL-1β . The observably decreased abundance of gamma-tocotrienol in SM cows was negatively associated with Proteobacteria and Lactobacillus_iners , while positively correlated with Paraprevotella and Coprococcus . With regards to the plasma metabolome in the current study, the relative abundance of linolenic acid metabolism related compounds, such as 9-hydroxy-10,12-octadecadienoic acid, monolinolein and alpha-linolenic acid, were significantly enhanced in SM cows. The increased abundance of 9-hydroxy-10,12-octadecadienoic acid(9-HODE) in plasma echoed with the elevated abundance of 9(S)-HPODE in feces. Alpha-linolenic acid, a precursor for the synthesis of DHA and EPA, has been certified to restrain the activation of NF-κB signaling, and reduce the expression of pro-inflammatory cytokines and oxidative stress to alleviate inflammation . The increased abundance of plasma alpha-linolenic acid in SM cows might be related to the defense against inflammation. The relative abundance of arachidonic acid metabolites related with inflammation in the plasma of SM cows were memorably increased, including 13,14-dihydro PGF1α, 12-oxo-10,15(Z)-phytodienoic acid, 5,6-dihydroxy PGF1α and 5,6-dehydro arachidonic acid. Among them, both of 13, 14-dihydro PGF1α and 5,6-dehydro arachidonic acid were positively correlated with Cyanobacteria and Lactobacillus_iners . Histamine plays a decisive role in immune response and neurotransmission, moreover, the histamine-secreting bacteria are notably enriched in the gut of patients with IBD . The significantly increased abundance of histamine in the plasma of SM cows were positively associated with Cyanobacteria and Lactobacillus_iners . Myristic acid, a medium-chain saturated fatty acid signally increased in the plasma of SM cows in this study, has been demonstrated to regulate the synthesis of TG in MAC-T cells through the ubiquitination pathway , as well as exacerbate the inflammation of adipose tissue induced by high fat diet in mice . Therefore, we presume that the enhancement of myristic acid in plasma of cows fed a high-concentrate diet during lactation chronically might be disadvantageous for the maintenance of udder health. Hippuric acid derived from the metabolism of benzoic acid by gut microbes, is significantly reduced in the blood and urine of patients with IBD . The colitis of mice induced by DSS is relieved by hippuric acid via increasing the abundance of beneficial gut bacteria and alleviating intestinal barrier damage . The significant increased plasma hippuric acid might be related to the anti-inflammatory self-regulation of the body. Bile acids has been verified to play a fundamental role in the modulation of intestinal microflora, carbohydrate and lipid metabolism, as well as host innate immunity . Deoxycholic acid, one of the most common secondary bile acids produced by intestinal microbes via the metabolism of cholic acid, ameliorate acute and chronic colitis in mice through activating G-protein coupled bile acid receptor 1 (GPBAR1), and downregulating the secretion of inflammatory cytokines and chemokines . In the present study, the relative abundance of deoxycholic acid in plasma of SM cows was prominently descend, that might be unfavourable for the body’s early defense against inflammation. Summarily, the enhancement in abundance of pro-inflammatory lipids implicated in linoleic acid, arachidonic acid and cholesterol metabolism, along with the reduction in the abundance of substances with anti-inflammatory and antioxidant activities in the gut and blood of dairy cows might be correlated with the occurrence of SM.
The content of TC, HDL-C and antioxidant capacity in serum of SM cows were significantly reduced compared with healthy cows. Composition of fecal microbiota and metabolic characteristics in feces and plasma differentiated between healthy and SM cows. The lipid metabolism capacity of intestinal microbial community in SM cows was prominently heightened. The decreased abundance of multiple intestinal symbiotic bacteria or potential probiotics ( Paraprevotella , Coprococcus , Succiniclasticum , Desulfovibrio and Bifidobacterium_pseudolongum ), and increased abundance of potential pro-inflammatory microbes ( Succinivibrio , Lactobacillus_iners ), coupled with the elevation of pro-inflammatory metabolites in feces (9(S)-HPODE, 25-hydroxycholesterol, dodecanedioic acid) and plasma (9-hydroxy-10,12-octadecadienoic acid, 13,14-dihydro PGF1α, 5,6-dehydro arachidonic acid, myristic acid, histamine), as well as reduction in the anti-inflammatory and antioxidative compounds (mandelic acid, gamma-tocotrienol, deoxycholic acid), might be detrimental to the maintenance of udder health and probably related with the development of SM in cows. Nevertheless, the precise regulatory mechanisms by which theses microorganisms and metabolites regulate SM in dairy cows remain largely indistinct, that need to be further clarified in the future research. Our study enhances the understanding of the relationship between gut microbiota, metabolic profiles and SM in dairy cows.
Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2 Supplementary Material 3 Supplementary Material 4 Supplementary Material 5 Supplementary Material 6
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Shifts in fungal communities drive soil profile nutrient cycling during grassland restoration | af2aa988-1529-4998-b0b2-9897000ebcf9 | 11898603 | Microbiology[mh] | Grazing exclusion, as a “nature-based” ecoengineering measure, is used globally to restore degraded grassland ecosystems . Soil microbes play crucial roles in ecological processes, including decomposing soil organic carbon (SOC) , facilitating soil nutrient cycling , and promoting plant growth , which are recognized as key factors driving vegetation restoration . While soil microbes in topsoil have been extensively studied in the context of vegetation restoration, there is a limited understanding of whether, how, and why the soil microbial community and its functions in the subsoil vary throughout this process. Investigating dynamic changes in the soil microbial community and its functions in the soil profile could enhance our knowledge of the vertical turnover of soil microbes following grassland restoration. Vegetation restoration impacts the soil microbe community and function through both direct and indirect effects on biotic and abiotic factors . In general, vegetation restoration increases above- and belowground biomass , thereby increasing SOC inputs and nutrient availability . Driven by changes in soil substrates and plant communities following vegetation restoration, soil microbial community composition and life strategies can adjust simultaneously with environmental changes . Moreover, the corresponding functions of soil microbes also shift, such as the decline in C degradation gene abundances along with long-term restoration, due to changes in litter characteristics and SOC . In general, bacteria excel in terms of environmental adaptability and the acquisition of nutrient sources , whereas fungi are superior in terms of enzyme degradation of SOC ; thus, bacterial and fungal communities exhibit different responses to restoration . However, the relative roles of bacterial and fungal communities in soil nutrient cycling during grassland restoration remain to be revealed. Previous studies have addressed subsoils as entities that are distinct from the surface due to different environmental peculiarities (e.g., inputs of fresh SOC, soil aeration, and substrate availability) . Microorganisms in topsoil utilize C sources more easily than those in subsoil because of the greater input of C from litter and root exudates, which results in a faster C decomposition rate . The subsoil is far more substrate limited, as less available substrate is delivered from plants . Thus, microbial biomass often decreases sharply with increasing soil depth, but the effects of soil microbes on biogeochemical processes persist throughout the soil profile . Following grassland restoration, plants progressively develop deeper and more complicated root systems with the replacement of plant species , impacting the input of fresh soil C and substrate availability in the soil profile . These substrates provide primary C and energy sources for subsoil microorganisms and increase the growth and evolution of these microbes . This further impacts the functions and structure of soil microbial communities ; however, it is unclear whether the microbial communities in the soil profile respond consistently to vegetation restoration. Therefore, elucidating the variations in the soil microbial community and functions in the soil profile could increase our understanding of the role that microbes play in the subsoil in SOC and nutrient accumulation during the vegetation restoration. A comprehensive understanding of the changes in soil microbial communities and functions in the soil profile following grassland restoration can help reveal the role of bacteria and fungi in promoting soil nutrient cycling. In this study, we investigated the changes in bacterial and fungal communities and nutrient cycling gene abundance in one grazed grassland and four grasslands with different fenced years (5, 15, 28, and 36 years). We hypothesize that (i) bacterial and fungal communities in the soil profile respond differently to grassland restoration; (ii) changes in the assembly processes of bacterial and fungal communities are driven differently by restoration years and soil physicochemical properties; and (iii) fungal communities might play a more important role in changes in nutrient cycling functional genes during grassland restoration.
Soil physicochemical properties and microbial community changes in the soil profile during vegetation restoration The vegetation coverage increased from 64.7% to 90.3% after 36 years of grazing exclusion , and the SOC increased by 76.0% in the topsoil (0–40 cm) and by 91.6% in the subsoil (40–100 cm). In addition, the total nitrogen (TN) content also increased from 1.69 to 2.92 g kg −1 in the topsoil and from 0.96 to 1.81 g kg −1 in the subsoil . Compared with those of the topsoil, the soil physicochemical properties of the subsoil showed more pronounced variations ( P < 0.01) across restoration years , and the differences in the soil physicochemical properties between the topsoil and the subsoil decreased as the vegetation restoration progressed . Soil depth and restoration years strongly impacted both the bacterial and fungal communities. The bacterial community appeared to be more strongly influenced by soil depth ( R 2 = 0.316, P < 0.001) than restoration year ( R 2 = 0.245, P < 0.001), whereas the fungal community was more sensitive to restoration year ( R 2 = 0.304, P < 0.001) than to soil depth ( R 2 = 0.164, P < 0.001) . The alpha diversity of the bacterial and fungal communities decreased as the soil depth increased . Interestingly, the differences in the microbial community spatial turnover rates and compositions among the different soil depths decreased with increasing restoration years , which exhibited varying correlations with the soil physicochemical properties . A total of 73% bacterial and 36% fungal amplicon sequence variants (ASVs) were detected in all the soil samples during vegetation restoration . Thus, we identified the important ASVs that correlated with restoration year using a random forest model with 10-fold cross-validation, and 397 bacterial species and 131 fungal species were distinguished as biomarker taxa in the model in order of the degree of discriminatory importance of the restoration year . The Shannon index of the restoration-discriminant bacteria was lower in the early stage (0 to 5 years) and then reached a steady value at the later stage (15 to 36 years), whereas the Shannon index of the restoration-discriminant fungi gradually increased from the early to the late restoration stage . Assembly process and life strategy shifts of the microbial community in the soil profile during restoration The bacterial assembly processes were driven primarily by dispersal limitation at 0–5 years (72.0%), whereas the variable selection increased to 58.4% at 5–15 years and 55.4% at 15–25 years . In contrast, fungal assembly processes were dominated by variable selection at 0–5 years (45.5%) and 5–15 years (46.8%), and then shifted to undominated processes at 25–36 years (65.8%) . Moreover, the deterministic assembly process of the bacterial and fungal communities decreased from 0 to 80 cm and then increased in the soil at 80–100 cm . The ribosomal RNA gene operon (rrn) copy number was used to distinguish the ecological strategies of bacterial communities, and a relatively high rrn copy number in bacteria is typically a feature of eutrophic bacteria ( r -strategy), whereas a relatively low rrn copy number can be a characteristic of oligotrophic bacteria ( K -strategy). The rrn copy numbers initially decreased from G0 to G5 but subsequently increased to G36 . Interestingly, the rrn copy numbers decreased and tended to favor the K -strategy in the subsoil . Furthermore, the ratio of labile (starch and hemicellulose) to recalcitrant carbon functional gene abundance (cellulose hydrolysis, chitin hydrolysis, pectin hydrolysis, and lignin hydrolysis) also exhibited the same trend as the rrn copy numbers of the microbial communities . Nutrient cycling functional gene abundance changes in the soil profile during restoration In the study, a total of 71 microbial functional genes related to C, N, P, and S cycling were detected via qPCR. The absolute abundance of functional genes first increased, and peaked at G15 and G28, and then decreased as the restoration progressed , which was consistent with the change in bacterial biomass . The diversity and composition of nutrient cycling genes significantly shifted with the restoration year, and the fungal diversity (Chao1 index) gradually increased with the restoration year . Among all types of nutrient cycling functional genes, there was a distinct increase in the C fixation function (from 18.4% to 30.1%) and a decrease in the C degradation function (from 21.8% to 12.1%) from G0 to G36 . The C degradation functions of starch and hemicellulose hydrolysis increased from 3.1% in G0 to 22.2% in G36, and 22.9% in G0 to 45.0% in G36. The lignin hydrolysis gene abundance decreased from 68.3% in G0 to 18.6% in G36 . Moreover, aerobic ammoxidation genes and organic N mineralization genes increased, whereas denitrification genes decreased and organic P mineralization in the P cycle increased following restoration . In addition, the discrepancies in microbial functions between topsoil and subsoil gradually diminished during restoration . Different responses of the microbial community and functional gene abundance in topsoil and subsoil to restoration Compared with those in the topsoil layer, the microbial communities and nutrient cycling functional genes in the subsoil were largely influenced by the restoration year . Similarly, the alpha diversity (Chao1) of the bacteria, fungi, and nutrient cycling genes in the subsoil layer responded more intensely to restoration than those in the topsoil . The relative proportion of nutrient cycling genes also showed more pronounced changes in the subsoil with restoration years . However, the microbial co-occurrence networks were more complex in the topsoil than in the subsoil, and greater number of edges, negative/positive edge rates, and relative modularity of bacteria and fungi were observed in the topsoil . Moreover, the Mantel test results also revealed a stronger relationship between the bacterial and fungal communities and the soil physicochemical properties in the subsoil . Driving mechanism of the changes in the soil microbial community structure and function during restoration Structural equation modeling (SEM) was employed to gain further insight into the factors driving the changes in the soil microbial community structure and function during vegetation restoration . The SEM revealed that soil depth (path coefficient = 0.67) plays a crucial role in shaping bacterial communities ( R 2 = 0.69) through both direct and indirect influences by altering soil physicochemical properties and the bacterial assembly process , whereas the fungal community ( R 2 = 0.62) is affected by both the restoration year (path coefficient = 0.44) and the soil depth (path coefficient = 0.42). The year of restoration influences the fungal community indirectly through its impact on fungal assembly, whereas soil depth directly influences the fungal community . SEM also revealed that nutrient cycling functional genes are driven primarily by changes in the fungal assembly process (path coefficient = 0.30), fungal communities (path coefficient = 0.24), and years of restoration (path coefficient = 0.19) , whereas shifts in the bacterial community negatively affect nutrient cycling.
The vegetation coverage increased from 64.7% to 90.3% after 36 years of grazing exclusion , and the SOC increased by 76.0% in the topsoil (0–40 cm) and by 91.6% in the subsoil (40–100 cm). In addition, the total nitrogen (TN) content also increased from 1.69 to 2.92 g kg −1 in the topsoil and from 0.96 to 1.81 g kg −1 in the subsoil . Compared with those of the topsoil, the soil physicochemical properties of the subsoil showed more pronounced variations ( P < 0.01) across restoration years , and the differences in the soil physicochemical properties between the topsoil and the subsoil decreased as the vegetation restoration progressed . Soil depth and restoration years strongly impacted both the bacterial and fungal communities. The bacterial community appeared to be more strongly influenced by soil depth ( R 2 = 0.316, P < 0.001) than restoration year ( R 2 = 0.245, P < 0.001), whereas the fungal community was more sensitive to restoration year ( R 2 = 0.304, P < 0.001) than to soil depth ( R 2 = 0.164, P < 0.001) . The alpha diversity of the bacterial and fungal communities decreased as the soil depth increased . Interestingly, the differences in the microbial community spatial turnover rates and compositions among the different soil depths decreased with increasing restoration years , which exhibited varying correlations with the soil physicochemical properties . A total of 73% bacterial and 36% fungal amplicon sequence variants (ASVs) were detected in all the soil samples during vegetation restoration . Thus, we identified the important ASVs that correlated with restoration year using a random forest model with 10-fold cross-validation, and 397 bacterial species and 131 fungal species were distinguished as biomarker taxa in the model in order of the degree of discriminatory importance of the restoration year . The Shannon index of the restoration-discriminant bacteria was lower in the early stage (0 to 5 years) and then reached a steady value at the later stage (15 to 36 years), whereas the Shannon index of the restoration-discriminant fungi gradually increased from the early to the late restoration stage .
The bacterial assembly processes were driven primarily by dispersal limitation at 0–5 years (72.0%), whereas the variable selection increased to 58.4% at 5–15 years and 55.4% at 15–25 years . In contrast, fungal assembly processes were dominated by variable selection at 0–5 years (45.5%) and 5–15 years (46.8%), and then shifted to undominated processes at 25–36 years (65.8%) . Moreover, the deterministic assembly process of the bacterial and fungal communities decreased from 0 to 80 cm and then increased in the soil at 80–100 cm . The ribosomal RNA gene operon (rrn) copy number was used to distinguish the ecological strategies of bacterial communities, and a relatively high rrn copy number in bacteria is typically a feature of eutrophic bacteria ( r -strategy), whereas a relatively low rrn copy number can be a characteristic of oligotrophic bacteria ( K -strategy). The rrn copy numbers initially decreased from G0 to G5 but subsequently increased to G36 . Interestingly, the rrn copy numbers decreased and tended to favor the K -strategy in the subsoil . Furthermore, the ratio of labile (starch and hemicellulose) to recalcitrant carbon functional gene abundance (cellulose hydrolysis, chitin hydrolysis, pectin hydrolysis, and lignin hydrolysis) also exhibited the same trend as the rrn copy numbers of the microbial communities .
In the study, a total of 71 microbial functional genes related to C, N, P, and S cycling were detected via qPCR. The absolute abundance of functional genes first increased, and peaked at G15 and G28, and then decreased as the restoration progressed , which was consistent with the change in bacterial biomass . The diversity and composition of nutrient cycling genes significantly shifted with the restoration year, and the fungal diversity (Chao1 index) gradually increased with the restoration year . Among all types of nutrient cycling functional genes, there was a distinct increase in the C fixation function (from 18.4% to 30.1%) and a decrease in the C degradation function (from 21.8% to 12.1%) from G0 to G36 . The C degradation functions of starch and hemicellulose hydrolysis increased from 3.1% in G0 to 22.2% in G36, and 22.9% in G0 to 45.0% in G36. The lignin hydrolysis gene abundance decreased from 68.3% in G0 to 18.6% in G36 . Moreover, aerobic ammoxidation genes and organic N mineralization genes increased, whereas denitrification genes decreased and organic P mineralization in the P cycle increased following restoration . In addition, the discrepancies in microbial functions between topsoil and subsoil gradually diminished during restoration .
Compared with those in the topsoil layer, the microbial communities and nutrient cycling functional genes in the subsoil were largely influenced by the restoration year . Similarly, the alpha diversity (Chao1) of the bacteria, fungi, and nutrient cycling genes in the subsoil layer responded more intensely to restoration than those in the topsoil . The relative proportion of nutrient cycling genes also showed more pronounced changes in the subsoil with restoration years . However, the microbial co-occurrence networks were more complex in the topsoil than in the subsoil, and greater number of edges, negative/positive edge rates, and relative modularity of bacteria and fungi were observed in the topsoil . Moreover, the Mantel test results also revealed a stronger relationship between the bacterial and fungal communities and the soil physicochemical properties in the subsoil .
Structural equation modeling (SEM) was employed to gain further insight into the factors driving the changes in the soil microbial community structure and function during vegetation restoration . The SEM revealed that soil depth (path coefficient = 0.67) plays a crucial role in shaping bacterial communities ( R 2 = 0.69) through both direct and indirect influences by altering soil physicochemical properties and the bacterial assembly process , whereas the fungal community ( R 2 = 0.62) is affected by both the restoration year (path coefficient = 0.44) and the soil depth (path coefficient = 0.42). The year of restoration influences the fungal community indirectly through its impact on fungal assembly, whereas soil depth directly influences the fungal community . SEM also revealed that nutrient cycling functional genes are driven primarily by changes in the fungal assembly process (path coefficient = 0.30), fungal communities (path coefficient = 0.24), and years of restoration (path coefficient = 0.19) , whereas shifts in the bacterial community negatively affect nutrient cycling.
Changes in edaphic factors drive the distinct responses of bacterial and fungal communities to grassland restoration Our results indicate that the soil microbial community shifted in the 0–100 cm soil profile following grassland restoration, which was driven by soil nutrient availability in the soil profile during vegetation restoration . However, we found that the fungal community was more sensitive to restoration than the bacterial community, which is consistent with previous studies , possibly because fungi have stronger associations with plants (e.g., by serving as plant symbionts) and are largely influenced by changes in plants during restoration . In addition, soil nutrients impact the response of microbes. We found that the bacterial communities were more strongly associated with the SOC and TN levels, which are more affected by soil depth than by restoration year . However, fungal communities presented a stronger correlation with NH 4 + , the primary nitrogen form utilized by fungi, which is less influenced by soil depth, indicating that fungi are highly dependent on soil nutrient availability . According to the size-plasticity hypothesis, smaller bacteria possess a broader ecological niche and exhibit a wider range of environmental adaptations than fungi . During G0 to G5 , minor environmental changes had a weak impact on the bacterial community, as demonstrated by the weaker variable selection observed in this stage ; thus, bacteria did not significantly change during the 0 to 5 year natural restoration period . After the accumulation of nutrient resources from the G5 to G15, the bacterial community is strongly influenced by environmental changes, as demonstrated by the sudden increase in variable selection in stages G5 to G15 and G15 to G28. Conversely, even minor environmental changes can exert strong selection pressure on the fungal community, leading to sustained changes, as evidenced by the highly variable selection of the fungal assembly process from 0 to 28 years . Grassland restoration enhances microbial C fixation and reduces C degradation gene abundance Increased nutrient availability also drives changes in soil microbial nutrient cycling gene abundance during restoration . Our results revealed that grassland restoration enhances the microbial C fixation capacity, reduces the C degradation capacity, and increases functional alpha diversity . The greater C degradation ability in the early restoration stage was attributed mainly to the requirement of microbial growth, which needs to break down recalcitrant organic matter to acquire C because of the low C availability and fixation . As restoration progresses, the increased plant C input promotes the redistribution of microbial functions and energy, reducing their capacity for C degradation and enhancing their ability to acquire other nutrients to maintain their growth; thus, a greater abundance of N- and P-cycling genes was observed in the later restoration stage . The increase in soil nutrient availability also altered the life strategies of the microorganisms. Consequently, we observed an increase in the ratio of labile to recalcitrant C functional genes and rrn copy numbers during restoration . These changes indicate that microbes shift from K- strategists to r -strategists and prefer to acquire easily degradable C . Interestingly, the abundance of functional genes and bacterial biomass increased after the initial 28 years of restoration but sharply decreased at 36 years , which could be attributed to a decreased plant community biomass and diversity . Differences in the microbial community and function between topsoil and subsoil during grassland restoration The results revealed that the microbial community diversity and function in subsoil were more sensitive to restoration than those in topsoil . After grazing exclusion, the increased plant biomass increased the soil nutrient input in both the topsoil and the subsoil , subsequently triggering simultaneous changes in both microbial community and functions , which further contributed to soil nutrient retention. The positive feedback loop between the microbial community and soil nutrients promotes the recovery of the grasslands during restoration . However, the low nutrient availability of the subsoil led to a stronger relationship between soil microbes and soil nutrients . Moreover, microbes in the subsoil are more inclined to cooperate rather than compete to maximize the utilization of limited resources and adapt to environmental pressures . Compared with the topsoil, the stronger relationships and cooperation among microorganisms enhance the recovery of soil microbes in the subsoil during restoration. In addition, vegetation restoration facilitates an increase in soil nutrients in the subsoil , which enhances the stability and consistency of the soil environment, leading to a reduced spatial turnover rate of the soil microorganisms, as well as a decrease in differences in microbial composition and functional genes among soil profiles over successive years . Nutrient cycling functional gene changes are driven mainly by the fungal community and related assembly processes Nutrient cycling is predominantly influenced by the fungal assembly process, fungal community composition, and restoration year . During restoration, the fungal assembly process experiences consistently high selection pressure due to changing environmental conditions, which are specifically related to resource availability. This ongoing selection pressure significantly influences the composition and structure of fungal communities. Fungi have an efficient extracellular enzyme system and a large mycelial network, providing them with an advantage in the decomposition of complex substrates (plant residues, cellulose, etc.) . Furthermore, fungal mycelia can form symbiotic relationships with plant roots, connecting plants and soil to establish an “expressway” for C transport. This allows fungi to actively participate in the input, stabilization, and decomposition of SOC . The powerful organic matter degradation and connectivity abilities of fungi position them as the main drivers of soil nutrient cycling, effectively promoting the nutrient release of plant residues and nutrient cycling in grassland ecosystems . In contrast, bacteria play a slightly inferior role in nutrient cycling. Bacterial DNA is in a free state within the cell, and an average of 42.5% of genes in the genome are affected by horizontal gene transfer ,which allows bacteria to dynamically adjust their functions to adapt to new environmental conditions without largely altering their community composition. This poses a challenge in accurately assessing the influence of the bacterial community on functional genes. Moreover, the low R 2 (0.11) for the nutrient cycling functional genes determined by the SEM indicates that the majority of the variability in the nutrient cycling function remains unexplained. In this study, we investigated the changes in the soil microbial community and nutrient cycling functional genes across soil profiles following vegetation restoration. Our findings indicate that soil depth predominantly influences bacterial communities, whereas fungal communities are highly sensitive to the duration of restoration. Notably, microbes in the subsoil recovered faster than those in the topsoil, which contributed to a reduction in differences in microbial community structure and the distribution of functional genes throughout the soil profile during the restoration process. Importantly, the fungal community assembly process played a pivotal role in driving changes in nutrient cycling functional genes, such as increased carbon fixation and nitrogen mineralization, in addition to a reduction in the abundance of genes involved in carbon degradation. These alterations contributed to increased SOC and nutrient accumulation. Overall, our results increase the understanding of the critical role of fungal communities in influencing changes in soil nutrient cycling genes, thereby facilitating nutrient accumulation in soil profiles during grassland restoration.
Our results indicate that the soil microbial community shifted in the 0–100 cm soil profile following grassland restoration, which was driven by soil nutrient availability in the soil profile during vegetation restoration . However, we found that the fungal community was more sensitive to restoration than the bacterial community, which is consistent with previous studies , possibly because fungi have stronger associations with plants (e.g., by serving as plant symbionts) and are largely influenced by changes in plants during restoration . In addition, soil nutrients impact the response of microbes. We found that the bacterial communities were more strongly associated with the SOC and TN levels, which are more affected by soil depth than by restoration year . However, fungal communities presented a stronger correlation with NH 4 + , the primary nitrogen form utilized by fungi, which is less influenced by soil depth, indicating that fungi are highly dependent on soil nutrient availability . According to the size-plasticity hypothesis, smaller bacteria possess a broader ecological niche and exhibit a wider range of environmental adaptations than fungi . During G0 to G5 , minor environmental changes had a weak impact on the bacterial community, as demonstrated by the weaker variable selection observed in this stage ; thus, bacteria did not significantly change during the 0 to 5 year natural restoration period . After the accumulation of nutrient resources from the G5 to G15, the bacterial community is strongly influenced by environmental changes, as demonstrated by the sudden increase in variable selection in stages G5 to G15 and G15 to G28. Conversely, even minor environmental changes can exert strong selection pressure on the fungal community, leading to sustained changes, as evidenced by the highly variable selection of the fungal assembly process from 0 to 28 years .
Increased nutrient availability also drives changes in soil microbial nutrient cycling gene abundance during restoration . Our results revealed that grassland restoration enhances the microbial C fixation capacity, reduces the C degradation capacity, and increases functional alpha diversity . The greater C degradation ability in the early restoration stage was attributed mainly to the requirement of microbial growth, which needs to break down recalcitrant organic matter to acquire C because of the low C availability and fixation . As restoration progresses, the increased plant C input promotes the redistribution of microbial functions and energy, reducing their capacity for C degradation and enhancing their ability to acquire other nutrients to maintain their growth; thus, a greater abundance of N- and P-cycling genes was observed in the later restoration stage . The increase in soil nutrient availability also altered the life strategies of the microorganisms. Consequently, we observed an increase in the ratio of labile to recalcitrant C functional genes and rrn copy numbers during restoration . These changes indicate that microbes shift from K- strategists to r -strategists and prefer to acquire easily degradable C . Interestingly, the abundance of functional genes and bacterial biomass increased after the initial 28 years of restoration but sharply decreased at 36 years , which could be attributed to a decreased plant community biomass and diversity .
The results revealed that the microbial community diversity and function in subsoil were more sensitive to restoration than those in topsoil . After grazing exclusion, the increased plant biomass increased the soil nutrient input in both the topsoil and the subsoil , subsequently triggering simultaneous changes in both microbial community and functions , which further contributed to soil nutrient retention. The positive feedback loop between the microbial community and soil nutrients promotes the recovery of the grasslands during restoration . However, the low nutrient availability of the subsoil led to a stronger relationship between soil microbes and soil nutrients . Moreover, microbes in the subsoil are more inclined to cooperate rather than compete to maximize the utilization of limited resources and adapt to environmental pressures . Compared with the topsoil, the stronger relationships and cooperation among microorganisms enhance the recovery of soil microbes in the subsoil during restoration. In addition, vegetation restoration facilitates an increase in soil nutrients in the subsoil , which enhances the stability and consistency of the soil environment, leading to a reduced spatial turnover rate of the soil microorganisms, as well as a decrease in differences in microbial composition and functional genes among soil profiles over successive years .
Nutrient cycling is predominantly influenced by the fungal assembly process, fungal community composition, and restoration year . During restoration, the fungal assembly process experiences consistently high selection pressure due to changing environmental conditions, which are specifically related to resource availability. This ongoing selection pressure significantly influences the composition and structure of fungal communities. Fungi have an efficient extracellular enzyme system and a large mycelial network, providing them with an advantage in the decomposition of complex substrates (plant residues, cellulose, etc.) . Furthermore, fungal mycelia can form symbiotic relationships with plant roots, connecting plants and soil to establish an “expressway” for C transport. This allows fungi to actively participate in the input, stabilization, and decomposition of SOC . The powerful organic matter degradation and connectivity abilities of fungi position them as the main drivers of soil nutrient cycling, effectively promoting the nutrient release of plant residues and nutrient cycling in grassland ecosystems . In contrast, bacteria play a slightly inferior role in nutrient cycling. Bacterial DNA is in a free state within the cell, and an average of 42.5% of genes in the genome are affected by horizontal gene transfer ,which allows bacteria to dynamically adjust their functions to adapt to new environmental conditions without largely altering their community composition. This poses a challenge in accurately assessing the influence of the bacterial community on functional genes. Moreover, the low R 2 (0.11) for the nutrient cycling functional genes determined by the SEM indicates that the majority of the variability in the nutrient cycling function remains unexplained. In this study, we investigated the changes in the soil microbial community and nutrient cycling functional genes across soil profiles following vegetation restoration. Our findings indicate that soil depth predominantly influences bacterial communities, whereas fungal communities are highly sensitive to the duration of restoration. Notably, microbes in the subsoil recovered faster than those in the topsoil, which contributed to a reduction in differences in microbial community structure and the distribution of functional genes throughout the soil profile during the restoration process. Importantly, the fungal community assembly process played a pivotal role in driving changes in nutrient cycling functional genes, such as increased carbon fixation and nitrogen mineralization, in addition to a reduction in the abundance of genes involved in carbon degradation. These alterations contributed to increased SOC and nutrient accumulation. Overall, our results increase the understanding of the critical role of fungal communities in influencing changes in soil nutrient cycling genes, thereby facilitating nutrient accumulation in soil profiles during grassland restoration.
Study site and soil sampling The study was conducted in the Yunwushan National Natural Grassland Protection Zone on the Loess Plateau (106°21′–106°27′E, 36°10′–36°17′N), China. The region experiences a semiarid climate, with a mean annual precipitation of 425 mm and an average annual temperature of 7°C. In August 2018, we selected a chronosequence of four restoration habitats fenced between 1984 and 2013, corresponding to 36 years (G36), 28 years (G28), 15 years (G15), and 5 years (G5) of restored grasslands, and a continuously grazed grassland was also included (G0). Each habitat was >1 ha in size, and we selected a 100 m long strip for sampling and established four replicate plots (2 × 2 m quadrats) at 20 m intervals. Five soil cores (5 cm in diameter) were randomly collected from the soil profile (0–20, 20–40, 40–60, 60–80, and 80–100 cm, referred to as A, B, C, D, and E, respectively) in each plot and were thoroughly mixed to produce a composite soil sample. All soil samples were sieved through a 2 mm screen to remove roots and other debris. Each bulk sample was then divided into two subsamples. One subsample was promptly stored at −80°C to allow for DNA extraction, while the other subsample was air dried to facilitate physicochemical analysis. Analysis of soil physicochemical properties The soil pH was measured using a pH meter after shaking the soil–water (1:2.5 [wt/vol]) suspension for 30 minutes. Air-dried soil passed through a 100 mesh sieve was used to measure the SOC content with dichromate oxidation, and the TN content using the Kjeldahl method . The soil samples were shaken in 50 mL of 1.0 mol l −1 KCl for 30 minutes to extract soil NH 4 + and NO 3 − , and the concentrations of NH 4 + and NO 3 − were determined after filtration using a continuous flow analytical system (Autoanalyzer 3, Bran + Luebbe, Germany) . DNA extraction, polymerase chain reaction amplification, and data processing Microbial DNA was extracted from a 0.5 g soil sample using an E.Z.N.A. soil DNA kit (Omega Biotek, Norcross, GA, USA). The V4 regions of the bacterial and archaeal 16S rRNA genes and ITS1 were amplified via polymerase chain reaction (PCR) using the Phusion High-Fidelity PCR Master Mix (New England Biolabs, New Ipswich, MA, USA). The PCR conditions consisted of an initial denaturation step at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 45 seconds, and a final extension step at 72°C for 10 minutes. Reads generated from 16S V4 and ITS1 sequencing were analyzed using QIIME 2 (v.2018.4), and the DADA2 algorithm was employed to dereplicate the reads via paired-end settings . For the ITS1 reads, the same dereplication algorithm was employed with single-end settings. This resulted in ASV tables with read counts. The taxonomic assignment of the representative ASVs for the 16S V4 region was performed using the SILVA 128 database and the naïve Bayes classifier within QIIME 2 . Phylogenetic trees were constructed in QIIME 2 using the MAFFT alignment method coupled with the FastTree algorithm. The sequencing data for the archaeal and bacterial 16S rRNA genes and the fungal ITS gene were deposited into the NCBI SRA database under the accession number PRJNA1138184 . Microbial nutrient cycling gene abundance detection by high-throughput qPCR High-throughput qPCR was used to detect the functional genes of the soil microbial community related to carbon, nitrogen, phosphorus, and sulfur cycling in the microbial samples , and a detailed list of 71 genes is shown in . Microbial DNA extracted from the samples was tested for total amount and purity. Qualified DNA samples and qPCR reagents were added to a 384-well plate as a sample source plate, and primers and qPCR reagents were added to another 384-well plate as an assay source plate. A SmartChip Multisample Nanodispenser was used to add the sample source plate and assay source plate reagents to the micropores of the high-throughput qPCR SmartChip MyDesign Chip (Takara Biomedical Technology, Clontech). qPCR and fluorescence signal detection were performed using a SmartChip Real-Time PCR System (WaferGen Biosystems, USA), and amplification and dissolution curves were automatically generated. Canco was employed to derive the threshold (Ct) values and detection rates for each gene in a sample, with 16S rRNA serving as the internal reference to normalize the data for the relative quantitative information of each gene. The absolute quantitative information for the 16S rRNA gene was obtained, whereas the absolute quantitative information for the other genes was obtained via conversion. Quality control was performed on the resulting Ct values, and genes satisfying the following conditions were discarded: (i) an amplification efficiency of less than 1.8 or more than 2.2; (ii) an amplified negative control; and (iii) Ct values greater than 31 (considered to indicate no amplification). Finally, the copy number of each gene was calculated as follows: c o p y n u m b e r = 10 ( 31 − C t ) / ( 10 / 3 ) . Statistical analysis The R software package (version 4.2) was used for the data analysis and visualization. Principal component analysis (PCA) and nonmetric multidimensional scaling (NMDS) were employed to assess the differences in the structures of the microbial communities and functional genes on the basis of the Bray–Curtis distance. We also tested the significance of the differences using permutational multivariate analysis of variance (PERMANOVA) (Adonis), analysis of similarities (ANOSIM), and the response permutation procedure (MRPP). Analysis of variance (ANOVA) was used to evaluate the effects of the restoration year on microbial alpha and beta diversity, as well as functional gene diversity. Post hoc comparisons were conducted using Tukey’s honestly significant difference (HSD) test, with a significance level of P < 0.05. The PCA, NMDS, Adonis, ANOSIM, MRPP, Mantel tests, alpha diversity, and distance matrices were determined with the “vegan” package. Furthermore, the spatial turnover rate was obtained by fitting the Bray–Curtis distance matrix with soil depth using the “ggplot2” package. ANOVA and Tukey’s HSD tests were performed with the “stats” package, while the letters indicating statistical significance were obtained using the “multcompView” package. The R package “randomForest” was used with default parameters to screen for restoration-discriminant fungal species by random forest classification . To determine the microbial community assembly process, we employed the null model, the β-nearest taxon index (βNTI), and Bray–Curtis-based Raup–Crick (RC Bray ) data for ecological inference . The βNTI and RC Bray values were calculated using the “comdistnt” and “vegan” packages in R, respectively. To understand the assembly process during the restoration process , we combined two adjacent restoration years into one restoration stage, resulting in four types of restoration progress: 0 to 5 years, 5 to 15 years, 15 to 28 years, and 28 to 36 years. The rRNA operon (rrn) copy number of each ASV was estimated from the rnnDB database . We calculated the abundance-weighted average rRNA operon copy numbers of the ASVs for each sample to assess the microbial life strategies. The co-occurrence network was constructed using the top 500 abundant bacterial and fungal species. We then calculated the SparCC correlation matrix, and edges with a SPARCC correlation coefficient > 0.6 and P < 0.01 were selected to build the network. The network construction process utilized the “ggclusternet” package . An SEM was constructed using the “piecewiseSEM” package . The soil physical and chemical properties were calculated on the basis of the Euclidean distance matrix, and the microbial structure and function were calculated on the basis of the Bray–Curtis distance matrix. The soil physicochemical properties were represented by a composite variable that included SOC, TN, TP, NO 3 − , NH 4 + , and pH. Similarly, the assembly mechanism was represented by a composite variable encompassing βNTI, βMNTD, and RCb. The direct path coefficient of a composite variable on the microbial community was taken to represent the direct effect. The indirect path coefficient was determined by identifying all the indirect paths of the composite variable and subsequently calculating the product of the coefficients for each indirect path. The effects of all paths were then aggregated to determine the overall indirect effect of the composite variable.
The study was conducted in the Yunwushan National Natural Grassland Protection Zone on the Loess Plateau (106°21′–106°27′E, 36°10′–36°17′N), China. The region experiences a semiarid climate, with a mean annual precipitation of 425 mm and an average annual temperature of 7°C. In August 2018, we selected a chronosequence of four restoration habitats fenced between 1984 and 2013, corresponding to 36 years (G36), 28 years (G28), 15 years (G15), and 5 years (G5) of restored grasslands, and a continuously grazed grassland was also included (G0). Each habitat was >1 ha in size, and we selected a 100 m long strip for sampling and established four replicate plots (2 × 2 m quadrats) at 20 m intervals. Five soil cores (5 cm in diameter) were randomly collected from the soil profile (0–20, 20–40, 40–60, 60–80, and 80–100 cm, referred to as A, B, C, D, and E, respectively) in each plot and were thoroughly mixed to produce a composite soil sample. All soil samples were sieved through a 2 mm screen to remove roots and other debris. Each bulk sample was then divided into two subsamples. One subsample was promptly stored at −80°C to allow for DNA extraction, while the other subsample was air dried to facilitate physicochemical analysis.
The soil pH was measured using a pH meter after shaking the soil–water (1:2.5 [wt/vol]) suspension for 30 minutes. Air-dried soil passed through a 100 mesh sieve was used to measure the SOC content with dichromate oxidation, and the TN content using the Kjeldahl method . The soil samples were shaken in 50 mL of 1.0 mol l −1 KCl for 30 minutes to extract soil NH 4 + and NO 3 − , and the concentrations of NH 4 + and NO 3 − were determined after filtration using a continuous flow analytical system (Autoanalyzer 3, Bran + Luebbe, Germany) .
Microbial DNA was extracted from a 0.5 g soil sample using an E.Z.N.A. soil DNA kit (Omega Biotek, Norcross, GA, USA). The V4 regions of the bacterial and archaeal 16S rRNA genes and ITS1 were amplified via polymerase chain reaction (PCR) using the Phusion High-Fidelity PCR Master Mix (New England Biolabs, New Ipswich, MA, USA). The PCR conditions consisted of an initial denaturation step at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 45 seconds, and a final extension step at 72°C for 10 minutes. Reads generated from 16S V4 and ITS1 sequencing were analyzed using QIIME 2 (v.2018.4), and the DADA2 algorithm was employed to dereplicate the reads via paired-end settings . For the ITS1 reads, the same dereplication algorithm was employed with single-end settings. This resulted in ASV tables with read counts. The taxonomic assignment of the representative ASVs for the 16S V4 region was performed using the SILVA 128 database and the naïve Bayes classifier within QIIME 2 . Phylogenetic trees were constructed in QIIME 2 using the MAFFT alignment method coupled with the FastTree algorithm. The sequencing data for the archaeal and bacterial 16S rRNA genes and the fungal ITS gene were deposited into the NCBI SRA database under the accession number PRJNA1138184 .
High-throughput qPCR was used to detect the functional genes of the soil microbial community related to carbon, nitrogen, phosphorus, and sulfur cycling in the microbial samples , and a detailed list of 71 genes is shown in . Microbial DNA extracted from the samples was tested for total amount and purity. Qualified DNA samples and qPCR reagents were added to a 384-well plate as a sample source plate, and primers and qPCR reagents were added to another 384-well plate as an assay source plate. A SmartChip Multisample Nanodispenser was used to add the sample source plate and assay source plate reagents to the micropores of the high-throughput qPCR SmartChip MyDesign Chip (Takara Biomedical Technology, Clontech). qPCR and fluorescence signal detection were performed using a SmartChip Real-Time PCR System (WaferGen Biosystems, USA), and amplification and dissolution curves were automatically generated. Canco was employed to derive the threshold (Ct) values and detection rates for each gene in a sample, with 16S rRNA serving as the internal reference to normalize the data for the relative quantitative information of each gene. The absolute quantitative information for the 16S rRNA gene was obtained, whereas the absolute quantitative information for the other genes was obtained via conversion. Quality control was performed on the resulting Ct values, and genes satisfying the following conditions were discarded: (i) an amplification efficiency of less than 1.8 or more than 2.2; (ii) an amplified negative control; and (iii) Ct values greater than 31 (considered to indicate no amplification). Finally, the copy number of each gene was calculated as follows: c o p y n u m b e r = 10 ( 31 − C t ) / ( 10 / 3 ) .
The R software package (version 4.2) was used for the data analysis and visualization. Principal component analysis (PCA) and nonmetric multidimensional scaling (NMDS) were employed to assess the differences in the structures of the microbial communities and functional genes on the basis of the Bray–Curtis distance. We also tested the significance of the differences using permutational multivariate analysis of variance (PERMANOVA) (Adonis), analysis of similarities (ANOSIM), and the response permutation procedure (MRPP). Analysis of variance (ANOVA) was used to evaluate the effects of the restoration year on microbial alpha and beta diversity, as well as functional gene diversity. Post hoc comparisons were conducted using Tukey’s honestly significant difference (HSD) test, with a significance level of P < 0.05. The PCA, NMDS, Adonis, ANOSIM, MRPP, Mantel tests, alpha diversity, and distance matrices were determined with the “vegan” package. Furthermore, the spatial turnover rate was obtained by fitting the Bray–Curtis distance matrix with soil depth using the “ggplot2” package. ANOVA and Tukey’s HSD tests were performed with the “stats” package, while the letters indicating statistical significance were obtained using the “multcompView” package. The R package “randomForest” was used with default parameters to screen for restoration-discriminant fungal species by random forest classification . To determine the microbial community assembly process, we employed the null model, the β-nearest taxon index (βNTI), and Bray–Curtis-based Raup–Crick (RC Bray ) data for ecological inference . The βNTI and RC Bray values were calculated using the “comdistnt” and “vegan” packages in R, respectively. To understand the assembly process during the restoration process , we combined two adjacent restoration years into one restoration stage, resulting in four types of restoration progress: 0 to 5 years, 5 to 15 years, 15 to 28 years, and 28 to 36 years. The rRNA operon (rrn) copy number of each ASV was estimated from the rnnDB database . We calculated the abundance-weighted average rRNA operon copy numbers of the ASVs for each sample to assess the microbial life strategies. The co-occurrence network was constructed using the top 500 abundant bacterial and fungal species. We then calculated the SparCC correlation matrix, and edges with a SPARCC correlation coefficient > 0.6 and P < 0.01 were selected to build the network. The network construction process utilized the “ggclusternet” package . An SEM was constructed using the “piecewiseSEM” package . The soil physical and chemical properties were calculated on the basis of the Euclidean distance matrix, and the microbial structure and function were calculated on the basis of the Bray–Curtis distance matrix. The soil physicochemical properties were represented by a composite variable that included SOC, TN, TP, NO 3 − , NH 4 + , and pH. Similarly, the assembly mechanism was represented by a composite variable encompassing βNTI, βMNTD, and RCb. The direct path coefficient of a composite variable on the microbial community was taken to represent the direct effect. The indirect path coefficient was determined by identifying all the indirect paths of the composite variable and subsequently calculating the product of the coefficients for each indirect path. The effects of all paths were then aggregated to determine the overall indirect effect of the composite variable.
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Assessment of oral hygiene parameters in association to eating behaviors and healthy eating self-efficacy in school-aged children | 65b93996-4f4a-4b23-a636-9444521f5589 | 11673282 | Dentistry[mh] | Dental caries, the most common chronic childhood disease, is a multifactorial, behavioral condition where diet is one of the most significant factors influencing oral health . The majority of evidence indicates that dietary sugar is the most significant risk factor for the development of caries . Dietary sugars are broken down by various bacteria involved in dental biofilm formation, producing acid byproducts that cause demineralization of tooth structure . Additionally, because feeding patterns, food choices, and motivations are interconnected, it is possible that eating behaviors may also be linked to the development of caries . Eating behavior are a broad category influenced by a various personal, social, cultural, environmental, and economic factors . Children often rate fatty and sugary foods as their favorites, while vegetables tend to be their least preferred . Vozza et al. indicated that over 80% of children consumed sweets or sugary drinks daily, while more than 38% did not eat fruit at all during the week, and 21% did not eat vegetables . Additionally, children’s eating behaviors are shaped by their appetite traits, such as their responsiveness to food cues like the smell or sight of food, and their sensitivity to feelings of fullness . Various instruments have been developed to assess children’s eating behavior, with the Children’s Eating Behavior Questionnaire (CEBQ) being one of the most comprehensive psychometric tools. This parent-reported assessment covers key aspects of eating behaviors that have been linked to the emergence of eating disorders and weight-related issues in children . Self-efficacy has been proven to be a significant factor in explaining a variety of health behaviors, including dietary habits. The Healthy Eating Self-Efficacy Scale (HESES), developed by Story et al., aims to measure an individual’s perceived competence in selecting and favoring healthier foods . Findings indicate that children’s healthy eating self-efficacy levels are determinants of their eating habits and body mass index values . As dental caries and obesity have common risk factors such as socioeconomic status, health awareness and dietary habits, having information about the relationship between eating behavior patterns and dental caries helps both to prevent dental caries and to correct chronic negative health conditions such as obesity . There is currently no study investigating the eating behavior patterns and healthy eating self-efficacy of Turkish children. Additionally, there is limited data on whether oral hygiene is influenced by these eating behavior patterns. This study aims to evaluate the oral hygiene status of school-aged children in relation to their eating behavior and healthy eating self-efficacy.
Study design and study population The Research Ethical Board of Altınbaş University, Faculty of Dentistry, approved all procedures involving human subjects, and the study was carried out in accordance with the principles outlined in the Declaration of Helsinki (2022/166). Written informed consent was obtained from all parents. This descriptive cross-sectional study was conducted between December 2022 and May 2023. Kline stated that structural equation modeling (SEM) is a large-sample technique and provided sample size guidelines for analyzing these models, suggesting that a sample of 100 is considered small, a sample of 100 to 200 is medium, and a sample of more than 200 is considered large . Accordingly, it was aimed to complete the study with at least 200 participants. The study was carried out with the participation of 225 children aged 7 to 9 years who applied to Altınbaş University Faculty of Dentistry, Department of Pediatric Dentistry, for treatment or control. Healthy children aged 7 to 9 years with no systemic diseases, who were cooperative for oral examination, could fully complete the scale, were included in our study, along with parents who completed the questionnaire in its entirety. Measurements There were two stages to the data collection process: (a) applying the Children’s Eating Behavior Questionnaire (CEBQ) to parents of children (b) oral examination of children and applying the Healthy Eating Self-Efficacy Scale (HESES) to children. Instruments Information on the economic status and gender of the children was collected. The income distribution of the participants’ families was categorized as low, medium, and high using the minimum wage in Turkey as the threshold value. Children’s eating behavior was evaluated using the CEBQ, which was administered to parents of the children, developed by Wardle et al. and translated and validated in Turkish by Yılmaz et al. . This survey consists of 35 questions divided into eight subscales; food responsiveness, emotional undereating, enjoyment of food, emotional overeating, desire to drink, satiety responsiveness, slowness in eating and food fussiness. Each item was rated on a 5-point Likert scale: “1”=Never, “2”=Rarely, “3”=Sometimes, “4”=Often, “5”=Always . The child’s healthy eating self-efficacy was assessed using the HESES, which was administered to children, developed by Story et al. and translated and validated in Turkish by Kabasakal et al. . This scale contains 9 questions about the nutritional preferences of children. Children were asked, ‘‘How difficult would it be for you to...’’ eat more of particular foods and less of other foods (for example, apples instead of French fries for an after-school snack). Each item was answered using 3 response options: “not at all difficult”, “rather difficult” and “very difficult” . Oral health examination A postgraduate student in pediatric dentistry conducted clinical oral examinations on children in dental clinic settings in accordance with the WHO guideline 2013 . The examinations were performed on dental chairs with dental light and equipment, including a WHO probe and a plain dental mirror. The clinical examination parameters were standardized using a prepared form. After 2 weeks, the patients were recalled, and the accuracy of the clinical findings was reassessed by the same researcher. The intraexaminer correlation was found to be high. The teeth were evaluated using the DMFS/dmfs (Decayed, Missing and Filled Surfaces) Index, in accordance with WHO criteria . To differentiate between teeth extracted due to caries and those that spontaneously exfoliated, the examiner asked children why they were missing teeth. The age of the children and the existence or absence of the opposing tooth were also taken into consideration. For primary teeth, dmfs was used, while DMFS was applied for permanent teeth. Plaque Index (PI) provided by Silness and Löe and Gingival Index (GI) provided by Löe and Silness were used for the periodontal examination. The Simplified Oral Hygiene Index (OHI-S) was applied to evaluate oral hygiene status. Each child’s six teeth -four posterior and two anterior- were examined and rated by the examiner. Statistical analysis Analysis of the data was carried out using IBM SPSS Statistics 26 and SmartPLS 3.3.2 package programs. A Partial Least Squares Structural Equation Modeling (PLS-SEM) was designed in the Smart PLS 3 program, utilizing variables such as gender, family income, oral hygiene indices, eating behavior in children and healthy eating self-efficacy in children. Path analysis was used to test the research hypotheses. This analysis employed PLS-SEM, a second generation multivariate statistical method. Differences between groups for categorical variables were assessed using the Independent Sample T test. The assumption of equality of variance was investigated with the Levene test. In cases where this assumption was not met, corrected t-test statistics were reported. Relationship between numerical variables were examined using the Pearson correlation test for those with normal distribution and the Spearman test for those with non-normal distribution. The significance level of 0.05 was accepted for all analyzes.
The Research Ethical Board of Altınbaş University, Faculty of Dentistry, approved all procedures involving human subjects, and the study was carried out in accordance with the principles outlined in the Declaration of Helsinki (2022/166). Written informed consent was obtained from all parents. This descriptive cross-sectional study was conducted between December 2022 and May 2023. Kline stated that structural equation modeling (SEM) is a large-sample technique and provided sample size guidelines for analyzing these models, suggesting that a sample of 100 is considered small, a sample of 100 to 200 is medium, and a sample of more than 200 is considered large . Accordingly, it was aimed to complete the study with at least 200 participants. The study was carried out with the participation of 225 children aged 7 to 9 years who applied to Altınbaş University Faculty of Dentistry, Department of Pediatric Dentistry, for treatment or control. Healthy children aged 7 to 9 years with no systemic diseases, who were cooperative for oral examination, could fully complete the scale, were included in our study, along with parents who completed the questionnaire in its entirety.
There were two stages to the data collection process: (a) applying the Children’s Eating Behavior Questionnaire (CEBQ) to parents of children (b) oral examination of children and applying the Healthy Eating Self-Efficacy Scale (HESES) to children.
Information on the economic status and gender of the children was collected. The income distribution of the participants’ families was categorized as low, medium, and high using the minimum wage in Turkey as the threshold value. Children’s eating behavior was evaluated using the CEBQ, which was administered to parents of the children, developed by Wardle et al. and translated and validated in Turkish by Yılmaz et al. . This survey consists of 35 questions divided into eight subscales; food responsiveness, emotional undereating, enjoyment of food, emotional overeating, desire to drink, satiety responsiveness, slowness in eating and food fussiness. Each item was rated on a 5-point Likert scale: “1”=Never, “2”=Rarely, “3”=Sometimes, “4”=Often, “5”=Always . The child’s healthy eating self-efficacy was assessed using the HESES, which was administered to children, developed by Story et al. and translated and validated in Turkish by Kabasakal et al. . This scale contains 9 questions about the nutritional preferences of children. Children were asked, ‘‘How difficult would it be for you to...’’ eat more of particular foods and less of other foods (for example, apples instead of French fries for an after-school snack). Each item was answered using 3 response options: “not at all difficult”, “rather difficult” and “very difficult” .
A postgraduate student in pediatric dentistry conducted clinical oral examinations on children in dental clinic settings in accordance with the WHO guideline 2013 . The examinations were performed on dental chairs with dental light and equipment, including a WHO probe and a plain dental mirror. The clinical examination parameters were standardized using a prepared form. After 2 weeks, the patients were recalled, and the accuracy of the clinical findings was reassessed by the same researcher. The intraexaminer correlation was found to be high. The teeth were evaluated using the DMFS/dmfs (Decayed, Missing and Filled Surfaces) Index, in accordance with WHO criteria . To differentiate between teeth extracted due to caries and those that spontaneously exfoliated, the examiner asked children why they were missing teeth. The age of the children and the existence or absence of the opposing tooth were also taken into consideration. For primary teeth, dmfs was used, while DMFS was applied for permanent teeth. Plaque Index (PI) provided by Silness and Löe and Gingival Index (GI) provided by Löe and Silness were used for the periodontal examination. The Simplified Oral Hygiene Index (OHI-S) was applied to evaluate oral hygiene status. Each child’s six teeth -four posterior and two anterior- were examined and rated by the examiner.
Analysis of the data was carried out using IBM SPSS Statistics 26 and SmartPLS 3.3.2 package programs. A Partial Least Squares Structural Equation Modeling (PLS-SEM) was designed in the Smart PLS 3 program, utilizing variables such as gender, family income, oral hygiene indices, eating behavior in children and healthy eating self-efficacy in children. Path analysis was used to test the research hypotheses. This analysis employed PLS-SEM, a second generation multivariate statistical method. Differences between groups for categorical variables were assessed using the Independent Sample T test. The assumption of equality of variance was investigated with the Levene test. In cases where this assumption was not met, corrected t-test statistics were reported. Relationship between numerical variables were examined using the Pearson correlation test for those with normal distribution and the Spearman test for those with non-normal distribution. The significance level of 0.05 was accepted for all analyzes.
Of the 225 children in the study, 128 (56.9%) were female and 97 (43.1%) were male. The mean age of children was 7.82 ± 0.73 years. Family income distribution showed that 0.4% were low income, 9.8% were medium income, and 89.8% were high income (Table ). The mean values for oral hygiene parameters (dmfs, DMFS, plaque index, gingival index and OHI-S), HESES and the eight subgroups of CEBQ are shown in Table . The overall mean score of the Healthy Eating Self-Efficacy Scale was 2.64 ± 0.31. According to this result, the answer given by the children to the self-efficacy scale was “It is not difficult at all”. Among the eight subgroups in the CEBQ, “enjoyment of food” had the lowest mean value at 2.55 ± 0.87, while “emotional overeating” had the highest at 3.38 ± 1.13. Children’s self-efficacy and subgroups of eating behaviors were not found to be statistically correlated ( p > 0.05). However, a negative correlation between self-efficacy score and the plaque index ( p < 0.001), OHI-S ( p < 0.001), dmfs ( p < 0.001), DMFS ( p = 0.002) and gingival index ( p < 0.001) was statistically significant. Therefore, as children’s healthy eating self-efficacy decreases, an increase is expected in DMFS, dmfs, plaque index, gingival index and OHI-S scores (Table ). No statistically significant relationship was found between food responsiveness, enjoyment of food, slowness in eating and desire to drink with oral hygiene parameters ( p > 0.05). However, a statistically significant positive effect of emotional overeating on DMFS was observed ( p = 0.036). This suggests that children wtih emotional overeating behavior are likely to have higher DMFS scores. In contrast, satiety responsiveness also showed a statistically significant positive effect on dmfs ( p = 0.038), indicating that children with high satiety responsiveness may also have higher dmfs scores. Emotional undereating had a statistically significant negative effect on dmfs ( p = 0.021), meaning children with emotional undereating are expected to have lower dmfs scores. Additionally, food fussiness was found to have a statistically significant positive effect on DMFS ( p = 0.034). Thus, children who are food fussy are likely to have higher DMFS scores as well (Table ). Family income has a statistically significant negative effect on emotional overeating ( p < 0.001) and the desire to drink ( p = 0.020). In contrast, it positively affects self-efficacy ( p < 0.001) and food fussiness ( p = 0.019). Therefore, children from high-income families are expected to have higher self-efficacy and food fussiness, along with lower levels of emotional overeating and desire to drink. Additionally, a significant negative effect of family income on dmfs was found ( p = 0.029), indicating that children from high-income families tend to have lower dmfs values. Gender did not significantly affect any oral hygiene parameters or eating behaviors ( p > 0.05) (Table ). Significant relationships between the two scales and the oral hygiene parameters were also reanalyzed with the Pearson correlation test (Table ).
There is a two-way relationship between nutrition and oral health. Healthy oral microflora, dental tissues, and orofacial tissues support adequate and correct nutrition, while a regular, balanced diet free from cariogenic foods indicates good oral health . Additionally, a well-balanced diet, providing the right amount of calories and nutrients, along with proper distribution throughout the day, supports optimal physical and cognitive development during children’s growth years . This study aimed to evaluate the effects of children’s eating behaviors and healthy eating self-efficacy on oral hygiene parameters. The findings in our study indicate that gender does not have a statistically significant effect on eating behavior, consistent with other previous research . Leuba et al. found that neither age nor gender had any effect on the seven factors of the CEBQ, despite the sample encompassing a broad age range from 2 to 6 years old, with a nearly equal gender distribution (47% girls and 53% boys) . Other studies reported no significant differences between genders for eating behavior, except in the desire to drink subscale . However, a study involving 8-year-old children in Brazil found that emotional overeating, satiety responsiveness, and emotional undereating were higher in girls, while the desire to drink was higher in boys . Sleddens et al. stated that in Dutch children aged 6–7 years, food fussiness and emotional overeating were higher in boys, while the enjoyment of food was higher in girls . Some previous studies suggest that eating behaviors in adolescents differ between genders , but it is unclear when this difference becomes apparent. Although there is no definitive data on the effect of gender on eating behaviors, age and environmental factors may also influence the results. Among the variables affecting eating behaviors; Dubois et al. noted that girls might be more self-conscious about their bodies than boys . Yılmaz et al. suggested that boys might be given more food throughout adolescence, a time of rapid growth, while girls might be more impacted by social pressure to eat less . Additionaly, different parental feeding strategies are critical in the development of eating behaviors in girls and boys . In present study, food responsiveness, enjoyment of food and slowness in eating had no statistically significant effect on oral hygiene parameters similar to the findings of Shqair et al. . In contrast to our study, Borrell García et al. reported that dental caries were statistically significantly higher in children with slowness in eating behavior . This result may be explained by the fact that the tooth surfaces of children who eat slowly naturally come into contact with food for a longer period of time. In the present study, satiety responsiveness was associated with a higher dmfs value, consistent with previous reports . According to Shqair et al., this outcome was caused by parents providing snacks or discretionary (high sugar) foods to entice children to eat more or providing food outside of meals since the children weren’t interested in eating . Additionally, a previous study indicated that food responsiveness, food fussiness, and enjoyment of food were associated with the incidence of early childhood caries in children . Children can react to emotional distress in two different ways. The first is a biologically natural response that involves a decrease in intestinal activity resulting in a reduction in food intake (loss of appetite when feeling saddness or angery). The second is overeating, according to psychosomatic theory, which which posits that individuals learn to cope with emotional distress based on early-life experiences . According to the results of our study, emotional undereating had a negative effect on the dmfs index, while emotional overeating had a positive effect on the DMFS index. This contradicts the study by Shqair et al., which found that emotional undereating and overeating had no effect on dental caries parameters . Nevertheless, previous studies suggested that emotional eating patterns, such as undereating and overeating, are linked to higher caries rates, and indicated a connection between stronger parental control and rising caries rates . According to our results, healthy eating self-efficiacy was not associated with the eating behavior subgroups. However, previous studies indicate that high self-efficacy can enhance the ability to change behavior . Kulik et al. found that nutritional behavior knowledge and healthy eating self-efficacy are effective in the developing healthy eating habits . Similarly, Saksvig et al. reported that increasing self-efficacy makes it easier to change and develop eating behaviors . As a result of our study, children’s scores on indices of oral hygiene decline as their self-efficacy for healthy eating increases. The association between self-efficacy and several healthy eating practices, such as the consumption of fruits and vegetables, is supported by previous research . However, assuming that self-efficacy always has a significant impact on eating behavior may not be accurate. Consumption of fruits and vegetables by children may also be influenced by a variety of psychosocial and environmental factors, such as parental feeding, dietary preferences, rules, and availability of healthy food . According to the study conducted by Shqair et al., emotional overeating was lower in children from high-income families, which coincides with the results of our study. In contrast, food fussiness was found to be lower, contrary to our findings . Rollins et al. reported that emotional eating is more prevelant in children from low-income families due to the emotional distress associated with poverty . Sirasa et al. found a positive relationship between family income and healthy food choice, in accordance with our study . The results of the present study showed that children from high-income families have a lower desire to drink. This desire to drink refers to children who constantly want something to drink. According to Nembhwani and Winnier, an increased desire to drink can lead to arise in early childhood caries . It is thought that the positive association between the desire to drink and caries may stem from children feeling thirsty or hungry, but it is more likely related to their desire to consume sugary drinks. A lower socioeconomic status is linked to poor eating habits, which include higher consumption of free or added sugars and other easily fermentable carbohydrates, crucial factors in dental caries . Additionally, low socioeconomic status indicates a more obesogenic food environment, unhealthy food consumption, and food inavailability, potentially increasing the risk of dental caries . According to our study, children from high-income families showed lower dmfs scores, consistent with previous reports . Kindergartens and schools play a crucial role in shaping eating-related behaviors. Effective education promotes knowledge about healthy eating, fosters positive attitudes, and encourages specific lifestyle choices. The early development of responsible eating habits is particularly important, as research indicates that these habits often carry over into adulthood . This study has a few limitations. Firstly, due to its cross-sectional design, it can only confirm the link between predictor variables and dependent variables, rather than establishing a cause-and-effect relationship. Secondly, the CEBQ questionnaire relies on self-reporting, meaning that parents’ subjective reports of their children’ eating habits are included. Thirdly, since the families participating in our study predominantly have high income levels and the sample consists only of children from central Istanbul, the findings may not be applicable to the overall country. Future studies with larger sample sizes and a more diverse distribution of socioeconomic levels and ages could provide more reliable results regarding the relationships between eating behavior and oral hygiene.
The findings indicate that emotional overeating, satiety responsiveness, and food fussiness are positively associated with higher dental caries scores (DMFS and dmfs), suggesting their role in caries development. Moreover, lower self-efficacy in healthy eating was correlated with poorer oral hygiene, as reflected in higher plaque and gingival indices, as well as greater dental caries. Family income emerged as a key factor, with children from higher-income families demonstrating better oral hygiene and healthier eating patterns. These results emphasize the need for targeted interventions addressing both dietary behaviors and oral hygiene, particularly in lower socioeconomic groups. While no gender differences were observed, the study underscores the importance of promoting healthy eating behaviors and self-efficacy to prevent dental caries. Given the study’s cross-sectional design and the homogeneity of the sample, further research with larger, more diverse populations is needed to establish causal relationships and inform effective preventive strategies.
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Compatibility and Stability of Ten Commonly Used Clinical Drugs in Pediatric Electrolyte Supplements Injection | 2093db96-fac8-407d-88e2-1fed9de5ed83 | 9112340 | Pediatrics[mh] | The purpose of liquid therapy is to maintain the stability of body fluid interval capacity, osmotic concentration and composition. It is an important and commonly used means of clinical rescue and treatment. In pediatric practice, medications are administered intravenously for faster action, liquid therapy or due to patient’s inability to swallow oral medication. However, the combination of drug and menstruum has the potential for physical and chemical reactions based on pharmaceutical formulation principles. The intravenous injections which directly entering the patient circulation may lead to morbidity or mortality. In order to ensure the safety, effectiveness and timeliness of intravenous medication, we should not only focus on the quality of drugs, but also strictly ensure the compatibility of drug and menstruum. Whether the solvent can be selected correctly is very important for drug stability and treatment. If the obvious appearance change is easy to be found during drug mixing, it will stop the injection immediately without damage. The slightly appearance change is easy to be ignored. Pediatric electrolyte supplements injection is compound preparation, which contains 37.5g glucose and 2.25g sodium chloride in 1000 mL. It is mainly used to supplement heat and body fluid, and commonly used in pediatrics in various hospitals. It is unknown for the compatibility and stability of some unstable and commonly used drugs in pediatric electrolyte supplements injection. As has been noted, cephalosporins are antibiotics with high efficiency, low side effects and wide clinical application. The structure contains unstable β- Lactam ring which is easy to hydrolyze in the presence of water. And acid, alkali and temperature rise can promote hydrolysis. Therefore, most of the cephalosporin injections are made in powder injection, which can be stored for a long time. It can be dissolved by injection solvent before clinical application. The study on the stability of vidarabine monophosphate in pediatric electrolyte supplements injection had been investigated, but the stability of mezlocillin sodium for injection, cefoperazone sodium and sulbactam sodium for injection, ceftazidime for injection, cefradine for injection, ceftriaxone sodium for injection, ambroxol hydrochloride for injection, cefazolin sodium for injection, cefathiamidine for injection, cefmetazole sodium for injection, cefoxitin sodium for injection with pediatric electrolyte supplements injection had not been reported. Reasonable compatibility promotes clinical rational drug use. It is not only related to the safety and effectiveness of clinical medication, but also about medication economic loss, health damage and even life loss. The objective of this study was to assess the compatibility and stability of ten drugs (mezlocillin sodium for injection, cefoperazone sodium and sulbactam sodium for injection, ceftazidime for injection, cefradine for injection, ceftriaxone sodium for injection, ambroxol hydrochloride for injection, cefazolin sodium for injection, cefathiamidine for injection, cefmetazole sodium for injection, cefoxitin sodium for injection) in pediatric electrolyte supplements injection stored for 6 hours at 25℃ avoiding from light. It may provide a scientific basis for clinical application of these combinations.
Materials and Reagents Mezlocillin, cefoperazone, sulbactam, ceftazidime, Cefradine, ceftriaxone sodium, ambroxol hydrochloride, cefazolin, cefathiamidine, cefmetazole, cefoxitin reference standards were purchased from China Institute for Food and Drug Control (Beijing, China). Pediatric electrolyte supplements injection (100mL) was obtained from Jiangsu Hengrui Pharmaceutical Co., Ltd. (Jiangsu, China). Mezlocillin sodium for injection (2.0g) was purchased from Sichuan pharmaceutical preparation Co., Ltd. (Chengdu, China). Cefoperazone sodium sulbactam sodium for injection (1.0g), ceftazidime for injection (1.0g) and ceftriaxone sodium for injection (0.5g) were purchased from Qilu Pharmaceutical Co., Ltd. (Jinan, China). Cefradine for injection (0.5g) and cefazolin sodium for injection were obtained from Shandong Lukang Pharmaceutical Co., Ltd. (Jining, China). Ambroxol hydrochloride for injection (15mg) was supplied by Shandong Yuxin Pharmaceutical Co., Ltd. (Linyi, China). Cefathiamidine for injection (0.5g) was obtained from Guangzhou Baiyun Pharmaceutical Group Co., Ltd. (Guangzhou, China). Cefmetazole sodium for injection (1.0g) was purchased from Sichuan Hexin Pharmaceutical Co., Ltd. (Chengdu, China). Cefoxitin sodium for injection (1.0g) was supplied by Sinopharm (Shenzhen) Pharmaceutical Co., Ltd. (Shenzhen, China). Methanol and acetonitrile were chromatographic purity and other chemicals used were of analytical grade, and totally purchased from Chengdu Hengxin Chemical Reagent Co., Ltd (Chengdu, China). Instrumentation Insoluble particles of each sample were detected by Particle analyzer (GWF-DS1, Tianjin Tianhe Analytical Instrument Co., Ltd.). The pH values of samples were measured with pH metre (Phs-3C+, Chengdu Century ark Technology Co., Ltd). The content of each sample was analyzed by high-performance liquid chromatography (1200, Agilent, USA). Ultrapure water was acquired by equipment (SSY-GDE-500L, Sichuan Shuisiyuan Environmental Technology Co., Ltd.) Insoluble Particles Detection and pH Measure Pediatric electrolyte supplements injection was used to mix with ten drugs according to the clinical drug concentration. The compatible solutions were analyzed at certain time intervals (0, 0.5, 1, 2, 4, 6 hours) after mixing, and stored avoiding from light at room temperature. Tests were according to the insoluble particle test method (general rule 0903, Part IV of Chinese Pharmacopoeia (2020 Edition)), and PH value determination method (general rule 0631, Part IV of Chinese Pharmacopoeia (2020 Edition)). Chromatographic Conditions The amount of active components were determined by HPLC using a Shimadzu C18 column (250mm×4.6mm, 5μm). The column was maintained at a temperature of 35°C, and at a flow rate of 1.0 mL/min while ceftazidime was 1.5 mL/min. For mezlocillin, the mobile phase was composed of phosphate buffer (potassium dihydrogen phosphate 4.9g and potassium dihydrogen phosphate 0.45g, dissolved with water to 1000mL) and acetonitrile in the ratio of 80:20 (v/v), and injection volume was 20μL, and the selected detection wavelength was 210nm. For cefoperazone and sulbactam, the mobile phase was composed of 0.005mol/L tetrabutylammonium hydroxide solution (add 1800mL of water to 6.6mL of 40% tetrabutylammonium hydroxide solution, adjust pH value to 4.0 with 1mol/L phosphoric acid, and then dilute to 2000mL) and acetonitrile in the ratio of 75:25 (v/v), and injection volume was 10μL, and the selected detection wavelength was 220nm. For ceftazidime, the mobile phase was composed of acetonitrile, pH 7.0 phosphate buffer (42.59g of anhydrous disodium hydrogen phosphate and 27.22g of potassium dihydrogen phosphate, diluted to 1000mL with water) and water in the ratio of 40:200:1760 (v/v/v), and injection volume was 20μL, and the selected detection wavelength was 254nm. For cefazolin, the mobile phase was composed of disodium hydrogen phosphate – citric acid solution (1.33g anhydrous disodium hydrogen phosphate and 1.12g citric acid, diluted to 1000mL with water) and acetonitrile in the ratio of 88:12 (v/v), and injection volume was 10 μL, and the selected detection wavelength was 254 nm. For ceftriaxone, the mobile phase was composed of 0.02 mol/L octylamine solution and acetonitrile in the ratio of 73:27 (v/v, pH 6.5), and injection volume was 20μL, and the selected detection wavelength was 254nm. For ambroxol hydrochloride, the mobile phase was composed of 0.01mol/l diammonium hydrogen phosphate solution (pH 7.0) and acetonitrile in the ratio of 50:50 (v/v), and injection volume was 20 μL, and the selected detection wavelength was 248 nm. For cefathiamidine, the mobile phase was composed of phosphate buffer (2.76 g of anhydrous disodium hydrogen phosphate and 1.29 g of citric acid, diluted to 1000mL with water) and acetonitrile in the ratio of 80:20 (v/v), and injection volume was 10μL, and the selected detection wavelength was 254nm. For cefradine, the mobile phase was composed of 0.027mol/ L disodium hydrogen phosphate solution containing 0.027 mol/L sodium octane sulfonate (pH 8.0) and methanol in the ratio of 75:25 (v/v), and injection volume was 10 μL, and the selected detection wavelength was 206 nm. For cefmetazole, the mobile phase was composed of ammonium dihydrogen phosphate solution (5.75 g of ammonium dihydrogen phosphate, add 730 mL of water to dissolve it, add 10% tetrabutyl ammonium hydroxide solution 19.2 mL), tetrahydrofuran and methanol in the ratio of 730:12.5:300 (v/v/v, pH 4.5), and injection volume was 20 μL, and the selected detection wavelength was 254 nm. For cefoxitin, the mobile phase was composed of water, acetonitrile and glacial acetic acid in the ratio of 81:19:1 (v/v/v), and injection volume was 10 μL, and the selected detection wavelength was 254 nm. Method Validation The validation of method was performed in terms of specificity, linearity, accuracy, intraday and interday precision. According to the corresponding content determination methods, the reference solution and pediatric electrolyte supplements injection were determined to investigate whether the blank sample solution interferes with the chromatographic peaks of the main drug components and whether the separation between main drug components and adjacent impurities meets the requirements. Calibration curves were constructed from a linear plot of peak area versus concentration of the reference standards for mezlocillin (0.02713–0.5426 mg/mL), cefoperazone (0.04823–0.9646 mg/mL), sulbactam (0.04646–0.9292 mg/mL), ceftazidime (0.02485–0.4970mg/mL), cefazolin (0.01997–0.3994 mg/mL), ceftriaxone (0.03737–0.7474mg/mL), ambroxol hydrochloride (0.006186–0.1237 mg/mL), cefathiamidine (0.02157–0.4313 mg/mL), cefradine (0.02684–0.5369 mg/mL), cefmetazole (0.02170–0.4340 mg/mL), and cefoxitin (0.02906–0.5812 mg/mL). According to the corresponding content determination methods, quality control samples were analyzed in sextuplicate (n = 6). They were calculated based on drug recovery for the accuracy, and calculated as coefficient of relative standard deviation (RSD %) for intraday/interday precisions. Stability of Compatible Solutions The reference control and compatible solutions were prepared at the following final concentrations: mezlocillin 0.15 mg/mL, cefoperazone 0.5 mg/mL, sulbactam 0.5 mg/mL, ceftazidime 0.15 mg/mL, cefazolin 0.1 mg/mL, ceftriaxone 0.22 mg/mL, ambroxol hydrochloride 30 μg/mL, cefathiamidine 0.1 mg/mL, cefradine 0.3 mg/mL, cefmetazole 0.1 mg/mL, cefoxitin 0.3 mg/mL. The compatible solutions were analyzed at certain time intervals (0, 0.5, 1, 2, 4, 6 hours) after mixing, and stored avoiding from light at room temperature. Analysis of the Data The initial concentration of compatible solutions was defined as 100%, and the subsequent samples’ concentration was expressed as percentage of the initial concentration. It was considered to be stable if they retained 95% of the initial concentrations.
Mezlocillin, cefoperazone, sulbactam, ceftazidime, Cefradine, ceftriaxone sodium, ambroxol hydrochloride, cefazolin, cefathiamidine, cefmetazole, cefoxitin reference standards were purchased from China Institute for Food and Drug Control (Beijing, China). Pediatric electrolyte supplements injection (100mL) was obtained from Jiangsu Hengrui Pharmaceutical Co., Ltd. (Jiangsu, China). Mezlocillin sodium for injection (2.0g) was purchased from Sichuan pharmaceutical preparation Co., Ltd. (Chengdu, China). Cefoperazone sodium sulbactam sodium for injection (1.0g), ceftazidime for injection (1.0g) and ceftriaxone sodium for injection (0.5g) were purchased from Qilu Pharmaceutical Co., Ltd. (Jinan, China). Cefradine for injection (0.5g) and cefazolin sodium for injection were obtained from Shandong Lukang Pharmaceutical Co., Ltd. (Jining, China). Ambroxol hydrochloride for injection (15mg) was supplied by Shandong Yuxin Pharmaceutical Co., Ltd. (Linyi, China). Cefathiamidine for injection (0.5g) was obtained from Guangzhou Baiyun Pharmaceutical Group Co., Ltd. (Guangzhou, China). Cefmetazole sodium for injection (1.0g) was purchased from Sichuan Hexin Pharmaceutical Co., Ltd. (Chengdu, China). Cefoxitin sodium for injection (1.0g) was supplied by Sinopharm (Shenzhen) Pharmaceutical Co., Ltd. (Shenzhen, China). Methanol and acetonitrile were chromatographic purity and other chemicals used were of analytical grade, and totally purchased from Chengdu Hengxin Chemical Reagent Co., Ltd (Chengdu, China).
Insoluble particles of each sample were detected by Particle analyzer (GWF-DS1, Tianjin Tianhe Analytical Instrument Co., Ltd.). The pH values of samples were measured with pH metre (Phs-3C+, Chengdu Century ark Technology Co., Ltd). The content of each sample was analyzed by high-performance liquid chromatography (1200, Agilent, USA). Ultrapure water was acquired by equipment (SSY-GDE-500L, Sichuan Shuisiyuan Environmental Technology Co., Ltd.)
Pediatric electrolyte supplements injection was used to mix with ten drugs according to the clinical drug concentration. The compatible solutions were analyzed at certain time intervals (0, 0.5, 1, 2, 4, 6 hours) after mixing, and stored avoiding from light at room temperature. Tests were according to the insoluble particle test method (general rule 0903, Part IV of Chinese Pharmacopoeia (2020 Edition)), and PH value determination method (general rule 0631, Part IV of Chinese Pharmacopoeia (2020 Edition)).
The amount of active components were determined by HPLC using a Shimadzu C18 column (250mm×4.6mm, 5μm). The column was maintained at a temperature of 35°C, and at a flow rate of 1.0 mL/min while ceftazidime was 1.5 mL/min. For mezlocillin, the mobile phase was composed of phosphate buffer (potassium dihydrogen phosphate 4.9g and potassium dihydrogen phosphate 0.45g, dissolved with water to 1000mL) and acetonitrile in the ratio of 80:20 (v/v), and injection volume was 20μL, and the selected detection wavelength was 210nm. For cefoperazone and sulbactam, the mobile phase was composed of 0.005mol/L tetrabutylammonium hydroxide solution (add 1800mL of water to 6.6mL of 40% tetrabutylammonium hydroxide solution, adjust pH value to 4.0 with 1mol/L phosphoric acid, and then dilute to 2000mL) and acetonitrile in the ratio of 75:25 (v/v), and injection volume was 10μL, and the selected detection wavelength was 220nm. For ceftazidime, the mobile phase was composed of acetonitrile, pH 7.0 phosphate buffer (42.59g of anhydrous disodium hydrogen phosphate and 27.22g of potassium dihydrogen phosphate, diluted to 1000mL with water) and water in the ratio of 40:200:1760 (v/v/v), and injection volume was 20μL, and the selected detection wavelength was 254nm. For cefazolin, the mobile phase was composed of disodium hydrogen phosphate – citric acid solution (1.33g anhydrous disodium hydrogen phosphate and 1.12g citric acid, diluted to 1000mL with water) and acetonitrile in the ratio of 88:12 (v/v), and injection volume was 10 μL, and the selected detection wavelength was 254 nm. For ceftriaxone, the mobile phase was composed of 0.02 mol/L octylamine solution and acetonitrile in the ratio of 73:27 (v/v, pH 6.5), and injection volume was 20μL, and the selected detection wavelength was 254nm. For ambroxol hydrochloride, the mobile phase was composed of 0.01mol/l diammonium hydrogen phosphate solution (pH 7.0) and acetonitrile in the ratio of 50:50 (v/v), and injection volume was 20 μL, and the selected detection wavelength was 248 nm. For cefathiamidine, the mobile phase was composed of phosphate buffer (2.76 g of anhydrous disodium hydrogen phosphate and 1.29 g of citric acid, diluted to 1000mL with water) and acetonitrile in the ratio of 80:20 (v/v), and injection volume was 10μL, and the selected detection wavelength was 254nm. For cefradine, the mobile phase was composed of 0.027mol/ L disodium hydrogen phosphate solution containing 0.027 mol/L sodium octane sulfonate (pH 8.0) and methanol in the ratio of 75:25 (v/v), and injection volume was 10 μL, and the selected detection wavelength was 206 nm. For cefmetazole, the mobile phase was composed of ammonium dihydrogen phosphate solution (5.75 g of ammonium dihydrogen phosphate, add 730 mL of water to dissolve it, add 10% tetrabutyl ammonium hydroxide solution 19.2 mL), tetrahydrofuran and methanol in the ratio of 730:12.5:300 (v/v/v, pH 4.5), and injection volume was 20 μL, and the selected detection wavelength was 254 nm. For cefoxitin, the mobile phase was composed of water, acetonitrile and glacial acetic acid in the ratio of 81:19:1 (v/v/v), and injection volume was 10 μL, and the selected detection wavelength was 254 nm.
The validation of method was performed in terms of specificity, linearity, accuracy, intraday and interday precision. According to the corresponding content determination methods, the reference solution and pediatric electrolyte supplements injection were determined to investigate whether the blank sample solution interferes with the chromatographic peaks of the main drug components and whether the separation between main drug components and adjacent impurities meets the requirements. Calibration curves were constructed from a linear plot of peak area versus concentration of the reference standards for mezlocillin (0.02713–0.5426 mg/mL), cefoperazone (0.04823–0.9646 mg/mL), sulbactam (0.04646–0.9292 mg/mL), ceftazidime (0.02485–0.4970mg/mL), cefazolin (0.01997–0.3994 mg/mL), ceftriaxone (0.03737–0.7474mg/mL), ambroxol hydrochloride (0.006186–0.1237 mg/mL), cefathiamidine (0.02157–0.4313 mg/mL), cefradine (0.02684–0.5369 mg/mL), cefmetazole (0.02170–0.4340 mg/mL), and cefoxitin (0.02906–0.5812 mg/mL). According to the corresponding content determination methods, quality control samples were analyzed in sextuplicate (n = 6). They were calculated based on drug recovery for the accuracy, and calculated as coefficient of relative standard deviation (RSD %) for intraday/interday precisions.
The reference control and compatible solutions were prepared at the following final concentrations: mezlocillin 0.15 mg/mL, cefoperazone 0.5 mg/mL, sulbactam 0.5 mg/mL, ceftazidime 0.15 mg/mL, cefazolin 0.1 mg/mL, ceftriaxone 0.22 mg/mL, ambroxol hydrochloride 30 μg/mL, cefathiamidine 0.1 mg/mL, cefradine 0.3 mg/mL, cefmetazole 0.1 mg/mL, cefoxitin 0.3 mg/mL. The compatible solutions were analyzed at certain time intervals (0, 0.5, 1, 2, 4, 6 hours) after mixing, and stored avoiding from light at room temperature.
The initial concentration of compatible solutions was defined as 100%, and the subsequent samples’ concentration was expressed as percentage of the initial concentration. It was considered to be stable if they retained 95% of the initial concentrations.
Description Within 6 hours, the properties of compatible liquids for ten drugs did not change significantly. Visual examination indicated that they were clear and transparent liquids, without color change, precipitation, crystallization and gas production. Insoluble Particles Detection For 10 kinds of compatible solutions, the number of insoluble particles at 0, 0.5, 1, 2, 4 and 6 h after mixing are shown in . According to the criteria of insoluble particles set by Chinese Pharmacopeia (Edition 2020), intravenous injection with a marked amount of 100mL contains the number of particles that are equal to or greater than 10 μm not exceed 25, and equal to or greater than 25 μm not exceed 3. In this experiment, the number of particles in the compatibility solution of mezlocillin sodium for injection and pediatric electrolyte supplements injection exceeded the standard requirements, and the other solutions met the standard requirements. pH Measure The pH values of prepared compatible solutions at 0, 0.5, 1, 2, 4 and 6 h after mixing are shown in . The pH of cefoperazone sodium for injection, sulbactam sodium, ceftriaxone for injection, ceftazidime for injection, ambroxol hydrochloride for injection, cefradine for injection, mezlocillin sodium for injection and cefmetazole sodium for injection did not change significantly after compatibility with pediatric electrolyte supplements injection. The pH value increased when cefazolin sodium for injection was compatible with pediatric electrolyte supplements injection, and decreased when cefathiamidine for injection was compatible with pediatric electrolyte supplements injection. According to the pharmaceutics, range of pH for injections is 4–9. The pH values of compatible solutions for ten drugs are within the allowable range, and the pH values of compatible solutions are within the requirements of each drug quality standard within 6 hours after compatibility. Validation of HPLC Method The HPLC methods of ten drugs were validated that the pediatric electrolyte supplements injection did not interfere with the chromatographic peak of the active drug, and the separation between the main drug and adjacent impurities met the requirements (shown in Supplementary Figure 1 ). Five-point standard curves were constructed for all ten drugs at varying concentrations and showed a good linear between the peak area vs the concentration with a correlation coefficient for all ten curves better than 0.9999 (shown in ). The intraday variations (n = 6), interday variations (n=12), and accuracy in mezlocillin, cefoperazone, sulbactam, ceftazidime, cefazolin, ceftriaxone, ambroxol hydrochloride, cefathiamidine, cefradine, cefmetazole, cefoxitin were shown in . The results showed that the proposed HPLC methods are simple, accurate and precise, which are useful for the determination of ten drugs in pediatric electrolyte supplements injection. The concentration of each drug in the compatible solutions was expressed as the percentage of initial concentration. As shown in , they retained >97% of their initial concentrations in the compatible solutions without precipitating degradation when stored avoiding from light at room temperature.
Within 6 hours, the properties of compatible liquids for ten drugs did not change significantly. Visual examination indicated that they were clear and transparent liquids, without color change, precipitation, crystallization and gas production.
For 10 kinds of compatible solutions, the number of insoluble particles at 0, 0.5, 1, 2, 4 and 6 h after mixing are shown in . According to the criteria of insoluble particles set by Chinese Pharmacopeia (Edition 2020), intravenous injection with a marked amount of 100mL contains the number of particles that are equal to or greater than 10 μm not exceed 25, and equal to or greater than 25 μm not exceed 3. In this experiment, the number of particles in the compatibility solution of mezlocillin sodium for injection and pediatric electrolyte supplements injection exceeded the standard requirements, and the other solutions met the standard requirements.
The pH values of prepared compatible solutions at 0, 0.5, 1, 2, 4 and 6 h after mixing are shown in . The pH of cefoperazone sodium for injection, sulbactam sodium, ceftriaxone for injection, ceftazidime for injection, ambroxol hydrochloride for injection, cefradine for injection, mezlocillin sodium for injection and cefmetazole sodium for injection did not change significantly after compatibility with pediatric electrolyte supplements injection. The pH value increased when cefazolin sodium for injection was compatible with pediatric electrolyte supplements injection, and decreased when cefathiamidine for injection was compatible with pediatric electrolyte supplements injection. According to the pharmaceutics, range of pH for injections is 4–9. The pH values of compatible solutions for ten drugs are within the allowable range, and the pH values of compatible solutions are within the requirements of each drug quality standard within 6 hours after compatibility.
The HPLC methods of ten drugs were validated that the pediatric electrolyte supplements injection did not interfere with the chromatographic peak of the active drug, and the separation between the main drug and adjacent impurities met the requirements (shown in Supplementary Figure 1 ). Five-point standard curves were constructed for all ten drugs at varying concentrations and showed a good linear between the peak area vs the concentration with a correlation coefficient for all ten curves better than 0.9999 (shown in ). The intraday variations (n = 6), interday variations (n=12), and accuracy in mezlocillin, cefoperazone, sulbactam, ceftazidime, cefazolin, ceftriaxone, ambroxol hydrochloride, cefathiamidine, cefradine, cefmetazole, cefoxitin were shown in . The results showed that the proposed HPLC methods are simple, accurate and precise, which are useful for the determination of ten drugs in pediatric electrolyte supplements injection. The concentration of each drug in the compatible solutions was expressed as the percentage of initial concentration. As shown in , they retained >97% of their initial concentrations in the compatible solutions without precipitating degradation when stored avoiding from light at room temperature.
Some commonly used drugs in clinic, such as antibiotics, are often produced as powder injections because of their poor stability in solution. They need to be compatible with the corresponding solvent. However, unreasonable compatibility will reduce the curative effect and increase the adverse reactions. Therefore, we investigated compatibility and stability of ten commonly used clinical drugs in pediatric electrolyte supplements injection which was a rare suitable and commonly used crystal solution for children. More than 90% of drugs are organic, weak electrolytes, especially those compounded, manufactured, or reconstituted as injections in predominantly ionized or salt form. Consequently, acid–base reactions are the most common causes of drug incompatibility. Some injections have a basic pH (>7) or high pKa and will theoretically cause precipitation when in low pH solutions such as 5% glucose (pH 4–4.5). It is well known that they are very prone to precipitation during dilution if the pH is allowed to drift too low. The inappropriate pH of solutions will accelerate drug decomposition or precipitate. Therefore, it can be used as a necessary reference to predict the compatibility change. In our study, the pH values of compatible solutions are within the requirements of each drug quality standard within 6h after compatibility. Insoluble particles widely exist in infusion products and are important factor leading to adverse reactions/events of intravenous administration. They can cause phlebitis, vascular embolism and pyrogen reaction, etc. The Pharmacopoeia of China, United States, Japan, United Kingdom and Europe have stipulated the detection and limiting standards for insoluble particles in injections. Except for mezlocillin sodium for injection, they meted the standards of Chinese Pharmacopoeia when the other nine drugs were compatible with pediatric electrolyte supplements injection. Because the amount of insoluble particles exceeds the standard, it is not recommended to combine mezlocillin sodium for injection with pediatric electrolyte supplements injection to avoid adverse drug reactions. Excessive insoluble particle could be explained by the following: the higher concentration of mezlocillin used in this study compared with other researches; dust particles produced during the preparation of mixture; incomplete dissolution of powder. The number of insoluble particles of ambroxol hydrochloride in pediatric electrolyte supplements injection was more than 25 at 0 hours. However, the number of insoluble particles at other time intervals (0.5, 1, 2, 4 and 6 h) was all not exceed 25. Since the injection solutions were not used at 0 hour after mixing in practice, and excessive insoluble particle could be explained by the incomplete dissolution of powder at 0 hour after mixing. Therefore, ambroxol hydrochloride and pediatric electrolyte supplements injection were compatible. We detect the content stability of 10 drugs in children’s electrolyte supplement injection within 6h by HPLC. The method is more accurate than UV spectrophotometry. There was no interference with the quantification of each drug and the combination of drugs. The determination results are not disturbed by other components. The results showed the percentages of ten drugs remaining in the drug admixtures were all higher than 97% within 6 hours. However, a limitation of this study is that it did not investigate the stability of related substances for ten drugs. Studies have shown that many drugs, such as cefmetazole sodium for injection, are prone to degradation after compatibility with children’s electrolyte supplement injection, which may reduce the content of the preparation and increase the related substances during storage, thus affecting the efficacy and increasing the incidence of adverse reactions. Therefore, in order to ensure the safety of clinical use, It should be used as soon as possible after compatibility. Furthermore, it was reported that higher drug concentrations have been associated with greater incompatibility than lower drug concentrations. Therefore, the compatibility and stability of ten commonly used clinical drugs in pediatric electrolyte supplements injection may be verified by multiple concentrations to provide more reliable decision basis for advisable utilization of drugs.
The clinical implications of our results are that nine diluted infusion solution containing, cefoperazone sodium and sulbactam sodium, ceftazidime, cefradine, ceftriaxone sodium, ambroxol hydrochloride, cefazolin sodium, cefathiamidine, cefmetazole sodium, cefoxitin sodium in pediatric electrolyte supplements injection may be pre-prepared and used up to at least 6hours at 25°C and protected from light. Admixture containing mezlocillin sodium in pediatric electrolyte supplements injection was not recommended for application. Considering the satisfactory stability results in the current study for these 10 chemicals, it can be concluded that the nine admixture can be safe for up to 6 h, and there are potential risks in the combined solution of mezlocillin sodium.
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Exploratory study of serum protein biomarkers for sudden cardiac arrest using protein extension assay: A case-control study | a86955a4-86e8-4aec-a667-439dfc522d9c | 11849859 | Biochemistry[mh] | Sudden cardiac arrest (SCA) is a significant health burden worldwide, and the survival rate has remained low for decades . The annual incidence of SCA is estimated to be 62 per 100,000 in the USA, 50–90 per 100,000 in European countries, and 40–90 per 100,000 in Asian countries . Because of the high fatality rate, identifying high-risk populations as candidates for preventive interventions is crucial for reducing its burden of disease . Currently, no widely utilized methods exist in clinical practice for detecting these high-risk populations. Various biomarkers, including genomic, proteomic, clinical and, imaging biomarkers, have been investigated in previous studies . However, these studies have various limitations, including low accuracy, limited sample size, and targeting of specific disease groups . Compared to other biomarkers, protein biomarkers are more cost-effective, provide rapid results, and enable real-time monitoring of disease states and treatment responses . Previous proteomic biomarker studies for SCA occurrence are often limited by their focus on commonly used biomarkers like C-reactive protein (CRP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP), and by often relying on samples collected long before or long after the cardiac event occurred . Recently, a study utilizing mass spectrometry proteomics analysis was conducted on 330 proteins using samples from SCA survivors and age- and gender-matched control groups, resulting in the proposal of 26 new protein biomarkers . However, many well-known proteins related to cardiovascular diseases were missed in the exploration, and the analysis included only survivors, with samples collected a median of 11 months after SCA. To address these limitations and improve the understanding of protein biomarkers in SCA, further research is needed to assess well-known cardiovascular-related proteins in more timely collected samples, ideally from both survivors and non-survivors, to enhance the accuracy and relevance of biomarker discovery. The aim of this study was to conduct an exploratory analysis to elucidate the association between well-recognized proteins related to cardiovascular, inflammatory and immune diseases and the occurrence of SCA using protein extension assay technique, and to evaluate their predictive power alongside traditional cardiovascular risk factors. Study design This case-controlled study is a part of the Cardiac Arrest Pursuit Trial with Unique Registration and Epidemiologic Surveillance (CAPTURES) project in Korea . This study aimed to identify the risk factors of SCA and develop preventive strategies against it, and has been ongoing since September 29, 2017. In this study, data from 17 participating hospitals from September 29, 2017 to April 30, 2022 were analysed. Ethics statements The study was approved by the ethics committees of all participating centers . All participants or their proxy provided written informed consent before taking part in the study and the study complied with the tenets of the Declaration of Helsinki. This study is registered at ClinicalTrials.gov (NCT03700203). No minors were included in the study. Populations SCA patients aged 20–79 years, who experienced cardiac arrest due to medical causes and were treated by emergency medical services before arrival at the emergency department (ED), were enrolled in the CAPTURES project. Patients with terminal illnesses, pregnancies, in hospice care, living alone, homeless, without reliable information sources, or with a ‘Do Not Resuscitate’ card were excluded. Among the enrolled patients, only SCA patients aged ≤ 65 years whose initial rhythm was shockable in the ED were included in this study because we wanted to focus primarily on relatively young SCA patients with a shockable rhythm and exclude patients with a long lapse of time from the cardiac arrest to ED arrival. Community-based voluntary controls were enrolled from two centers representing metropolitan and non-metropolitan areas. All controls were recruited in collaboration with public health centers or community centers where the project was promoted. One or two controls matched for age, sex, and urbanization level of residence were recruited in each case. Sample collection Structured questionnaires, physical examination, routine laboratory analysis, and blood sampling were conducted for the patients and controls Blood samples (20 mL) were drawn and split into an EDTA tube and two serum-separating tubes (SSTs). The SSTs were centrifuged within 2 hours of sampling. After refrigerated storage of blood samples at 2–8 °C, all blood samples were sent to an external laboratory (Seoul Clinical Laboratories, Seoul, Republic of Korea) for storage and future study. Blood samples were sent to the laboratory once daily on weekdays. For the patients, blood sample extraction was recommended during the initial management, but blood samples collected within 24 h of the ED visit were also included in the study. Lactate was included in the routine laboratory analysis for patients. Protein analysis We used three Olink target panels: the Cardiovascular II (version 5007) 96-Plex panel, Cardiovascular III (version 6114) 96-Plex panel, and Immuno-Oncology (version 3113) panel. The Cardiovascular panels cover proteins associated with biological functions linked to cardiovascular and inflammatory diseases, while the Immuno-Oncology panel covers proteins associated with cancer, the immune system, and systemic inflammation. Serum protein levels were measured using proximity extension immunoassay (PEA) (Olink Proteomics, Uppsala, Sweden). The Olink PEA technology uses a dual-recognition DNA-coupled immunoassay that rapidly allows for protein identification with high sensitivity and specificity. Proteomic level assessments have been described in detail previously . Protein levels were measured on a relative scale and presented as normalised protein expression (NPX), which is an arbitrary unit on a log2 scale. A high NPX value corresponds to a high protein concentration. The levels of different proteins cannot be compared using NPX. However, using inter-plate controls (IPC), any systematic differences across different plates were adjusted; therefore, a consistent comparison of the same protein levels across different plates was possible. The IPC consists of a pool of 92 antibodies, each with unique DNA-tags, and is included in triplicate on each plate. The IPC serves as a synthetic sample, expected to give a high signal across all assays, and the median of the IPC triplicates is used to normalize each assay, correcting for potential variation between runs and plates. Except for the use of IPC in triplicate, the samples themselves were not measured in replicate. Each panel analyzed 92 proteins, totaling 276 proteins with 18 overlapping. NPX values from the panel with the fewest quality control flags were kept for overlaps. The limit of detection (LOD) of each protein was estimated based on the concentration in the negative controls in each sample plate. Proteins were excluded if more than 25% of the measurements were below the LOD. For the remaining proteins, the values below the LOD were replaced by the respective LOD. Since the samples were randomized before analysis, the placement of case and control samples within the plate was random. Statistical analysis All analyses were performed using the R environment for statistical computing, version 4.2.1. The association between proteins and SCA was assessed using a two-sided rank-based Spearman test. We labeled SCA as 1 and the control as 0 and calculated Spearman’s correlation coefficient for each protein. Power analysis was carried out, and we have 0.95 power at 0.05 significance level to detect correlation of 0.591. Therefore, proteins with a correlation exceeding the cutoff (|Spearman’s correlation coefficient|>0.591) were extracted. Among them, we further extracted proteins with low post-cardiac arrest changes, because SCA causes systemic ischemia and inflammation, which affect the levels of various proteins. The procedure was performed in two steps. First, from the 40 SCA patients with confirmed lactate levels, we extracted proteins with no or weak correlation between lactate and protein levels (|Spearman’s correlation coefficient|<0.1). The lactate level is a sensitive marker of cellular hypoxia, including cardiac arrest . Second, from the 20 SCA patients with confirmed arrest time and blood sampling performed within 60 min of SCA onset, we extracted proteins with no or weak correlation between onset-to-sampling time and protein level (|Spearman’s correlation coefficient|<0.1). A full list of proteins and the results of each extraction step are available in and . The distribution of extracted proteins according to SCA was plotted using boxplot, and t-test was used to compare protein levels between groups. A heatmap was also used to visualize the distribution of biomarkers, and a hierarchical cluster analysis was performed. The heatmap reorders the rows and columns of the dataset to place data with similar profiles close to one another. Subsequently, ranges of similar values were assigned specific color codes, and each entry in the data matrix was displayed graphically as one specific colour according to its degree of expression. We also performed a Gene Ontology (GO) Slim summary to simplify the interpretation of the gene ontology analysis . GO is a widely used bioinformatics tool that provides a standardised vocabulary for describing genes and their products . GO Slim is a subset of the full GO dataset, which includes a number of terms selected from each of the three main GO categories (biological process, molecular function, and cellular component). For exploratory analysis, we plotted the relationship between age, BNP, and protein levels in the SCA group and control groups using a scatterplot and smooth line with a fitted linear line since age is an important demographic factor and BNP is a well-known risk factor for cardiac arrest . To evaluate predictive performance of extracted proteins, we calculated area under the receiver operating characteristic curve (AUROC). Multivariable logistic regression models were constructed using extracted proteins, with six traditional risk factors (age, sex, diabetes, hypertension, myocardial infarction, stroke) included as independent variables. Multivariable logistic regression models were also constructed based on BNP, with traditional risk factors and extracted proteins added separately. DeLong’s test was utilized to assess whether there is a statistically significant difference between each ROC curve of the models . This case-controlled study is a part of the Cardiac Arrest Pursuit Trial with Unique Registration and Epidemiologic Surveillance (CAPTURES) project in Korea . This study aimed to identify the risk factors of SCA and develop preventive strategies against it, and has been ongoing since September 29, 2017. In this study, data from 17 participating hospitals from September 29, 2017 to April 30, 2022 were analysed. The study was approved by the ethics committees of all participating centers . All participants or their proxy provided written informed consent before taking part in the study and the study complied with the tenets of the Declaration of Helsinki. This study is registered at ClinicalTrials.gov (NCT03700203). No minors were included in the study. SCA patients aged 20–79 years, who experienced cardiac arrest due to medical causes and were treated by emergency medical services before arrival at the emergency department (ED), were enrolled in the CAPTURES project. Patients with terminal illnesses, pregnancies, in hospice care, living alone, homeless, without reliable information sources, or with a ‘Do Not Resuscitate’ card were excluded. Among the enrolled patients, only SCA patients aged ≤ 65 years whose initial rhythm was shockable in the ED were included in this study because we wanted to focus primarily on relatively young SCA patients with a shockable rhythm and exclude patients with a long lapse of time from the cardiac arrest to ED arrival. Community-based voluntary controls were enrolled from two centers representing metropolitan and non-metropolitan areas. All controls were recruited in collaboration with public health centers or community centers where the project was promoted. One or two controls matched for age, sex, and urbanization level of residence were recruited in each case. Structured questionnaires, physical examination, routine laboratory analysis, and blood sampling were conducted for the patients and controls Blood samples (20 mL) were drawn and split into an EDTA tube and two serum-separating tubes (SSTs). The SSTs were centrifuged within 2 hours of sampling. After refrigerated storage of blood samples at 2–8 °C, all blood samples were sent to an external laboratory (Seoul Clinical Laboratories, Seoul, Republic of Korea) for storage and future study. Blood samples were sent to the laboratory once daily on weekdays. For the patients, blood sample extraction was recommended during the initial management, but blood samples collected within 24 h of the ED visit were also included in the study. Lactate was included in the routine laboratory analysis for patients. We used three Olink target panels: the Cardiovascular II (version 5007) 96-Plex panel, Cardiovascular III (version 6114) 96-Plex panel, and Immuno-Oncology (version 3113) panel. The Cardiovascular panels cover proteins associated with biological functions linked to cardiovascular and inflammatory diseases, while the Immuno-Oncology panel covers proteins associated with cancer, the immune system, and systemic inflammation. Serum protein levels were measured using proximity extension immunoassay (PEA) (Olink Proteomics, Uppsala, Sweden). The Olink PEA technology uses a dual-recognition DNA-coupled immunoassay that rapidly allows for protein identification with high sensitivity and specificity. Proteomic level assessments have been described in detail previously . Protein levels were measured on a relative scale and presented as normalised protein expression (NPX), which is an arbitrary unit on a log2 scale. A high NPX value corresponds to a high protein concentration. The levels of different proteins cannot be compared using NPX. However, using inter-plate controls (IPC), any systematic differences across different plates were adjusted; therefore, a consistent comparison of the same protein levels across different plates was possible. The IPC consists of a pool of 92 antibodies, each with unique DNA-tags, and is included in triplicate on each plate. The IPC serves as a synthetic sample, expected to give a high signal across all assays, and the median of the IPC triplicates is used to normalize each assay, correcting for potential variation between runs and plates. Except for the use of IPC in triplicate, the samples themselves were not measured in replicate. Each panel analyzed 92 proteins, totaling 276 proteins with 18 overlapping. NPX values from the panel with the fewest quality control flags were kept for overlaps. The limit of detection (LOD) of each protein was estimated based on the concentration in the negative controls in each sample plate. Proteins were excluded if more than 25% of the measurements were below the LOD. For the remaining proteins, the values below the LOD were replaced by the respective LOD. Since the samples were randomized before analysis, the placement of case and control samples within the plate was random. All analyses were performed using the R environment for statistical computing, version 4.2.1. The association between proteins and SCA was assessed using a two-sided rank-based Spearman test. We labeled SCA as 1 and the control as 0 and calculated Spearman’s correlation coefficient for each protein. Power analysis was carried out, and we have 0.95 power at 0.05 significance level to detect correlation of 0.591. Therefore, proteins with a correlation exceeding the cutoff (|Spearman’s correlation coefficient|>0.591) were extracted. Among them, we further extracted proteins with low post-cardiac arrest changes, because SCA causes systemic ischemia and inflammation, which affect the levels of various proteins. The procedure was performed in two steps. First, from the 40 SCA patients with confirmed lactate levels, we extracted proteins with no or weak correlation between lactate and protein levels (|Spearman’s correlation coefficient|<0.1). The lactate level is a sensitive marker of cellular hypoxia, including cardiac arrest . Second, from the 20 SCA patients with confirmed arrest time and blood sampling performed within 60 min of SCA onset, we extracted proteins with no or weak correlation between onset-to-sampling time and protein level (|Spearman’s correlation coefficient|<0.1). A full list of proteins and the results of each extraction step are available in and . The distribution of extracted proteins according to SCA was plotted using boxplot, and t-test was used to compare protein levels between groups. A heatmap was also used to visualize the distribution of biomarkers, and a hierarchical cluster analysis was performed. The heatmap reorders the rows and columns of the dataset to place data with similar profiles close to one another. Subsequently, ranges of similar values were assigned specific color codes, and each entry in the data matrix was displayed graphically as one specific colour according to its degree of expression. We also performed a Gene Ontology (GO) Slim summary to simplify the interpretation of the gene ontology analysis . GO is a widely used bioinformatics tool that provides a standardised vocabulary for describing genes and their products . GO Slim is a subset of the full GO dataset, which includes a number of terms selected from each of the three main GO categories (biological process, molecular function, and cellular component). For exploratory analysis, we plotted the relationship between age, BNP, and protein levels in the SCA group and control groups using a scatterplot and smooth line with a fitted linear line since age is an important demographic factor and BNP is a well-known risk factor for cardiac arrest . To evaluate predictive performance of extracted proteins, we calculated area under the receiver operating characteristic curve (AUROC). Multivariable logistic regression models were constructed using extracted proteins, with six traditional risk factors (age, sex, diabetes, hypertension, myocardial infarction, stroke) included as independent variables. Multivariable logistic regression models were also constructed based on BNP, with traditional risk factors and extracted proteins added separately. DeLong’s test was utilized to assess whether there is a statistically significant difference between each ROC curve of the models . Demographic findings During the study period, a total of 1,228 SCA patients and 2,065 controls were enrolled in CAPTURES project. Among the SCA cases included, 60 patients aged ≤ 65 years with a shockable initial rhythm at the ED were identified. Among the 60 SCA patients with a shockable rhythm, a random sample of 42 patients was analyzed with 42 matched controls. For the patient group, 42 cases were collected from 13 centers as follows: 5, 4, 7, 5, 2, 2, 1, 2, 5, 1, 4, 1, and 3 cases, respectively. For the control group, 26 and 10 cases were collected from two centers, respectively. The demographic characteristics of the 42 patients and 42 controls are shown in . Each group had 35 males (83.3%) with a median (IQR) age of 56 (60–61) years. The number of comorbidities was significantly higher in the cases . Among 42 cases, survival to admission and survival to discharge were 36 (85.7%) and 23 (54.8%), respectively. Coronary angiography was performed in 23 (54.8%) cases, and percutaneous coronary intervention was performed in 13 (31.0%) cases. Among the 35 patients with confirmed arrest time and blood sampling time, the median arrest-to-sampling time was 55 minutes (IQR: 35–105). Among the 40 patients with confirmed initial lactate levels, the median lactate level was 11.3 mmol/L (IQR: 10.1–14.7). Biomarker extraction and exploration Among the 258 distinct proteins, 12 proteins were excluded for analysis because more than 25% of the measurements were below the LOD. Of the remaining 246 proteins, 97 showed a strong correlation with SCA, exceeding the cutoff (|Spearman’s correlation coefficient|>0.591). Among these 97 proteins, 44 showed weak or no correlation with lactate levels, and 12 showed weak or no correlation with onset-to-sampling time. Two proteins (AXL receptor tyrosine kinase [AXL] and TIMP Metallopeptidase inhibitor 4 [TIMP-4]) met all the criteria for biomarker extraction ( and ). In the GO Slim summary, both proteins were related to the extracellular space in the cellular component category . Similarly, in the GO Slim summary for 97 proteins strongly correlated with sudden cardiac arrest, the top cellular component category was also related to the extracellular space . The distribution of extracted proteins according to SCA was plotted in . Both proteins had higher NPX levels in the SCA group compared to the control group (both p < 0.001). In the SCA group, AXL’s NPX values ranged from 7.7 to 10, while TIMP-4’s NPX values ranged from 2.6 to 4.9, with the difference between the maximum and minimum values being less than an NPX of 3 for both. The NPX values of the two proteins collected from each center are presented in . The results of hierarchical cluster analysis were plotted using a heatmap. The heatmap also showed that the levels of the two proteins were higher in patients than in the controls. Examining the largest cluster in the heatmap by group, 25 (59.5%) in the SCA group and 22 (52.3%) in the control group were in the same cluster . In the exploratory analysis, the values of these two proteins tended to be higher in patients than in controls for all age groups and BNP levels . In the case of BNP, the overall level was lower in controls than in patients, but the level of the two proteins was higher in patients than in controls when the BNP level was low in both groups . The AUROC (95% confidence interval [CI]) of AXL model and TIMP-4 model were 0.893 (0.820–0.967) and 0.867 (0.792–0.942), respectively. The predictive performance of the AXL model and the TIMP-4 model was similar (p for comparison = 0.593). However, the NPX values of AXL and TIMP-4 did not showed a strong correlation (correlation coefficient [95% CI]: 0.543 [0.372–0.679]), and when both AXL and TIMP-4 were included in the model, the predictive performance was significantly higher than that of each extracted protein model (AUROC [95% CI] 0.944 [0.895–0.994] for AXL with TIMP-4 model (p for comparison = 0.026 for AXL model and p for comparison = 0.031 for TIMP-4 model). The AUROC of the baseline model using six traditional risk factors was 0.692 (95% Confidence interval [CI], 0.578–0.806). The addition of AXL, TIMP-4 or both showed a significantly higher predictive power compared to baseline model (AUROC [95% CI] 0.891 [0.817–0.964] for baseline with AXL model and 0.910 [0.910–0.997] for baseline with TIMP-4 model, and 0.954 [0.910–0.997] for baseline with AXL and TIMP-4 model, respectively, all p < 0.01 comparison to the baseline model). When both proteins were added to the model, there was a significant difference in AUROC compared to the baseline with AXL model ( p = 0.007), but there was no significant difference in AUROC compared to the baseline with TIMP-4 model ( p = 0.121) . The AUROC of the BNP model was 0.787 (95% CI, 0.688–0.885). While the addition of six traditional risk factors to BNP did not significantly enhance predictive power (AUROC [95% CI] 0.788 [0.689–0.888] for BNP with six traditional risk factors model, p for comparison = 0.072), the inclusion of AXL or TIMP–4 significantly improved the predictive performance compared to the BNP model (AUROC [95% CI] 0.918 [0.853–0.983] and p for comparison = 0.029 for BNP with AXL model and 0.914 [0.850 + 0.978] and p for comparison = 0.005 for BNP with TIMP-4 model) . During the study period, a total of 1,228 SCA patients and 2,065 controls were enrolled in CAPTURES project. Among the SCA cases included, 60 patients aged ≤ 65 years with a shockable initial rhythm at the ED were identified. Among the 60 SCA patients with a shockable rhythm, a random sample of 42 patients was analyzed with 42 matched controls. For the patient group, 42 cases were collected from 13 centers as follows: 5, 4, 7, 5, 2, 2, 1, 2, 5, 1, 4, 1, and 3 cases, respectively. For the control group, 26 and 10 cases were collected from two centers, respectively. The demographic characteristics of the 42 patients and 42 controls are shown in . Each group had 35 males (83.3%) with a median (IQR) age of 56 (60–61) years. The number of comorbidities was significantly higher in the cases . Among 42 cases, survival to admission and survival to discharge were 36 (85.7%) and 23 (54.8%), respectively. Coronary angiography was performed in 23 (54.8%) cases, and percutaneous coronary intervention was performed in 13 (31.0%) cases. Among the 35 patients with confirmed arrest time and blood sampling time, the median arrest-to-sampling time was 55 minutes (IQR: 35–105). Among the 40 patients with confirmed initial lactate levels, the median lactate level was 11.3 mmol/L (IQR: 10.1–14.7). Among the 258 distinct proteins, 12 proteins were excluded for analysis because more than 25% of the measurements were below the LOD. Of the remaining 246 proteins, 97 showed a strong correlation with SCA, exceeding the cutoff (|Spearman’s correlation coefficient|>0.591). Among these 97 proteins, 44 showed weak or no correlation with lactate levels, and 12 showed weak or no correlation with onset-to-sampling time. Two proteins (AXL receptor tyrosine kinase [AXL] and TIMP Metallopeptidase inhibitor 4 [TIMP-4]) met all the criteria for biomarker extraction ( and ). In the GO Slim summary, both proteins were related to the extracellular space in the cellular component category . Similarly, in the GO Slim summary for 97 proteins strongly correlated with sudden cardiac arrest, the top cellular component category was also related to the extracellular space . The distribution of extracted proteins according to SCA was plotted in . Both proteins had higher NPX levels in the SCA group compared to the control group (both p < 0.001). In the SCA group, AXL’s NPX values ranged from 7.7 to 10, while TIMP-4’s NPX values ranged from 2.6 to 4.9, with the difference between the maximum and minimum values being less than an NPX of 3 for both. The NPX values of the two proteins collected from each center are presented in . The results of hierarchical cluster analysis were plotted using a heatmap. The heatmap also showed that the levels of the two proteins were higher in patients than in the controls. Examining the largest cluster in the heatmap by group, 25 (59.5%) in the SCA group and 22 (52.3%) in the control group were in the same cluster . In the exploratory analysis, the values of these two proteins tended to be higher in patients than in controls for all age groups and BNP levels . In the case of BNP, the overall level was lower in controls than in patients, but the level of the two proteins was higher in patients than in controls when the BNP level was low in both groups . The AUROC (95% confidence interval [CI]) of AXL model and TIMP-4 model were 0.893 (0.820–0.967) and 0.867 (0.792–0.942), respectively. The predictive performance of the AXL model and the TIMP-4 model was similar (p for comparison = 0.593). However, the NPX values of AXL and TIMP-4 did not showed a strong correlation (correlation coefficient [95% CI]: 0.543 [0.372–0.679]), and when both AXL and TIMP-4 were included in the model, the predictive performance was significantly higher than that of each extracted protein model (AUROC [95% CI] 0.944 [0.895–0.994] for AXL with TIMP-4 model (p for comparison = 0.026 for AXL model and p for comparison = 0.031 for TIMP-4 model). The AUROC of the baseline model using six traditional risk factors was 0.692 (95% Confidence interval [CI], 0.578–0.806). The addition of AXL, TIMP-4 or both showed a significantly higher predictive power compared to baseline model (AUROC [95% CI] 0.891 [0.817–0.964] for baseline with AXL model and 0.910 [0.910–0.997] for baseline with TIMP-4 model, and 0.954 [0.910–0.997] for baseline with AXL and TIMP-4 model, respectively, all p < 0.01 comparison to the baseline model). When both proteins were added to the model, there was a significant difference in AUROC compared to the baseline with AXL model ( p = 0.007), but there was no significant difference in AUROC compared to the baseline with TIMP-4 model ( p = 0.121) . The AUROC of the BNP model was 0.787 (95% CI, 0.688–0.885). While the addition of six traditional risk factors to BNP did not significantly enhance predictive power (AUROC [95% CI] 0.788 [0.689–0.888] for BNP with six traditional risk factors model, p for comparison = 0.072), the inclusion of AXL or TIMP–4 significantly improved the predictive performance compared to the BNP model (AUROC [95% CI] 0.918 [0.853–0.983] and p for comparison = 0.029 for BNP with AXL model and 0.914 [0.850 + 0.978] and p for comparison = 0.005 for BNP with TIMP-4 model) . In this exploratory study, differences in serum protein profiles of 42 SCA cases with medical causes, aged 20 to 65 years, and whose initial rhythm was shockable on admission to the ED, compared to 42 community-based age- and sex-matched controls, were evaluated using a PEA protein assay. Among 246 proteins that met the quality criteria, 97 showed a strong correlation, satisfying sufficient power in this study’s sample size. When extracting proteins unlikely to show post-cardiac arrest changes based on their levels in relation to lactate and sampling time, two proteins (AXL and TIMP-4) were identified. Both proteins demonstrated enhanced discrimination power when added to traditional risk factors in multivariable analysis. AXL is a cell surface receptor that is involved in signal transduction, from the extracellular matrix (ECM) into the cytoplasm, associated with cell proliferation, adhesion, migration and survival. AXL is an inhibitor of the innate immune response, and is associated with a variety of pathological processes including cancer and autoimmune disorders . AXL also drives cardiac remodelling by regulating endothelial cells, vascular smooth muscle cells, cardiomyocytes, and potentially, fibroblasts . A study using a rat model reported that AXL level increases in the early stages of left ventricular remodelling with pressure overload, with no further increase in heart failure . TIMP-4 inhibits the activity of matrix metalloproteinases (MMP). MMPs play a crucial role in extracellular matrix remodelling and are involved in various physiological processes including tissue development, wound healing, and the malignant conversion of tumour cells . TIMP-4 are the most abundant TIMP protein within the myocardium. A previous study reported that the TIMP-4 level increased soon after acute myocardial infarction (AMI) and was positively correlated with left ventricular volume changes . In animal model-based studies, an increase in TIMP-4 was observed in compensated left ventricular hypertrophy, but in heart failure, TIMP-4 level or activity had decreased . TIMP-4 was also negatively correlated with atrial fibrosis and ECM changes in the atria of rheumatic heart disease with atrial fibrillation . We found that the biomarker analysis results of both proteins were related to the ECM region . In addition, we found that all two proteins were directly associated with cardiac remodelling. Cardiac remodelling is one of the main causes of cardiac arrhythmia, ventricular dysfunction, and sudden death . In particular, a previous study has reported an important relationship between cardiac remodelling and arrhythmia, whereby the acquired changes in cardiac structure or function can promote the occurrence of cardiac arrhythmia (arrhythmogenic cardiac remodelling) . The heart can be electrically remodelled by various stimuli in the absence of structural remodelling . Aging itself can cause functional cardiac changes before structural remodelling . In this study, we found that the levels of the two protein biomarkers identified were higher in SCA patients than in controls, under a low BNP level scenario . We also found a significant improvement in predictive performance for SCA when BNP was combined with the extracted protein, compared to the BNP model . These findings suggest that functional or molecular changes in the heart prior to prominent structural changes may affect the risk of cardiac arrest, and that the two biomarkers we discovered might help detect these changes. A recent study analyzed 330 proteins of 20 SCA survivors and 40 control participants using a TripleTOF® 6600 mass spectrometer with a data-independent acquisition technique, and reported 26 protein biomarkers associated with SCA, of which 20 differentiated SCA from coronary artery disease . In this study, the extracellular matrix was included among the top identified biological processes, which is consistent with our results. This study enhances its validity by conducting additional replication analyses using an additional 29 cases and 57 controls. However, direct comparison with our study is limited because only nine proteins overlap with those analyzed in the current research, and neither AXL nor TIMP-4 were analyzed. In the GO Slim summary for 97 proteins strongly correlated with sudden cardiac arrest, we also found that extracellular components accounted for a significant proportion in the cellular component category. However, the top GO terms in the biological process category were related to inflammatory response and apoptosis-related pathways, while in the molecular function category, the top GO terms were identical protein binding, cytokine activity, and zinc ion binding. This may reflect various pathogenesis mechanisms associated with the occurrence of SCA. Further research targeting various proteins is still necessary. Limitations This study had several limitations. First, the case-control design was used to explore differences in blood test results between SCA patients and controls. Given the unexpected nature of SCA, a case-control design was employed to generate hypotheses more efficiently. Our findings need to be verified in larger cohorts. Second, blood sampling was performed after SCA occurred, which means the samples could be influenced by post-cardiac arrest changes. We reduced this effect by using early post-SCA samples and additional analysis with lactate levels and arrest-to-sampling time. The timing of biomarker measurement is also a concern in previous studies, with samples collected months before or after SCA, leading to interpretation difficulties . We minimized the temporal gap to SCA, but retrospective proximity may still influence data, requiring caution. In addition, single-time sampling without repeats limited the identification of post-cardiac arrest effects. Third, only pre-specified proteins were analyzed, excluding other known or unknown proteins. Fourth, only Korean patients with shockable rhythm aged ≤ 65 years were included, requiring caution in interpreting and applying the results. Fifth, AXL and TIMP-4 may be influenced by confounding effects due to their association with other conditions related to SCA. While we included diabetes, hypertension, myocardial infarction, and stroke in our multivariable model, the small sample size limited our ability to adequately adjust for other potential comorbidities. Lastly, because this study was conducted with a retrospective design, it was inherently limited to identifying associations among the variables examined. As a result, it is not possible to draw definitive conclusions regarding causal relationships from the findings. This study had several limitations. First, the case-control design was used to explore differences in blood test results between SCA patients and controls. Given the unexpected nature of SCA, a case-control design was employed to generate hypotheses more efficiently. Our findings need to be verified in larger cohorts. Second, blood sampling was performed after SCA occurred, which means the samples could be influenced by post-cardiac arrest changes. We reduced this effect by using early post-SCA samples and additional analysis with lactate levels and arrest-to-sampling time. The timing of biomarker measurement is also a concern in previous studies, with samples collected months before or after SCA, leading to interpretation difficulties . We minimized the temporal gap to SCA, but retrospective proximity may still influence data, requiring caution. In addition, single-time sampling without repeats limited the identification of post-cardiac arrest effects. Third, only pre-specified proteins were analyzed, excluding other known or unknown proteins. Fourth, only Korean patients with shockable rhythm aged ≤ 65 years were included, requiring caution in interpreting and applying the results. Fifth, AXL and TIMP-4 may be influenced by confounding effects due to their association with other conditions related to SCA. While we included diabetes, hypertension, myocardial infarction, and stroke in our multivariable model, the small sample size limited our ability to adequately adjust for other potential comorbidities. Lastly, because this study was conducted with a retrospective design, it was inherently limited to identifying associations among the variables examined. As a result, it is not possible to draw definitive conclusions regarding causal relationships from the findings. Using blood samples from 42 SCA patients and 42 controls, we evaluated the serum levels of 246 proteins, identifying AXL and TIMP-4 as potential SCA biomarkers. Both proteins showed a significant association with SCA and enhanced predictive power with traditional risk factors in multivariable analysis. Our findings suggest that these biomarkers, involved in cardiac remodelling and extracellular matrix processes, may aid early detection and risk assessment of SCA. However, the study’s limitations, including its case-control design, single-time sampling, and small sample size, necessitate validation in future studies. Identifying patients with minimal post-cardiac arrest changes could be one potential approach. Selecting patients with witnessed cardiac arrest who have both a short time from arrest to return of spontaneous circulation and a short time to blood sampling could help minimize post-cardiac arrest changes for analysis. Alternatively, analyzing blood samples already collected from cohorts where SCA occurrence is monitored could provide an opportunity to investigate the relationship between identified biomarkers and SCA. Future research should also explore additional biomarkers and verify AXL and TIMP-4’s utility in diverse populations to solidify their clinical role in SCA prevention and management. S1 Table Institutional Review Board (IRB) Numbers of participating hospitals. (DOCX) S2 Table Full list of proteins in the analysis with protein selection criteria. (DOCX) S3 Table Full list of proteins in the analysis with correlation coefficients at each criteria. (DOCX) S1 Fig Goslim summary for biological process, molecular function, and cellular component for AXL and TIMP-4 proteins. AXL Receptor Tyrosine Kinase; TIMP Metallopeptidase Inhibitor 4. (DOCX) S2 Fig Goslim summary for biological process, molecular function, and cellular component for 97 proteins with strong correlation with sudden cardiac arrest, exceeding the cutoff (|Spearman’s correlation coefficient|>0.516). (DOCX) S3 Fig Distribution of AXL and TIMP-4 in patient groups by center. AXL Receptor Tyrosine Kinase; TIMP Metallopeptidase Inhibitor 4. (DOCX) |
Simultaneous Intra-articular and Extra-articular Corrective Osteotomies Using a Patient-Matched Surgical Guide and Plate for Malunion After Distal Radius Fractures: A Report of Two Cases | fc49fdea-2814-4619-9e41-e1495d35a796 | 11882271 | Surgical Procedures, Operative[mh] | Case 1 A 64-year-old woman had a left distal radial fracture (AO type C1). Initial conservative splint treatment resulted in malunion. She was referred to our hospital because of wrist pain and restricted ROM. Grip strength was reduced to 12 kg on the affected side (50% of the unaffected side). Radiography revealed a volar tilt (VT) of 38°, radial inclination (RI) of 12°, and ulnar variance (UV) of +5 mm. CT showed an intra-articular step-off of up to 5 mm and volar dislocation of the carpal bones (Figure ). Case 2 A 78-year-old woman presented with a left distal radial fracture (AO type C1). Initial conservative treatment was a cast, which resulted in malunion. Subsequently, she reported of wrist pain and restricted ROM and was referred to our institution for malunion treatment. Her grip strength had reduced to 5 kg on the affected side (30% of the unaffected side). Radiography revealed a VT of −30°, RI of 18°, and UV of +5 mm. CT revealed an intra-articular irregularity characterized by a 4-mm step-off and a 7-mm gap at the sigmoid notch (Figure ). Simulation and Surgery For deformity evaluation and corrective osteotomy planning, three-dimensional (3D) bone models were created from CT images using software (BoneSimulator; Teijin Nakashima Medical). In Case 1, the extra-articular deformity, evaluated against a mirrored image of the unaffected side, showed radial flexion, 0.5°; palmar flexion, 19.2°; pronation, 8.9°; and intra-articular step-off, 5 mm (Figure ). Corrective simulation was performed based on the mirrored image (Figure , A). A patient-matched plate (Accurio deformity correction system; Teijin Nakashima Medical) was designed to fit the volar bone surface after correction and provide sufficient fixation strength for the insertion of multiple screws, including into the intra-articular bone fragment. The extra-articular surgical guide was shaped to fit the bone surface and had an osteotomy slit and predrilled screw holes. The intra-articular surgical guide had multiple drilling sleeves to divide the malunited fragment using an extra-articular approach, thus eliminating intra-articular step-offs. To precisely define the placement of the guide, it was designed to fit both the volar bone surface and the osteotomy plane (Figure , B–D). The corrective osteotomy was conducted 7 months after the initial injury. Extra-articular osteotomy was conducted first by setting the extra-articular surgical guide on the radius, predrilling screw holes for the patient-matched plate, and then cutting the bone using a slit (Figure , A). Subsequently, intra-articular osteotomy was conducted. An intra-articular surgical guide was fitted to the distal bone fragments, both the volar bone surface and the osteotomy plane. Multiple drill holes were made through the drill sleeves using a 1.2-mm diameter Kirschner wire, and the fragment was divided along the drill holes (Figure , B). The separated fragments were realigned using fluoroscopy and fixed with two double-threaded headless screws (Double Threaded Screw; MEIRA) to correct the intra-articular deformity. Finally, a patient-matched plate was placed by inserting screws into the predrilled holes, automatically completing the correction and fixation of the extra-articular deformity (Figure , C). The defect was grafted with an iliac bone and an artificial bone. Postoperative radiography showed that the extra-articular deformity was corrected to a VT of 14°, RI of 25°, and UV of 0 mm (Figure , A and B). On postoperative CT, the intra-articular step-off was reduced from 5 to 0 mm, with correction of the volar displacement of the carpal bones (Figure , C). Bone fusion was confirmed without corrective loss 4 months postoperatively. At the latest follow-up, 1 year postoperatively, ROM had improved from 60° to 75° for flexion, 40° to 70° for extension, 80° to 90° for pronation, and 40° to 80° for supination (Figure ). Activity-related pain was reduced from a visual analog scale score of 30/100 to 14/100 mm, and grip strength was increased to 80% of the unaffected side strength. The patient resumed normal activities without impairment. The same procedures were performed for Case 2. A 3D CT scan revealed an extra-articular deformity with 26° of radial flexion, 49° of dorsal flexion, and 14° of pronation and an intra-articular deformity with a 4-mm step-off and a 7-mm gap (Figure ). Given the dorsal location of the intra-articular deformity, an intra-articular osteotomy guide was specifically designed for dorsal application. Corrective osteotomy was conducted 6 months after the initial injury. An approach from the volar side was first used to perform the extra-articular osteotomy. Subsequently, extra-articular reduction was performed using a patient-matched plate, and iliac and artificial bone were grafted at the defect. The dorsal side was exposed, and osteotomy was performed using an intra-articular cutting guide. The bone fragment was reduced with pressure against the articular surface and fixed using double-threaded headless screws. Postoperative radiography showed a VT of 18°, RI of 20°, and UV of +1 mm, indicating improvement (Figure , A and B). The intra-articular deformity was reduced from a 4-mm step-off and 7-mm gap to 0 mm for both (Figure , C). Bone fusion was confirmed without corrective loss 6 months postoperatively. The ROM improved from 25° to 60° for flexion, 75° to 80° for extension, 55° to 80° for pronation, and 55° to 85° for supination. Activity-related pain reduced from a visual analog scale score of 80/100 to 10/100 mm, and grip strength increased to 75% of the unaffected side strength.
A 64-year-old woman had a left distal radial fracture (AO type C1). Initial conservative splint treatment resulted in malunion. She was referred to our hospital because of wrist pain and restricted ROM. Grip strength was reduced to 12 kg on the affected side (50% of the unaffected side). Radiography revealed a volar tilt (VT) of 38°, radial inclination (RI) of 12°, and ulnar variance (UV) of +5 mm. CT showed an intra-articular step-off of up to 5 mm and volar dislocation of the carpal bones (Figure ).
A 78-year-old woman presented with a left distal radial fracture (AO type C1). Initial conservative treatment was a cast, which resulted in malunion. Subsequently, she reported of wrist pain and restricted ROM and was referred to our institution for malunion treatment. Her grip strength had reduced to 5 kg on the affected side (30% of the unaffected side). Radiography revealed a VT of −30°, RI of 18°, and UV of +5 mm. CT revealed an intra-articular irregularity characterized by a 4-mm step-off and a 7-mm gap at the sigmoid notch (Figure ).
For deformity evaluation and corrective osteotomy planning, three-dimensional (3D) bone models were created from CT images using software (BoneSimulator; Teijin Nakashima Medical). In Case 1, the extra-articular deformity, evaluated against a mirrored image of the unaffected side, showed radial flexion, 0.5°; palmar flexion, 19.2°; pronation, 8.9°; and intra-articular step-off, 5 mm (Figure ). Corrective simulation was performed based on the mirrored image (Figure , A). A patient-matched plate (Accurio deformity correction system; Teijin Nakashima Medical) was designed to fit the volar bone surface after correction and provide sufficient fixation strength for the insertion of multiple screws, including into the intra-articular bone fragment. The extra-articular surgical guide was shaped to fit the bone surface and had an osteotomy slit and predrilled screw holes. The intra-articular surgical guide had multiple drilling sleeves to divide the malunited fragment using an extra-articular approach, thus eliminating intra-articular step-offs. To precisely define the placement of the guide, it was designed to fit both the volar bone surface and the osteotomy plane (Figure , B–D). The corrective osteotomy was conducted 7 months after the initial injury. Extra-articular osteotomy was conducted first by setting the extra-articular surgical guide on the radius, predrilling screw holes for the patient-matched plate, and then cutting the bone using a slit (Figure , A). Subsequently, intra-articular osteotomy was conducted. An intra-articular surgical guide was fitted to the distal bone fragments, both the volar bone surface and the osteotomy plane. Multiple drill holes were made through the drill sleeves using a 1.2-mm diameter Kirschner wire, and the fragment was divided along the drill holes (Figure , B). The separated fragments were realigned using fluoroscopy and fixed with two double-threaded headless screws (Double Threaded Screw; MEIRA) to correct the intra-articular deformity. Finally, a patient-matched plate was placed by inserting screws into the predrilled holes, automatically completing the correction and fixation of the extra-articular deformity (Figure , C). The defect was grafted with an iliac bone and an artificial bone. Postoperative radiography showed that the extra-articular deformity was corrected to a VT of 14°, RI of 25°, and UV of 0 mm (Figure , A and B). On postoperative CT, the intra-articular step-off was reduced from 5 to 0 mm, with correction of the volar displacement of the carpal bones (Figure , C). Bone fusion was confirmed without corrective loss 4 months postoperatively. At the latest follow-up, 1 year postoperatively, ROM had improved from 60° to 75° for flexion, 40° to 70° for extension, 80° to 90° for pronation, and 40° to 80° for supination (Figure ). Activity-related pain was reduced from a visual analog scale score of 30/100 to 14/100 mm, and grip strength was increased to 80% of the unaffected side strength. The patient resumed normal activities without impairment. The same procedures were performed for Case 2. A 3D CT scan revealed an extra-articular deformity with 26° of radial flexion, 49° of dorsal flexion, and 14° of pronation and an intra-articular deformity with a 4-mm step-off and a 7-mm gap (Figure ). Given the dorsal location of the intra-articular deformity, an intra-articular osteotomy guide was specifically designed for dorsal application. Corrective osteotomy was conducted 6 months after the initial injury. An approach from the volar side was first used to perform the extra-articular osteotomy. Subsequently, extra-articular reduction was performed using a patient-matched plate, and iliac and artificial bone were grafted at the defect. The dorsal side was exposed, and osteotomy was performed using an intra-articular cutting guide. The bone fragment was reduced with pressure against the articular surface and fixed using double-threaded headless screws. Postoperative radiography showed a VT of 18°, RI of 20°, and UV of +1 mm, indicating improvement (Figure , A and B). The intra-articular deformity was reduced from a 4-mm step-off and 7-mm gap to 0 mm for both (Figure , C). Bone fusion was confirmed without corrective loss 6 months postoperatively. The ROM improved from 25° to 60° for flexion, 75° to 80° for extension, 55° to 80° for pronation, and 55° to 85° for supination. Activity-related pain reduced from a visual analog scale score of 80/100 to 10/100 mm, and grip strength increased to 75% of the unaffected side strength.
Malunion after distal radial fractures is a notable complication in approximately 5.3% of cases. The malunion rate after conservative treatment is particularly high (15.6% to 37.5%). , Malunion has a substantial effect on clinical outcomes, with extra-articular deformities of >10° dorsal tilt and shortening of >3 mm, contributing to wrist dysfunction. Intra-articular deformities with a step-off >2 mm contribute to osteoarthritic changes. For symptomatic malunion, corrective osteotomy is performed to restore normal anatomy. , Various reports have described this corrective osteotomy method for malunion after distal radial fractures. Patient-matched surgical guides from preoperative 3D CT have been introduced to achieve accurate osteotomy correction. Regarding treatment outcomes, extra-articular deformities can be corrected with an accuracy of 1 mm and 1°, and for intra-articular deformities, step-offs can be reduced to approximately 1 mm. , , However, when corrective osteotomy is required for both intra-articular and extra-articular deformities, treatment becomes challenging. Few studies report the simultaneous correction of both intra-articular and extra-articular deformities , , because the simultaneous procedure is associated with a risk of necrosis of the intra-articular bone fragments, particularly with the addition of osteotomy sites. The use of patient-matched surgical guides allows osteotomies to be performed from outside the joint without damaging the joint capsule, resulting in the preservation of soft tissue attached to the bone fragments. Furthermore, the simultaneous fixation of multiple bone fragments is complex. Meticulous attention is needed for plate placement and fixation between bone fragments to secure multiple bone pieces. The patient-matched surgical guides enable osteotomy and screw hole creation as planned preoperatively. In addition, by using a patient-matched plate, screws can be positioned at any location according to the preoperative plan, and the plate can be shaped to fit the bone surface after reduction, thereby simplifying the complexity of the reduction. Patient-matched plates closely fitted to the bone surface also minimize damage to soft tissues and interference with the flexor tendons. In our cases, satisfactory correction angles were achieved for extra-articular deformities and intra-articular deformities were also reduced to <1 mm. These results suggest that performing both intra-articular and extra-articular corrective osteotomies simultaneously using PMIs may achieve a level of precision comparable with that achieved with these procedures separately. In addition, improvements were observed in both postoperative ROM and pain relief, and bone fusion was achieved within 6 months postoperatively, without bone fragment necrosis. Consequently, we believe that simultaneous corrective osteotomy using PMIs for both intra-articular and extra-articular deformities after distal radius fractures is a highly effective treatment method. This study had some limitations. First, the relatively short follow-up period limited the assessment of long-term outcomes. Second, even if an osteotomy is performed from outside a joint without damaging the joint capsule, multiple drilling into small bone fragments could lead to osteonecrosis, especially in patients with osteoporosis. In cases with fairly small bone fragments or osteoporosis, it is crucial to determine the number of screws considering stability and the risk of osteonecrosis. Furthermore, osteoporotic patients are at risk of collapse even if they do not develop osteonecrosis. Therefore, it is essential to design plates that offer robust support to the volar surface of intra-articular fragments and provide subchondral support with screws. Concurrently, the use of anabolic agents, such as teriparatide, is recommended for the treatment of osteoporosis. Moreover, PMI creation requires notable time and cost, which may not be feasible in clinical settings. Approximately 2 weeks are needed to manufacture the instruments, with the patient-matched surgical guide and plate costing approximately 600 and 2000 USD, respectively. , However, because corrective osteotomies for both intra-articular and extra-articular deformities are inherently complex procedures, our method seems a viable option for patients with severely impaired wrist function after intra-articular and extra-articular fractures, who may require salvage surgery such as arthrodesis. We believe that as this technology becomes more affordable and widely adopted, its range of applications will expand markedly.
Simultaneous corrective osteotomy using PMIs was conducted for intra-articular and extra-articular malunions after distal radial fractures. Both patients achieved satisfactory outcomes, suggesting that PMIs are useful surgical options for corrective intra-articular and extra-articular osteotomy.
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Influence of tapentadol and oxycodone on the spinal cord and brain using electrophysiology: a randomized, placebo‐controlled trial | 5c21181b-058f-488c-a356-6ce127f51188 | 9796052 | Physiology[mh] | Tapentadol is an opioid analgesic that theoretically employs its analgesic effect by combining moderate μ‐opioid receptor agonistic affinity with noradrenaline reuptake inhibition. It is relevant to investigate the mechanisms of tapentadol on the central and peripheral nervous systems in order to strengthen the clinical foundation for pain management. The nociceptive withdrawal reflex has been used to objectively assess drug‐induced effects on nociceptive processing and has previously been used to determine the analgesic properties of opioids. Tapentadol affects the number of reflexes observed using the nociceptive withdrawal reflex. Both oxycodone and tapentadol affect cortical measures. This study replicates preclinical studies suggesting that oxycodone and tapentadol activate different pain control mechanisms and support clinical findings. INTRODUCTION Strong opioids are predominantly μ receptor agonists. Tapentadol is an opioid analgesic that theoretically employs its analgesic effect by combining moderate μ‐opioid receptor agonistic affinity with noradrenaline reuptake inhibition. It has been shown to reduce some of the typical opioid‐induced side effects compared to equianalgesic doses of classic opioids and has been investigated preclinically. , Tapentadol has been found to be an effective and generally well‐tolerated treatment in a broad range of chronic pain conditions. However, it is relevant to investigate the central and peripheral mechanisms of tapentadol in order to strengthen the clinical foundation for pain management. The nociceptive withdrawal reflex (NWR) is a spinal polysynaptic reflex which is the basis for the protective mechanism against possible limb damage. , The NWR has been used to objectively assess drug‐induced effects on nociceptive processing, and has previously been used to determine the analgesic properties of opioids. , , Using a stimulation under the sole of the foot and recording at the tibialis anterior has previously been proven to result in a more tolerable stimulation and good between‐session reliability. The reflex threshold, onset latency and area under the rectified curve (AUC) can quantify the NWR and have been shown to change after opioid administration. Additionally, the number of NWRs elicited has recently been used to quantify the difference between people with diabetic peripheral polyneuropathy and healthy controls. Using the NWR it is possible to investigate the spinal and cortical level of the central nervous system by simultaneously recording electromyography (EMG), and somatosensory evoked potentials (SEPs) using electroencephalography (EEG). On the supraspinal level, SEPs have an excellent temporal resolution and, combined with brain source localization methods, it is possible to estimate the underlying brain sources. Analysis of the supraspinal level of NWR contributes to a deeper understanding of treatments with analgesics. We performed a randomized, double‐blinded, placebo‐controlled, cross‐over study to investigate the hypothesis that spinal and supraspinal effects would result after 14‐day treatment with oxycodone and tapentadol in healthy subjects. The aims were to investigate the treatment effects on (1) the spinal level by measuring the number of elicited NWRs, as well as to quantify the NWR using latency and AUC of the EMG response, and (2) the supraspinal level by measuring latencies and amplitudes of the SEPs and inverse modelling of the cortical signals. METHODS 2.1 Subjects and ethics The study was carried out at Mech‐Sense, Department of Gastroenterology and Hepatology, Aalborg University Hospital, Aalborg, Denmark. Participants gave written, informed consent before participating in the study and were free to withdraw from the study at any time. The study was approved by The North Denmark Region Committee on Health Research Ethics (N‐20170009) and the Danish Medicines Agency, registered at www.clinicaltrialsregister.eu (EudraCT number: 2017‐000141‐52), monitored by the Good Clinical Practice unit at Aalborg and Aarhus University Hospitals, Denmark, and conducted according to the Declaration of Helsinki. Data in this present study is a subset of a larger study with the main objectives to investigate the effects of tapentadol and oxycodone on the central, autonomic and enteric nervous systems. Other data based on this trial are available in Refs , , , , . 2.2 Study design In total, 21 healthy male subjects completed this randomized, double‐blinded, placebo‐controlled, cross‐over study. A sample size of 20 was calculated based on previous studies using the same experimental models, and so 21 subjects were considered sufficient to reach the goal of this study. Each trial arm lasted 14 days and was administered in a randomized order. To remove potential risks of bias, all subjects were opioid‐naïve (have not taken opioid doses for 1 week or more) and could not use any medications (analgesic, herbal or over the counter) within 48 hours before the start of the study as well as for the duration of the study. A treatment period of 14 days was chosen to ensure adequate treatment since previous studies have indicated that the noradrenaline reuptake inhibitor modulation of tapentadol slowly increases and reaches its maximal effect after 2 weeks. NWR measurements were obtained prior to the first dose and after the last dose. Participants were treated with tapentadol extended‐release tablets 50 mg (Palexia; Grunenthal GmbH, Aachen, Germany), oxycodone extended‐release tablets 10 mg (OxyContin; Mundipharma A/S, Vedbæk, Denmark), and placebo tablets (Hospital Pharmacy Aarhus, Aarhus University Hospital, Aarhus, Denmark) administered orally BID for 14 days. The subjects were administered tapentadol (50 mg) or oxycodone (10 mg), based on previous clinical studies deeming these to be equipotent. Nomenclature related to drugs and molecular targets conforms to the IUPHAR/BPS Guide. To minimize the risk of addiction, only people who had no previous or current history of abuse or addiction were included, as these factors have shown to result in a frequency of abuse of 0.19% in people with chronic pain. Additionally, all subjects were required to fill out a Subjective Opiate Withdrawal Scale questionnaire 3 days after receiving the last dose in all study periods to monitor whether any degree of dependence was developing. Medication was masked to similar resemblance using DBcaps® (red colour, size AA, 13.07–14.44 × 9.39 mm, Capsugel®), which do not affect the drug release properties of the original tablets. , A single tablet was ingested on Day 1 after baseline recordings (evening dose) and Day 14 before the post‐intervention recording (morning dose). Subjects continued their normal daily lives between recordings. To ensure dosing compliance, the subjects filled in a medication diary on ingestion of each pill. Furthermore, all pill containers were returned and the remaining pills, if any, were accounted for. The “wash‐out” period between treatments was at least 1 week. All medication was dispensed by The Hospital Pharmacy Aarhus, Aarhus University Hospital, Aarhus, Denmark. All recordings were performed before first ingestion of medication on Day 1 (baseline) or after the last ingestion of medication on Day 14 (post‐intervention). The NWR was elicited by electrical stimulation of the plantar skin (site of innervation of the medial plantar nerve). The cathode was placed at the arch of the sole of the right foot (15 × 15 mm, Neuroline 700; Ambu A/S, Denmark), and the anode was placed on the foot dorsum (50 × 90 mm, Synapse; Ambu A/S, Denmark). The electrical stimulations were delivered by an electrical stimulator (Noxitest IES 230, Aalborg, Denmark) consisting of a constant current burst of five square‐wave pulses with 1 ms duration and 5 ms between pulses, which was felt as one single stimulus. A custom‐made software program (Center for Sensory‐Motor Interaction, Aalborg University, Denmark) was used to manage the electrical stimulations. The perception threshold (PT) and reflex threshold (RT) were identified by manually increasing the stimulus intensity with steps of 1 mA. The PT was reached at the stimulation intensity in mA when the first sensation was felt. The RT was identified using the staircase method described in detail previously. Once the RT was found, the intensity needed to elicit a reflex measured in mA was noted and the subject was asked to rate the pain intensity corresponding to stimulus of the RT using a numeric rating scale (NRS) ranging from 0–10, where 0 = no pain, 1 = first sensation of pain, and 10 = maximum imaginable pain. After the RT was identified and rated, the participant received 18 stimuli at three different stimulus intensities with an inter‐stimulus interval of 8–12 s. These stimulations were delivered at low intensity (1.3 × RT), medium intensity (1.6 × RT), and high intensity (2.0 × RT), with six stimuli at each intensity in a randomized order. A visualization of the experimental setup is presented in Figure . 2.2.1 Electromyography During stimulations, EMG data were obtained from the ipsilateral tibialis anterior muscle. The skin was prepared using sandpaper and alcohol to clean the skin before placing two surface electrodes, where one electrode (15 × 15 mm, Neuroline 700; Ambu A/S, Ballerup, Denmark) was placed on the belly of the tibialis anterior muscle, and one electrode (50 × 90 mm, Synapse; Ambu A/S) the ground electrode, was placed just below the patella. Pre‐processing was performed by bandpass filtering the signal between 5 and 500 Hz using a zero‐phase digital 12th order Butterworth filter. 2.2.2 Electroencephalography EEG data from the scalp were recorded during the NWR. A 62‐channel surface electrode EEG cap using the 10–20 system (MEQNordic A/S, Jyllinge, Denmark) was placed on the head, and impedance was kept below 5 kΩ. An electrode located between AFz and Fz was used as the reference electrode. EEG data were recorded in continuous mode with open filters. The sampling rate was 1000 Hz (SynAmp, Neuroscan, El Paso, TX, USA). Recordings were stored offline for further analysis. 2.3 Data analysis The EMG and EEG data were analysed in MATLAB (R 2019a Mathworks Inc., Natick, MA, USA). 2.3.1 Electromyography The EMG analysis was performed on single sweeps and quantified using the interval peak z ‐score. The z‐score was defined by the highest peak in the rectified reflex window minus a pre‐stimulus mean divided by the standard deviation (SD) of the same pre‐stimulus area. The pre‐stimulus window was taken from 100 to 10 ms before the stimulation, while the reflex window was taken from 70 to 160 ms post‐stimulus. In all cases, a rectified AUC was calculated in the reflex window. A peak interval z ‐score was set to 6 based on the method described by Herm et al. If any part of the reflex window had a z ‐score above 6, it was interpreted as an elicited NWR. When successfully elicited, the latency was defined at the first point where the rectified EMG trace had a z ‐score above 6. 2.3.2 Electroencephalography The EEG preprocessing was performed using MATLAB and EEGLAB toolbox (version 14.1.2; Schwartz Center for Computational Neuroscience, Institute for Neural Computation, University of California, San Diego, CA). The data were bandpass filtered between 1 and 30 Hz. Each recording was manually investigated to identify bad channels and artifacts. These were interpolated using the EEGLAB spherical interpolation method. Lastly, data were epoched from 50 ms before stimulus to 950 ms after the stimulation and averaged for further analysis. Latencies and peak‐to‐peak amplitudes of the evoked potentials were assessed. The SEP response at the central electrode (Cz) to electrical stimulation has a triphasic shape, see Figures and . The three peaks (P1, N2 and P2) were identified on an average trace for each subject. The Cz electrode was used because of its central location and maximal SEP amplitude due to the electrical stimulation on the foot and its cortical location on the sensory cortex. Brain source network analysis was performed to study the underlying brain sources generating the SEPs using inverse modelling (BESA research 5.3; MEGIS Software GmbH, Gräfelfing, Germany). The model for the baseline analysis was based on an average of all baseline recordings to get an indication of the location and number of sources. The post‐intervention brain source networks were created based on the group‐average of each condition. Standardized low‐resolution brain electromagnetic tomography sLORETA , was used to guide the location of the sources for the individual subjects. Once the dipoles were placed in BESA, the model fit was obtained by fixing their locations but allowing their orientations to move freely. The percentage of data that the model could not explain was expressed as the residual variance. The residual variances were sought to be below 10%. All coordinates are reported in Talairach coordinates. 2.4 Statistical analyses All data were assessed for normal distribution using Q‐Q plots and histograms. In cases of a non‐normal distribution, data were log‐transformed. For all analyses, the change over time (baseline/post‐intervention) was investigated, and the subject was included as a random factor in all analyses. A multilevel mixed‐effects linear regression model with the factors treatment (oxycodone/tapentadol/placebo) and time with subject as a random factor was used to analyse changes from baseline to post‐intervention of: the PT and RT of the medial plantar nerve, the subjective pain intensity ratings, the AUC and latency of EMG, the latency and peak‐to‐peak amplitude of SEPs and location of dipoles of the inverse modelling. To test the binary outcome of eliciting an NWR (yes/no), a mixed‐effects logistic regression model was used against the stimulation intensities (categorical: low, medium and high), treatment (oxycodone/tapentadol/placebo), to investigate the change over time. A repeated‐measures analysis of variance was used with factors treatment (oxycodone/tapentadol/placebo) and time to test if there was a difference in the residual variance of the inverse models between baseline and post‐intervention. Bonferroni post hoc analysis was used to correct for multiple comparisons when analysing the treatment effects when the models showed significant results in time (baseline/post‐intervention). All statistical analysis was performed in Stata (StataCore LLC, College Station, TX, version 16.1). P ‐values < 0.05 were considered significant. 2.5 Nomenclature of targets and ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org , and are permanently archived in the Concise Guide to PHARMACOLOGY 2021. , Subjects and ethics The study was carried out at Mech‐Sense, Department of Gastroenterology and Hepatology, Aalborg University Hospital, Aalborg, Denmark. Participants gave written, informed consent before participating in the study and were free to withdraw from the study at any time. The study was approved by The North Denmark Region Committee on Health Research Ethics (N‐20170009) and the Danish Medicines Agency, registered at www.clinicaltrialsregister.eu (EudraCT number: 2017‐000141‐52), monitored by the Good Clinical Practice unit at Aalborg and Aarhus University Hospitals, Denmark, and conducted according to the Declaration of Helsinki. Data in this present study is a subset of a larger study with the main objectives to investigate the effects of tapentadol and oxycodone on the central, autonomic and enteric nervous systems. Other data based on this trial are available in Refs , , , , . Study design In total, 21 healthy male subjects completed this randomized, double‐blinded, placebo‐controlled, cross‐over study. A sample size of 20 was calculated based on previous studies using the same experimental models, and so 21 subjects were considered sufficient to reach the goal of this study. Each trial arm lasted 14 days and was administered in a randomized order. To remove potential risks of bias, all subjects were opioid‐naïve (have not taken opioid doses for 1 week or more) and could not use any medications (analgesic, herbal or over the counter) within 48 hours before the start of the study as well as for the duration of the study. A treatment period of 14 days was chosen to ensure adequate treatment since previous studies have indicated that the noradrenaline reuptake inhibitor modulation of tapentadol slowly increases and reaches its maximal effect after 2 weeks. NWR measurements were obtained prior to the first dose and after the last dose. Participants were treated with tapentadol extended‐release tablets 50 mg (Palexia; Grunenthal GmbH, Aachen, Germany), oxycodone extended‐release tablets 10 mg (OxyContin; Mundipharma A/S, Vedbæk, Denmark), and placebo tablets (Hospital Pharmacy Aarhus, Aarhus University Hospital, Aarhus, Denmark) administered orally BID for 14 days. The subjects were administered tapentadol (50 mg) or oxycodone (10 mg), based on previous clinical studies deeming these to be equipotent. Nomenclature related to drugs and molecular targets conforms to the IUPHAR/BPS Guide. To minimize the risk of addiction, only people who had no previous or current history of abuse or addiction were included, as these factors have shown to result in a frequency of abuse of 0.19% in people with chronic pain. Additionally, all subjects were required to fill out a Subjective Opiate Withdrawal Scale questionnaire 3 days after receiving the last dose in all study periods to monitor whether any degree of dependence was developing. Medication was masked to similar resemblance using DBcaps® (red colour, size AA, 13.07–14.44 × 9.39 mm, Capsugel®), which do not affect the drug release properties of the original tablets. , A single tablet was ingested on Day 1 after baseline recordings (evening dose) and Day 14 before the post‐intervention recording (morning dose). Subjects continued their normal daily lives between recordings. To ensure dosing compliance, the subjects filled in a medication diary on ingestion of each pill. Furthermore, all pill containers were returned and the remaining pills, if any, were accounted for. The “wash‐out” period between treatments was at least 1 week. All medication was dispensed by The Hospital Pharmacy Aarhus, Aarhus University Hospital, Aarhus, Denmark. All recordings were performed before first ingestion of medication on Day 1 (baseline) or after the last ingestion of medication on Day 14 (post‐intervention). The NWR was elicited by electrical stimulation of the plantar skin (site of innervation of the medial plantar nerve). The cathode was placed at the arch of the sole of the right foot (15 × 15 mm, Neuroline 700; Ambu A/S, Denmark), and the anode was placed on the foot dorsum (50 × 90 mm, Synapse; Ambu A/S, Denmark). The electrical stimulations were delivered by an electrical stimulator (Noxitest IES 230, Aalborg, Denmark) consisting of a constant current burst of five square‐wave pulses with 1 ms duration and 5 ms between pulses, which was felt as one single stimulus. A custom‐made software program (Center for Sensory‐Motor Interaction, Aalborg University, Denmark) was used to manage the electrical stimulations. The perception threshold (PT) and reflex threshold (RT) were identified by manually increasing the stimulus intensity with steps of 1 mA. The PT was reached at the stimulation intensity in mA when the first sensation was felt. The RT was identified using the staircase method described in detail previously. Once the RT was found, the intensity needed to elicit a reflex measured in mA was noted and the subject was asked to rate the pain intensity corresponding to stimulus of the RT using a numeric rating scale (NRS) ranging from 0–10, where 0 = no pain, 1 = first sensation of pain, and 10 = maximum imaginable pain. After the RT was identified and rated, the participant received 18 stimuli at three different stimulus intensities with an inter‐stimulus interval of 8–12 s. These stimulations were delivered at low intensity (1.3 × RT), medium intensity (1.6 × RT), and high intensity (2.0 × RT), with six stimuli at each intensity in a randomized order. A visualization of the experimental setup is presented in Figure . 2.2.1 Electromyography During stimulations, EMG data were obtained from the ipsilateral tibialis anterior muscle. The skin was prepared using sandpaper and alcohol to clean the skin before placing two surface electrodes, where one electrode (15 × 15 mm, Neuroline 700; Ambu A/S, Ballerup, Denmark) was placed on the belly of the tibialis anterior muscle, and one electrode (50 × 90 mm, Synapse; Ambu A/S) the ground electrode, was placed just below the patella. Pre‐processing was performed by bandpass filtering the signal between 5 and 500 Hz using a zero‐phase digital 12th order Butterworth filter. 2.2.2 Electroencephalography EEG data from the scalp were recorded during the NWR. A 62‐channel surface electrode EEG cap using the 10–20 system (MEQNordic A/S, Jyllinge, Denmark) was placed on the head, and impedance was kept below 5 kΩ. An electrode located between AFz and Fz was used as the reference electrode. EEG data were recorded in continuous mode with open filters. The sampling rate was 1000 Hz (SynAmp, Neuroscan, El Paso, TX, USA). Recordings were stored offline for further analysis. Electromyography During stimulations, EMG data were obtained from the ipsilateral tibialis anterior muscle. The skin was prepared using sandpaper and alcohol to clean the skin before placing two surface electrodes, where one electrode (15 × 15 mm, Neuroline 700; Ambu A/S, Ballerup, Denmark) was placed on the belly of the tibialis anterior muscle, and one electrode (50 × 90 mm, Synapse; Ambu A/S) the ground electrode, was placed just below the patella. Pre‐processing was performed by bandpass filtering the signal between 5 and 500 Hz using a zero‐phase digital 12th order Butterworth filter. Electroencephalography EEG data from the scalp were recorded during the NWR. A 62‐channel surface electrode EEG cap using the 10–20 system (MEQNordic A/S, Jyllinge, Denmark) was placed on the head, and impedance was kept below 5 kΩ. An electrode located between AFz and Fz was used as the reference electrode. EEG data were recorded in continuous mode with open filters. The sampling rate was 1000 Hz (SynAmp, Neuroscan, El Paso, TX, USA). Recordings were stored offline for further analysis. Data analysis The EMG and EEG data were analysed in MATLAB (R 2019a Mathworks Inc., Natick, MA, USA). 2.3.1 Electromyography The EMG analysis was performed on single sweeps and quantified using the interval peak z ‐score. The z‐score was defined by the highest peak in the rectified reflex window minus a pre‐stimulus mean divided by the standard deviation (SD) of the same pre‐stimulus area. The pre‐stimulus window was taken from 100 to 10 ms before the stimulation, while the reflex window was taken from 70 to 160 ms post‐stimulus. In all cases, a rectified AUC was calculated in the reflex window. A peak interval z ‐score was set to 6 based on the method described by Herm et al. If any part of the reflex window had a z ‐score above 6, it was interpreted as an elicited NWR. When successfully elicited, the latency was defined at the first point where the rectified EMG trace had a z ‐score above 6. 2.3.2 Electroencephalography The EEG preprocessing was performed using MATLAB and EEGLAB toolbox (version 14.1.2; Schwartz Center for Computational Neuroscience, Institute for Neural Computation, University of California, San Diego, CA). The data were bandpass filtered between 1 and 30 Hz. Each recording was manually investigated to identify bad channels and artifacts. These were interpolated using the EEGLAB spherical interpolation method. Lastly, data were epoched from 50 ms before stimulus to 950 ms after the stimulation and averaged for further analysis. Latencies and peak‐to‐peak amplitudes of the evoked potentials were assessed. The SEP response at the central electrode (Cz) to electrical stimulation has a triphasic shape, see Figures and . The three peaks (P1, N2 and P2) were identified on an average trace for each subject. The Cz electrode was used because of its central location and maximal SEP amplitude due to the electrical stimulation on the foot and its cortical location on the sensory cortex. Brain source network analysis was performed to study the underlying brain sources generating the SEPs using inverse modelling (BESA research 5.3; MEGIS Software GmbH, Gräfelfing, Germany). The model for the baseline analysis was based on an average of all baseline recordings to get an indication of the location and number of sources. The post‐intervention brain source networks were created based on the group‐average of each condition. Standardized low‐resolution brain electromagnetic tomography sLORETA , was used to guide the location of the sources for the individual subjects. Once the dipoles were placed in BESA, the model fit was obtained by fixing their locations but allowing their orientations to move freely. The percentage of data that the model could not explain was expressed as the residual variance. The residual variances were sought to be below 10%. All coordinates are reported in Talairach coordinates. Electromyography The EMG analysis was performed on single sweeps and quantified using the interval peak z ‐score. The z‐score was defined by the highest peak in the rectified reflex window minus a pre‐stimulus mean divided by the standard deviation (SD) of the same pre‐stimulus area. The pre‐stimulus window was taken from 100 to 10 ms before the stimulation, while the reflex window was taken from 70 to 160 ms post‐stimulus. In all cases, a rectified AUC was calculated in the reflex window. A peak interval z ‐score was set to 6 based on the method described by Herm et al. If any part of the reflex window had a z ‐score above 6, it was interpreted as an elicited NWR. When successfully elicited, the latency was defined at the first point where the rectified EMG trace had a z ‐score above 6. Electroencephalography The EEG preprocessing was performed using MATLAB and EEGLAB toolbox (version 14.1.2; Schwartz Center for Computational Neuroscience, Institute for Neural Computation, University of California, San Diego, CA). The data were bandpass filtered between 1 and 30 Hz. Each recording was manually investigated to identify bad channels and artifacts. These were interpolated using the EEGLAB spherical interpolation method. Lastly, data were epoched from 50 ms before stimulus to 950 ms after the stimulation and averaged for further analysis. Latencies and peak‐to‐peak amplitudes of the evoked potentials were assessed. The SEP response at the central electrode (Cz) to electrical stimulation has a triphasic shape, see Figures and . The three peaks (P1, N2 and P2) were identified on an average trace for each subject. The Cz electrode was used because of its central location and maximal SEP amplitude due to the electrical stimulation on the foot and its cortical location on the sensory cortex. Brain source network analysis was performed to study the underlying brain sources generating the SEPs using inverse modelling (BESA research 5.3; MEGIS Software GmbH, Gräfelfing, Germany). The model for the baseline analysis was based on an average of all baseline recordings to get an indication of the location and number of sources. The post‐intervention brain source networks were created based on the group‐average of each condition. Standardized low‐resolution brain electromagnetic tomography sLORETA , was used to guide the location of the sources for the individual subjects. Once the dipoles were placed in BESA, the model fit was obtained by fixing their locations but allowing their orientations to move freely. The percentage of data that the model could not explain was expressed as the residual variance. The residual variances were sought to be below 10%. All coordinates are reported in Talairach coordinates. Statistical analyses All data were assessed for normal distribution using Q‐Q plots and histograms. In cases of a non‐normal distribution, data were log‐transformed. For all analyses, the change over time (baseline/post‐intervention) was investigated, and the subject was included as a random factor in all analyses. A multilevel mixed‐effects linear regression model with the factors treatment (oxycodone/tapentadol/placebo) and time with subject as a random factor was used to analyse changes from baseline to post‐intervention of: the PT and RT of the medial plantar nerve, the subjective pain intensity ratings, the AUC and latency of EMG, the latency and peak‐to‐peak amplitude of SEPs and location of dipoles of the inverse modelling. To test the binary outcome of eliciting an NWR (yes/no), a mixed‐effects logistic regression model was used against the stimulation intensities (categorical: low, medium and high), treatment (oxycodone/tapentadol/placebo), to investigate the change over time. A repeated‐measures analysis of variance was used with factors treatment (oxycodone/tapentadol/placebo) and time to test if there was a difference in the residual variance of the inverse models between baseline and post‐intervention. Bonferroni post hoc analysis was used to correct for multiple comparisons when analysing the treatment effects when the models showed significant results in time (baseline/post‐intervention). All statistical analysis was performed in Stata (StataCore LLC, College Station, TX, version 16.1). P ‐values < 0.05 were considered significant. Nomenclature of targets and ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org , and are permanently archived in the Concise Guide to PHARMACOLOGY 2021. , RESULTS For this study, 23 subjects were screened, and 22 subjects were included. Twenty‐one subjects completed the study (one subject did not comply with the protocol and was excluded). The demographics for the 21 subjects were: age (24.9 ± 2.7 years), height (181.3 ± 6.3 cm), weight (83.2 ± 9.9 kg), body mass index (25.3 ± 2.5 kg/m 2 ). The AUC measure was log‐transformed to fulfil a normal distribution. All measures were assessed at the baseline time (all P > .05). Between baseline and post‐intervention for each individual intervention, an overall decrease of PT by −0.21 mA ( P = .84), and increase of RT by 0.07 mA ( P = .9) were observed; the overall subjective pain scores decreased by −0.16 ( P = .35). For the logistic regression, an increased odds ratio of eliciting an NWR was shown with increasing stimulus intensities: medium 2.55 ( P < .001, 95% CI 1.86, 3.49) and high 2.74 ( P < .001, 95% CI 1.99, 3.75). The overall odds of observing a reflex decreased over time for the treatments ( P = .001), and the post hoc analysis showed the change to be in the tapentadol arm −0.89 ( P = .001, 95% CI −1.46, −0.32). The number of reflexes observed using the interval z ‐score is visualized in Figure . The drug effect on the NWR did not differ from baseline to post‐intervention for the latency ( P = .234) or AUC ( P = .051). The data are presented in Figure . For the cortical measures of latency at the Cz electrode, the N1 peak was significantly different ( P = .008), and the post hoc analysis revealed an increase in the oxycodone arm of 12.52 mS ( P = .003, 95% CI 3.37, 21.69). The P1 and P2 peaks did not differ between baseline and post‐intervention (P1: P = .687, P2: P = .732). For the peak‐to‐peak amplitudes, neither the P1‐N1 ( P = .944) nor N1‐P2 ( P = .915) amplitudes differed across time. A grand average of the oxycodone trace is shown in Figure . In addition, analysis of: PT, RT, drug effect on the NWR (latency, AUC), and cortical measures (latency, peak‐to‐peak amplitude) were performed between post‐dose treatments by subtracting the baseline measure from the post‐dose measure and comparing them. No differences were found (all P > .05). 3.1 Inverse modelling Four dipoles were selected to describe the brain activity adequately. The locations of the dipoles are shown in Figure . For dipole 1, the y ‐coordinate changed over time ( P < .001). The post hoc test revealed the change to be across all treatment arms (placebo: P = .012, 95% CI −23.10, −2.10, oxycodone: P < .001, 95% CI −36.32, −15.24, tapentadol: P = .001, 95% CI −26.58, −5.48). In dipoles 2–3, the y ‐coordinates changed overall ( P = .001), where the post hoc analysis did not reveal any differences between treatments. The z ‐coordinates differed over time ( P < .001); the post hoc analysis showed a change of the oxycodone ( P = .022, 95% CI −9.20, −0.51) and tapentadol ( P = .001, 95% CI −10.88, −2.17) arms. The x ‐coordinate did not change over time. No coordinates changed over time for dipole 4. Data are presented in Table and Figure . No differences in the residual variance were observed between baseline and post‐intervention (all P > .05). In addition, dipole locations were analysed between post‐dose treatments by subtracting the baseline measure from the post‐dose measure and comparing them. No differences were found (all P > .05). Inverse modelling Four dipoles were selected to describe the brain activity adequately. The locations of the dipoles are shown in Figure . For dipole 1, the y ‐coordinate changed over time ( P < .001). The post hoc test revealed the change to be across all treatment arms (placebo: P = .012, 95% CI −23.10, −2.10, oxycodone: P < .001, 95% CI −36.32, −15.24, tapentadol: P = .001, 95% CI −26.58, −5.48). In dipoles 2–3, the y ‐coordinates changed overall ( P = .001), where the post hoc analysis did not reveal any differences between treatments. The z ‐coordinates differed over time ( P < .001); the post hoc analysis showed a change of the oxycodone ( P = .022, 95% CI −9.20, −0.51) and tapentadol ( P = .001, 95% CI −10.88, −2.17) arms. The x ‐coordinate did not change over time. No coordinates changed over time for dipole 4. Data are presented in Table and Figure . No differences in the residual variance were observed between baseline and post‐intervention (all P > .05). In addition, dipole locations were analysed between post‐dose treatments by subtracting the baseline measure from the post‐dose measure and comparing them. No differences were found (all P > .05). DISCUSSION We explored the effects of oxycodone (μ receptor agonist) and tapentadol (μ receptor agonist and noradrenaline reuptake inhibitor) using the NWR and the corresponding EMG and EEG signals. At the spinal level, tapentadol decreased the odds of eliciting an NWR. No other differences were demonstrated in EMG. At the cortical level, oxycodone increased the latency of the N1 component of the SEPs at the vertex. The inverse modelling revealed that the insula components changed during both oxycodone and tapentadol treatments. These findings suggests that oxycodone mainly affects cortical pain processing, whereas tapentadol modulates analgesia at the cortical and spinal levels. 4.1 Sensory properties and electromyographic findings Tapentadol treatment decreased the odds of eliciting the NWR. Tapentadol employs its analgesic in part with noradrenaline reuptake inhibition. This has been shown in preclinical experiments to affect the brain stem's descending nerve tracts, which can inhibit the incoming nociceptive barrage at the spinal level. Previous studies have shown that low doses of opioids did not affect the NWR. , , Willer found a linear increasing relationship between intravenous morphine administration and the related pain of the NWR. In addition to this, Bossard et al. found that the nociceptive withdrawal reflex was reduced after a combination of morphine and ketamine , but not after individual administration of the drugs. Lelic et al. investigated oxycodone and venlafaxine (a serotonin and norepinephrine reuptake inhibitor) and demonstrated that venlafaxine affected the NWR while oxycodone did not. This could suggest that tapentadol's noradrenaline reuptake inhibitor mechanisms similarly affect the NWR. Lastly, an increased odds ratio of eliciting an NWR with increasing stimulus intensities was observed. This was expected, as higher stimulation intensities result in larger reflex amplitudes. Arguissain et al. found significantly larger amplitude reflexes when comparing above‐threshold stimulations (1.5 × RT and 2 × RT) to lower stimulations (0.5 × RT and 0.75 × RT). The analgesic effects of oxycodone and tapentadol were also tested in the current trial and they were found to decrease perceived pain by 7–11% in oxycodone and 6–9% in tapentadol after submerging the hand in cold water. 4.2 Cortical changes at the scalp The only change observed in the SEPs was an increase in the latency of the N1 component in the oxycodone arm. This indicates altered responses of the later cortical signal, possibly due to decreased phase‐locking of signals after administration of opioids. The N1 component is in part driven by sources in the secondary somatosensory cortex, insula and the anterior cingulate cortex. Generally, changes in SEP amplitudes correlate with clinical effect, while latencies of the SEPs are infrequently reported and with no coherent pattern. The fact that there was no change in the tapentadol arm can also be attributed to the dual effect of tapentadol, where less μ‐opioid receptor agonistic effect was needed for the same level of analgesia, and thus tapentadol does not affect the cortical signal to the same extent as oxycodone. These findings were collaborated in a study from the same trial which found changes in the processing of pain in oxycodone and tapentadol, but suggest that the oxycodone treatment has a larger cortical effect. 4.3 Inverse modelling Four dipoles were chosen resulting in a residual variance of less than 10% in the baseline measures. The anterior cingulate cortex component of the inverse modelling moved caudally across all treatment arms, and the insula dipoles moved caudally in both the oxycodone and tapentadol arms. The anterior cingulate cortex and insula are involved in the processing of sensory stimuli, and while these networks do not only process pain‐specific cortical processes, their magnitude has been shown to correlate with the magnitude of the perceived saliency of a stimulation. The anterior cingulate cortex and insular cortex have been reported to play a role in previous pain studies. , , , The insula component only changed in the active treatments, suggesting a drug effect on the insula, probably due to the μ‐opioid receptor agonist in both treatments. Previously, inverse modelling has also detected changes after opioid analgesia. These findings are supported by another study from the same trial which found changes in functional connectivity in areas related to pain processing in oxycodone and tapentadol compared to placebo. The dipole in the midcingulate area did not change in any treatment. 4.4 Limitations The experimental pain model was investigated in healthy young men without considering the differences in pain sensitivity and analgesic effect between men and women. , , The rationale for selecting healthy young men was to test these complex experimental models in a homogeneous group. The choice to use a homogeneous group and a repeated measures design allowed the number of subjects to be relatively small; however, it does come with a risk of type 2 error. This contrasts a population with chronic pain and multiple comorbidities that often affect the reliability of the outcomes, and more subjects would be needed to investigate mechanistic effects. Matthey et al. investigated milnacipran, a serotonin‐norepinephrine reuptake inhibitor, on a fibromyalgia population using the NWR. They found that the milnacipran had a supraspinal analgesic effect, that did not change RT, but reported higher doses associated with higher pain reduction. Only minor changes were observed using EEG, which seems counterintuitive to several changes found using inverse modelling. Only one electrode at the vertex was analysed, which also corresponds with the only dipole in the inverse model that did not change location. The inverse modelling approximates the underlining cortical source generation and is not the only possible method to assess the effect of opioids on the central nervous system. Imaging methods, such as functional magnetic resonance imaging, have a higher spatial resolution. However, it does not have the temporal resolution of EEG and is less suitable for comparing spinal and supraspinal effects. The observed changes could be related to drug effects such as analgesic effects but also side effects. In conclusion, a decrease in the number of reflexes was observed only for the tapentadol treatment, on the spinal level. This could be due to the effects on the brainstem's noradrenaline reuptake inhibition. No other features of the NWR changed for any treatment. At the brain level, decreased latencies of the N1 component of the SEPs were identified only after the oxycodone intervention. Furthermore, the inverse modelling of dipolar sources in the insula component changed for both oxycodone and tapentadol treatments, indicating that both opioids affect cortical measures. This study replicates preclinical studies suggesting that the two opioids activate different pain control mechanisms and support clinical findings. Sensory properties and electromyographic findings Tapentadol treatment decreased the odds of eliciting the NWR. Tapentadol employs its analgesic in part with noradrenaline reuptake inhibition. This has been shown in preclinical experiments to affect the brain stem's descending nerve tracts, which can inhibit the incoming nociceptive barrage at the spinal level. Previous studies have shown that low doses of opioids did not affect the NWR. , , Willer found a linear increasing relationship between intravenous morphine administration and the related pain of the NWR. In addition to this, Bossard et al. found that the nociceptive withdrawal reflex was reduced after a combination of morphine and ketamine , but not after individual administration of the drugs. Lelic et al. investigated oxycodone and venlafaxine (a serotonin and norepinephrine reuptake inhibitor) and demonstrated that venlafaxine affected the NWR while oxycodone did not. This could suggest that tapentadol's noradrenaline reuptake inhibitor mechanisms similarly affect the NWR. Lastly, an increased odds ratio of eliciting an NWR with increasing stimulus intensities was observed. This was expected, as higher stimulation intensities result in larger reflex amplitudes. Arguissain et al. found significantly larger amplitude reflexes when comparing above‐threshold stimulations (1.5 × RT and 2 × RT) to lower stimulations (0.5 × RT and 0.75 × RT). The analgesic effects of oxycodone and tapentadol were also tested in the current trial and they were found to decrease perceived pain by 7–11% in oxycodone and 6–9% in tapentadol after submerging the hand in cold water. Cortical changes at the scalp The only change observed in the SEPs was an increase in the latency of the N1 component in the oxycodone arm. This indicates altered responses of the later cortical signal, possibly due to decreased phase‐locking of signals after administration of opioids. The N1 component is in part driven by sources in the secondary somatosensory cortex, insula and the anterior cingulate cortex. Generally, changes in SEP amplitudes correlate with clinical effect, while latencies of the SEPs are infrequently reported and with no coherent pattern. The fact that there was no change in the tapentadol arm can also be attributed to the dual effect of tapentadol, where less μ‐opioid receptor agonistic effect was needed for the same level of analgesia, and thus tapentadol does not affect the cortical signal to the same extent as oxycodone. These findings were collaborated in a study from the same trial which found changes in the processing of pain in oxycodone and tapentadol, but suggest that the oxycodone treatment has a larger cortical effect. Inverse modelling Four dipoles were chosen resulting in a residual variance of less than 10% in the baseline measures. The anterior cingulate cortex component of the inverse modelling moved caudally across all treatment arms, and the insula dipoles moved caudally in both the oxycodone and tapentadol arms. The anterior cingulate cortex and insula are involved in the processing of sensory stimuli, and while these networks do not only process pain‐specific cortical processes, their magnitude has been shown to correlate with the magnitude of the perceived saliency of a stimulation. The anterior cingulate cortex and insular cortex have been reported to play a role in previous pain studies. , , , The insula component only changed in the active treatments, suggesting a drug effect on the insula, probably due to the μ‐opioid receptor agonist in both treatments. Previously, inverse modelling has also detected changes after opioid analgesia. These findings are supported by another study from the same trial which found changes in functional connectivity in areas related to pain processing in oxycodone and tapentadol compared to placebo. The dipole in the midcingulate area did not change in any treatment. Limitations The experimental pain model was investigated in healthy young men without considering the differences in pain sensitivity and analgesic effect between men and women. , , The rationale for selecting healthy young men was to test these complex experimental models in a homogeneous group. The choice to use a homogeneous group and a repeated measures design allowed the number of subjects to be relatively small; however, it does come with a risk of type 2 error. This contrasts a population with chronic pain and multiple comorbidities that often affect the reliability of the outcomes, and more subjects would be needed to investigate mechanistic effects. Matthey et al. investigated milnacipran, a serotonin‐norepinephrine reuptake inhibitor, on a fibromyalgia population using the NWR. They found that the milnacipran had a supraspinal analgesic effect, that did not change RT, but reported higher doses associated with higher pain reduction. Only minor changes were observed using EEG, which seems counterintuitive to several changes found using inverse modelling. Only one electrode at the vertex was analysed, which also corresponds with the only dipole in the inverse model that did not change location. The inverse modelling approximates the underlining cortical source generation and is not the only possible method to assess the effect of opioids on the central nervous system. Imaging methods, such as functional magnetic resonance imaging, have a higher spatial resolution. However, it does not have the temporal resolution of EEG and is less suitable for comparing spinal and supraspinal effects. The observed changes could be related to drug effects such as analgesic effects but also side effects. In conclusion, a decrease in the number of reflexes was observed only for the tapentadol treatment, on the spinal level. This could be due to the effects on the brainstem's noradrenaline reuptake inhibition. No other features of the NWR changed for any treatment. At the brain level, decreased latencies of the N1 component of the SEPs were identified only after the oxycodone intervention. Furthermore, the inverse modelling of dipolar sources in the insula component changed for both oxycodone and tapentadol treatments, indicating that both opioids affect cortical measures. This study replicates preclinical studies suggesting that the two opioids activate different pain control mechanisms and support clinical findings. There are no conflicts of interest to declare. A.M.D. and A.D. conceived the idea for the study. A.M.D., A.D. and M.N. designed the study. R.B.N. and T.M.H. conducted the study and wrote the manuscript. C.D.M. provided statistical advice. All authors participated in the interpretation of the study results and approved the final version of the manuscript. |
Beyond the assistance: additional exposure situations to COVID-19 for healthcare workers | 4c3f75c5-8a5c-46ee-a37f-1e739c01458f | 7174833 | Preventive Medicine[mh] | None declared. None. |
Abusive Head Trauma Module in the Child Abuse Pediatrics Curriculum for Physicians (CAP-CuP): Rehma's Story | 87e823a3-6187-4b37-9229-67da969e1013 | 11882959 | Pediatrics[mh] | By the end of this activity, learners will be able to: 1. Define abusive head trauma, including the most common mechanisms and injuries. 2. Describe the frequency and reasons for missed cases of abusive head trauma. 3. Summarize the medical, behavioral, and psychological sequelae of abusive head trauma.
Abusive head trauma (AHT; formerly referred to as Shaken Baby Syndrome) is the most fatal cause of head injuries under the age of 2 years and the leading cause of death from child abuse under the age of 5 years. , It may involve shaking, head impact, or both inflicted upon a young child. It can result in physical injuries as well as altered brain function affecting level of alertness, tone, breathing, and movement. , The majority of victims of abuse head trauma sustain long-term sequelae including developmental, behavioral, and neurological disability. While AHT can be accompanied by visible signs and symptoms, such as bruising or seizures, it can also occur without any external visible indicators. Furthermore, despite widespread consensus within the medical community about the validity of the diagnosis of AHT, there is a small but vocal group of physicians sparking false controversy in legal settings. Thus, it is more important than ever that physicians remain aware of the common signs, symptoms, sentinel injuries, histories, and risk factors that may indicate the need to pursue a workup for AHT. Signs and symptoms of child abuse are undertaught in medical education. – Medical students, residents, and physicians in practice have demonstrated both knowledge deficits , and insight into the need for more education in this area. , – The American Academy of Pediatrics has provided a set of study materials for child abuse, but these are restricted to subscribing members and have not been updated since 2015. Previous MedEdPORTAL publications on AHT include a simulation case from 2017 and a PedsCase from 2010. Both are designed for use by medical students. The simulation case includes a didactic that summarizes many different forms and mimics of child physical abuse, and does not focus solely on AHT. The PedsCase pairs a case description of AHT with a podcast and multiple-choice questions. The case description uses names for the patient and mother that are traditionally associated with Black race, , and prior literature has suggested that there are racial disparities in the medical evaluation of AHT. Teaching materials are needed that address and counter implicit biases about child abuse being more likely in Black families. This interactive module on AHT, designed for use by medical students and physicians who treat children, offers an updated review of the literature and expands upon prior work , by exploring the modern complexities of a tumultuous medicolegal landscape and an equity-focused sociocultural climate. One unique aspect of this module is its exploration of racial disparity in the diagnosis of AHT. It highlights a 1999 study in which the diagnosis of AHT was more likely to be missed in White infants and therefore more likely to be suspected in infants of color. Equity in health care requires medical providers to be mindful of implicit biases and how they may perpetuate discrimination in medical decision-making. Although this data comes from an older study, it is consistent with newer literature that demonstrates overrepresentation of Black and Brown children in child abuse evaluations and the child welfare system. , In addition, 16 of the 23 module references were published after the most recent MedEdPORTAL content on AHT. This module benefits from the design elements of a larger interactive curriculum, minimal equipment and personnel needed for implementation, flexibility of format (i.e., large audience, small group, or self-paced completion), and the use of storytelling to maximize its educational impact. The purpose of this interactive module on AHT was to provide a comprehensive yet succinct review that is accessible, organized, engaging, and versatile to various medical audiences.
The AHT module was created in 2022 with a dual purpose: First, there was a need to update the AHT didactic material in the Mayo Clinic Alix School of Medicine Disruptions in Development course, and second, the author of the module (Katie Johnson) sought to develop a core curriculum in child abuse pediatrics for physicians. (The Child Abuse Pediatrics Curriculum for Physicians [CAP-CuP] ), which would include a module on AHT. Because AHT was a relatively undertaught topic in medical education, , it was decided that one module could be built to serve both audiences. In other words, this module was built to be suitable for teaching medical students, residents, and physicians in practice about AHT. The AHT module was created by a child abuse pediatrician (Katie Johnson) who had experience with AHT from a clinical, educational, legal, and research perspective. , The module was reviewed and edited by two board-certified child abuse pediatricians with similar experience (Caitlin Crumm, Emily Brown), one of whom (Caitlin Crumm) was pursuing additional training in pediatric emergency medicine. The module was built to incorporate elements of the science of learning, including the generation of responses (e.g., multiple choice, true/false, and free response) before the answers were revealed, audiovisual material to vary the cadence of the presentation, and the weaving of a child's story into the material to make it more meaningful. Rehma Sabir was a child who died of AHT while in the care of her babysitter on her first birthday. Rehma's story was shared in three parts, interspersed with information on the epidemiology, clinical presentation, differential diagnosis, recommended workup, and short- and long-term sequelae of AHT. The sharing of Rehma's story in this open-access module was supported by her parents as well as the Cable News Network (CNN), the producer of the special report Justice for Rehma, after purchase of a copyright agreement. The AHT module was built in PowerPoint and was optimally implemented with the use of a quiz-based polling software, such as Kahoot! or PollEverywhere, though polling software was not required for implementation. Optional Kahoot! links as well as a presenter script were included in the PowerPoint slide notes as well as the technical guide to facilitate accessibility, accuracy, and reliability in the presentation of this material. When polling software was not used, participants could use a pen and paper or a personal cell phone or computer to record their answers. Pilot testing of this module included presentation to large audiences; small groups; and individual, self-paced completion by residents. Large-group presentations included a virtual education conference for pediatric residents at Mayo Clinic (10–15 attendees) and a virtual grand rounds for Mayo Clinic emergency medical services first responders (50–75 attendees), the latter of whom received a modified version that emphasized the first-responder role. The small-group and self-paced completion of this module was implemented with medical students, residents, and fellows completing the Mayo Clinic child abuse pediatrics elective. Specifically, these learners included fourth-year medical students, a fellow in pediatric hospital medicine, a resident in emergency medicine, and multiple residents in pediatrics, family medicine, and psychiatry. In all pilot tests, qualitative feedback was positive about the accessibility and effectiveness of the material for learners from a variety of training backgrounds. The AHT module was evaluated in three different formats: individual, self-paced completion by medical students and physicians from a variety of specialties and institutions, a small multidisciplinary audience at a Child and Adolescent Neurology (CAN) educational conference, and a large group of first-year medical students in the Mayo Clinic Alix School of Medicine in a course on neuropsychology and development (called Disruptions in Development). All versions took about 45 minutes to complete. The individuals completing the module in a self-paced format were from a variety of training levels, specialties, and institutions. They received access to the module from child abuse pediatricians at their institutions who had specifically requested the link after an oral presentation about the CAP-CuP curriculum at a child maltreatment conference (the Ray E. Helfer Society annual meeting in 2024). Most of the participants were pediatric residents, but participants also included family medicine residents, fourth-year medical students, a medicine/pediatrics resident, a pediatric hospital medicine fellow, and a child abuse pediatrician. Submission of learner demographic data by the child abuse pediatricians who had requested the module was voluntary. For the CAN educational conference and large-group medical student presentation, the optional Kahoot! link for large groups ( and ) was used. The presenter was a child abuse pediatrician and primary author of the module (Katie Johnson). The CAN audience was hybrid (i.e., some attended in person, and some attended via Zoom) and included child and adolescent neurology residents, child and adolescent neurology attendings, a child psychiatry resident, a medical student, and a nurse. The Disruptions in Development course audience was entirely in person and consisted of all first-year medical students. The following materials were needed: • Presenter: a computer with internet connection, a projector screen, and the ability to share visual and audio content; Zoom capability was needed for the hybrid presentation • Participants: a cell phone or personal computer with internet connection; Zoom capability was needed for those attending the hybrid presentation remotely In all delivery formats, the AHT module included a pre- and postmodule assessment consisting of five questions: one evaluating self-rated confidence in recognizing and managing AHT on a 10-point Likert-type scale (1 = not at all confident , 10 = completely confident ), and four evaluating knowledge about AHT . Participation of attendees was voluntary and anonymous. Attendees who opted to participate were not required to respond to every question, and thus, the number of respondents for each question varied. Descriptive statistics were calculated and included the median and interquartile range (IQR) of the confidence-level question (question 1) and the proportion of correct responses to the multiple-choice and true/false questions (questions 2–5). These metrics were calculated separately for each presentation format and also pooled for the two multidisciplinary audiences composed of primarily physicians (i.e., the CAN audience and individual participants). This project was approved by the Mayo Clinic Institutional Review Board and Education Research Committee.
A total of 24 multidisciplinary participants completed the individual self-paced module, 10 multidisciplinary participants attended the CAN educational conference, and 62 first-year medical students attended the Disruptions in Development course presentation. For the live presentations, total attendee numbers were not available. It is the author's observation that most (if not all) of the attendees participated in the game-based module. Among the largest group (the first-year medical students), the median confidence level pertaining to AHT demonstrated a modest increase from 3 (IQR: 2.00–5.00) to 6 (IQR: 6.00–7.75; ). The proportion of medical students answering knowledge questions correctly increased for every question, as follows: 27% (14 of 52) to 84% (43 of 51), 50% (26 of 52) to 90% (46 of 51), 2% (1 of 52) to 90% (44 of 49), and 79% (41 of 52) to 96% (47 of 49; ). Among the multidisciplinary audiences composed primarily of physicians (i.e., the CAN education conference and individual participants), the baseline confidence and knowledge scores tended to be higher, but the percentage of correct responses still increased for every question. Specifically, the multidisciplinary CAN group—which included two CAN residents, one CAN attending, one psychiatry resident, one medical student, one nurse, and four attendees who did not provide demographic data—had a median confidence level of 5 (IQR: 5.00–6.50) in the premodule assessment and 7 (IQR: 7.00–8.00) in the postmodule assessment. The percentages of correct responses to each knowledge question before and after the module were as follows: 57% (4 of 7) to 100% (7 of 7), 71% (5 of 7) to 100% (7 of 7), 25% (2 of 8) to 86% (6 of 7), and 63% (5 of 8) to 100% (7 of 7). The individual participants completing the self-paced module—which included 10 pediatric residents, three family medicine residents, three fourth-year medical students, one medicine/pediatrics resident, one pediatric hospital medicine fellow, one child abuse pediatrician, and five for which no demographic data was available—had a median confidence level of 6 (IQR: 5.00–6.00) in the premodule assessment and 7 (IQR: 5.75–8.00) in the postmodule assessment. The percentages of correct responses to each knowledge question before and after the module were as follows: 70% (16 of 23) to 90% (18 of 20), 83% (19 of 23) to 95% (19 of 20), 30% (7 of 23) to 100% (20 of 20), and 70% (16 of 23) to 100% (20 of 20). The data for the latter two groups, which were both multidisciplinary and composed primarily of physicians, were pooled for comparison to the first-year medical student group. This pooling yielded a premodule confidence level of 6 (IQR: 5.00–6.00) and a postmodule confidence level of 7 (IQR: 6.00–8.00; ). The percentage of correct responses in the pooled data set before and after the module were as follows: 67% (20 of 30) to 93% (25 of 27), 80% (24 of 30) to 96% (26 of 27), 29% (9 of 31) to 96% (26 of 27), and 68% (21 of 31) to 100% (27 of 27; ). The greatest improvement for all audiences was observed for question 4, which asked participants to identify the percentage of AHT diagnoses missed by the first physician they see. The answer to this question was presented in the context of a slide about racial disparities (i.e., physicians are more likely to miss the diagnosis of AHT in White infants, and therefore more likely to suspect the diagnosis in infants of color).
This versatile, interactive module about AHT was associated with increased confidence and knowledge for a variety of medical audiences and various delivery formats, including self-paced completion by residents, small-group participation by multidisciplinary attendees at a CAN educational conference, and large-group participation by first-year medical students. All presentation formats covered a succinct, comprehensive, interactive, and case-based presentation about AHT in under 45 minutes. This module may serve as an effective precursor to either the simulation or the PedsCase previously published in MedEdPORTAL . Given its timely incorporation of medicolegal and health equity perspectives on AHT, it may enhance the debrief following either of these teaching tools. Lessons learned from this project included how to navigate copyright permissions with a cable news network including both primary and third-party permissions for use of audiovisual material in an open-access, Creative Commons-licensed educational product. In the earliest version of this module, Rehma's story was shared in one 9-minute segment at the beginning of the module. Because of the need to remove certain segments of the video with copyright-protected third-party content, the video was divided into three segments, and the decision was made to intersperse these segments throughout the presentation. This turned out to be a favorable format for keeping the audience engaged by varying the presentation style between a live presenter and audiovisual media. In addition, the opportunity to connect with Rehma Sabir's father prior to implementation of this module served as a reminder that the support of families when using patient stories in education cannot be overstated. Limitations of this project include its heterogeneity in audience specialty, training background, and delivery format, although this could be viewed as a strength of the module's versatility as well. While the physician audiences provided some secondary data ( n = 34), the heterogeneity of the audience and limitations in demographic data rendered by voluntary submission and anonymous participation made it difficult to draw meaningful conclusions about the effectiveness of the module. Nevertheless, we observed that this mixed physician group had higher baseline confidence levels and knowledge scores than the first-year medical students, and similar increases in confidence and knowledge for each question, suggesting reliability in effect. This project is limited in its evaluation method, which included short-term recall and self-rated confidence. These are both relatively weak forms of evaluation, leaving the long-term clinical impact of this module unknown. Nevertheless, we used science of learning principles woven together with a poignant patient story to support meaningful, enduring learning. Next steps for this module may include wider implementation and testing with various medical audiences, such as emergency medicine providers, pediatricians, pediatric intensivists, and advanced practice practitioners. All of these groups are stakeholders who may have the opportunity to recognize, appropriately manage, and testify regarding AHT in the best interest of safeguarding children.
Abusive Head Trauma Module.pptx Abusive Head Trauma - Technical Guide and Script.docx Abusive Head Trauma - Assessment.docx All appendices are peer reviewed as integral parts of the Original Publication.
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Development and validation of a sensitive liquid chromatography tandem mass spectrometry assay for the simultaneous determination of ten kinase inhibitors in human serum and plasma | 4c2bcb78-4c34-4fbc-bbca-64ec70ad482e | 7644392 | Pharmacology[mh] | Almost 20 years after the approval of imatinib, oral anticancer therapy has become an integral part in the treatment regimens of various malignancies and other diseases such as graft versus host disease (GvHD) and even COVID-19 . One group of oral anticancer drugs comprises so-called kinase inhibitors (KI) such as osimertinib and afatinib which share a similar mechanism of action by inhibiting protein kinases in malignant cells. Protein kinases play a key role in cellular signaling pathways regulating cell proliferation and differentiation or programmed cell death. Their effect is mediated by catalyzing the phosphorylation of protein residues (e.g., tyrosine residues). Genetic alterations may lead to improper regulation, promoting the development of cancer or other diseases . Osimertinib for instance is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that binds to certain mutant forms of EGFR that predominate in non-small cell lung cancer (NSCLC) tumors . Afatinib as another example is a selective and irreversible ErbB family blocker such as EGFR and the human epidermal growth factor receptor 2 and is approved for the treatment of locally advanced or metastatic NSCLC . Considering the complex pharmacokinetics (PK) of KI, a high level of interindividual variability in drug exposure due to varying absorption, distribution, metabolism, and excretion has been demonstrated for many KI . As KI are administered orally, drug formulation, food intake, stomach pH, solubility, disturbed gastrointestinal adsorption, or first-pass hepatic metabolism may lead to variability in oral bioavailability. Moreover, almost all KI are predominantly metabolized by isoenzymes of the cytochrome P450 family (CYP) . Variability in expression and activity of CYP enzymes contribute to interindividual differences in drug metabolism and drug clearance . This effect is particularly enhanced by concomitant administration of CYP inducers or inhibitors and thus common in patients with polypharmacotherapy such as cancer and GvHD patients . In addition, polymorphisms in genes encoding drug transporters (e.g., ABC transporter) involved in the absorption and excretion of many KI influence influx and efflux processes and subsequently drug distribution and exposure . A targeted mechanism of action must not be confused with a total lack of side effects. KI therapy comes along with a wide range of mild to severe adverse events (AE), often of the augmented type, which may be enhanced by drug overexposure . Intolerable AE and toxicity frequently lead to dose reductions or treatment discontinuations endangering treatment efficacy . On the other hand, tumors progress within months after initially responding to targeted therapy in many cases. At least in some cases, this behavior might be driven by subtherapeutic concentrations, triggering the development of resistance mechanisms . In contrast to intravenous chemotherapy, non-adherence might be an additional factor leading to varying drug exposure. For many KI, large differences in plasma concentrations between patients have been described . One strategy to prevent under- or overexposure of drug concentrations and monitor adherence is therapeutic drug monitoring (TDM). For some agents, TDM has already proven to be feasible and useful . Strong evidence exists for imatinib in chronic myelogenous leukemia . For alectinib , axitinib, crizotinib , trametinib , and vemurafenib , a PK target associated with either efficacy or toxicity has been established but has not been evaluated in clinical studies yet . However, for most kinase inhibitors, no information about the potential benefit of TDM is available, as exposure-response relationships and consequently PK targets have not been established. To further investigate concentration-dependent effects and the potential use of TDM for KI in daily clinical routine, the aim of this work was the development and validation of a simple and time-saving liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of an analyte panel comprising the KI afatinib (AFA), axitinib (AXI), bosutinib (BOS), cabozantinib (CAB), dabrafenib (DAB), lenvatinib (LEN), nilotinib (NIL), osimertinib (OSI), ruxolitinib (RUX), and trametinib (TRA). The assay was so far successfully applied to analyze serum levels of one patient taking AFA and two patients taking OSI.
Chemicals Axitinib and lenvatinib were purchased from Alsachim (Illkirch-Graffenstaden, France), and afatinib, bosutinib monohydrate, cabozantinib malate, dabrafenib mesylate, osimertinib mesylate, and ruxolitinib were acquired from Carbosynth Ltd. (Berkshire, UK). Nilotinib was bought from LC Laboratories (Woburn, MA, USA) and trametinib from Ark Pharm (Arlington Heights, IL, USA). 2 H 6 -Afatinib, 2 H 9 -bosutinib, 2 H 9 -dabrafenib, 2 H 5 -lenvatinib, 13 C 2 H 3 -osimertinib, and 13 C 2 H 6 -trametinib were obtained from Alsachim (Illkirch-Graffenstaden, France), and 13 C 2 H 3 -axitnib, 2 H 4 -cabozantinib, 2 H 6 -nilotinib, and 2 H 4 -ruxolitinib were purchased from Biozol Diagnostica GmbH (Eching, Germany). Ammonium bicarbonate was bought from Sigma-Aldrich Inc. (Steinheim, Germany). Acetonitrile, methanol, water (all LC-MS-grade), and dimethyl sulfoxide were purchased from Merck KGaA (Darmstadt, Germany). Blank human plasma was provided by the Institute for Transfusion Medicine and Haemotherapy of the University of Wuerzburg Medical Center. Stock solutions and working solutions Stock solutions of all analytes were separately prepared at a concentration of 1 mg/mL (calculated as free base) in dimethyl sulfoxide (DMSO). Two independent stock solutions were used for the preparation of calibration standards and quality control (QC) samples. The stock solutions were combined and diluted with methanol to obtain working solutions. Stock solutions for all internal standards (IS) were separately prepared at a concentration of 1 mg/mL in DMSO and were diluted with DMSO to prepare an IS working stock solution at a concentration of 20 μg/mL. Subsequently, the mixture was diluted with acetonitrile (ACN) to yield the precipitating agent at an IS concentration of 50 ng/mL (for each IS). Stock solutions were stored at − 80 °C in 2.0 mL polypropylene tubes and working solutions were stored at − 20 °C in glass tubes (0.5 mL). AXI and DAB were stored in amber-colored tubes (2.0 mL) due to their light sensitivity . Calibration standards and quality control samples Nine hundred fifty microliters of blank human plasma was spiked with 50 μL calibration standard working solution to obtain the highest calibration standard, containing all KI but OSI. The highest QC sample was established by spiking 50 μL of the separately prepared working solution to 950 μL of blank human plasma. QC low (QC-L), QC middle (QC-M), QC high (QC-H), and the remaining target calibrators were obtained by serial dilution with human plasma. To determine the lower limit of quantification (LLOQ), QC-LLOQ was prepared equivalently. Aliquots of 50 μL for each concentration level were stored at − 20 °C. Both validation and analytical runs contained nine calibration levels over the range (one replicate per level). All validation runs, except for stability assessment runs, consisted of five replicates of each QC level (in total n = 20 per run). Stability assessment runs and analytical runs included QC-H, QC-M, and QC-L in triplicates (in total n = 9 per run). OSI calibration standards and QC samples were first prepared in methanol in a 20-fold higher concentration compared to the final concentrations in human plasma. Nine hundred fifty microliters of blank human plasma was then spiked with 50 μL of each concentration level to obtain final calibrators and QC samples. The calibration range for CAB, DAB, NIL, and OSI was 6–1500 ng/mL. The concentrations for QC-LLOQ, QC-L, QC-M, and QC-H were 6, 15, 600, and 1200 ng/mL, respectively. AFA, AXI, BOS, LEN, RUX, and TRA calibration was applied in the range of 2–500 ng/mL. QC samples for these analytes were prepared at concentrations of 2, 5, 200, and 400 ng/mL for QC-LLOQ, QC-L, QC-M, and QC-H, respectively. Dilution of QC-H yielded QC-M (1:2), which was used to prepare an intermediate (level 1) (1:4). Another intermediate (level 2) was prepared from this solution (1:3 for CAB, DAB, NIL OSI, or 2:5 for AFA, AXI, BOS, LEN, RUX, TRA, respectively). Dilution of intermediate level 2 was used to prepare QC-L (1:4) which then again was diluted to QC-LLOQ (2:5). Collection of patient samples Immediately after sample collection by venipuncture, patient blood samples were centrifuged for 10 min at 4500 rcf at 18 °C. Serum was subsequently isolated and aliquots of 300 μL were stored at − 80 °C. Light protection of AXI samples was implemented by packaging samples in opaque plastic bags for transportation. Samples were thawed at room temperature prior to processing while being protected from daylight. OSI samples were thawed in the fridge (8 °C). Sample preparation For each validation and analytical run, freshly prepared QC samples and calibrators were used. Samples were prepared by protein precipitation with ACN as precipitation agent. One hundred fifty microliters of ice-cooled ACN containing IS was added to 50 μL sample, calibrator, or QC sample. The samples were vortexed for 15 s and centrifuged for 5 min at 4 °C and 11,000 rcf. Fifty microliters of supernatant was subsequently transferred to another tube containing 450 μL mobile phase A and was vortexed for an additional 10 s. Three hundred microliters was transferred to an autosampler vial with polypropylene insert. Forty microliters was injected into the chromatographic system. Chromatographic equipment and conditions Chromatographic separation was carried out using an Agilent 1290 Infinity LC System equipped with binary pump, autosampler, and thermostatted column compartment (Waldbronn, Germany). Chromatographic separation was achieved using a Waters XBridge® Phenyl 3.5 μm (2.1 × 50 mm) column and an eluent consisting of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A and methanol-water (9:1, v/v) containing 10 mM ammonium bicarbonate as phase B. To degas mobile phases and for further purification, vacuum filtration with a Millipore filtering system consisting of a ground joint flask, glass funnel, glass frit base, and clamp (Millipore Corporation, Billerica, MA, USA) using Sartorius™ SARTOLON polyamide membrane filter, 0.45 μm (Sartorius Stedim Biotech GmbH, Goettingen, Germany), was used. Gradient elution was applied at a flow rate of 400 μL/min using the following time program: 0–0.5 min 60% B, 0.5–2.00 min linear increase to 80% B, 2.00–5.00 min 80% B, 5.00–5.25 min linear decline to 60% B and remained at 60% B for the last 2 min. ACN-water (9:1, v/v) was used as needle wash solution. The temperature of the autosampler was kept at 10 °C. The column temperature was set to ambient temperature (controlled at 18 °C by air conditioning). Acquisition time was 7.0 min per run. Mass spectrometer and conditions For detection, a Sciex QTRAP 4500 MD mass spectrometer (Framingham, MA, USA) meeting the legal requirements for medical devices (MD) was used. Pneumatically assisted electrospray ionization (ESI) in positive mode and multiple reaction monitoring (MRM) were configured for ionization and fragmentation, respectively. The linear ion trap was not used in this analysis. Source parameters were set as follows: ion spray gas 3000 Volt, nebulizer gas 30 AU, collision gas medium, curtain gas 30 AU, temperature 400 °C, heater gas 30 AU. The mass spectrometer settings for each compound are illustrated in Electronic Supplementary Material (ESM) Table . Data processing and illustrations Analytical results were recorded and processed using Analyst Software 1.6.3 MD (Sciex, Framingham, MA, USA). Validation results were processed using Microsoft Excel Version 13.36 (Microsoft Corporation, Redmond, WA, USA). Patient data were analyzed using R Version 3.6.0 (R Foundation, Austria). Plots were generated using the “ggplot2” package in R. Chemical structures were illustrated with ChemDraw Version 19.0.1.332 (PerkinElmer Informatics, Waltham, MA, USA). Method characterization The assay was validated according to the European Medical Agency (EMA) (2011) and US Food and Drug Administration (2018) regulatory guidelines on bioanalytical method validation . Method robustness The method’s robustness was monitored daily by comparing the intensity of the analytes’ and IS’ signal for each concentration and compound. Retention times of each analyte were compared to previous runs. The system’s pressure during analysis and prior to analysis, variations in pressure, and its possible sources were also monitored. Another lot of column and solvents were used to check for differences in intensity and retention times. The pressure during a run was also randomly compared between samples and to previous runs for the same concentration. To ensure the accuracy of freshly prepared calibrators and QC samples, they were measured against previous calibrators or QC samples. Robustness of the method was also evaluated by the guidelines’ requirements of intra-day and inter-day accuracy and precision. Imprecision was calculated to monitor daily variations and was not expected to be < 15%. Calibration curves, linearity, and sensitivity The ratio of analyte peak area and isotopically labeled IS was defined as response and the concentration as the independent variable. The upper limit of quantitation (ULOQ) was defined as the concentration of the highest calibration level with accuracy between 85 and 115% ( n = 5) and a precision within 15%. LLOQ was defined as the concentration of the lowest calibrator with an accuracy between 80 and 120% ( n = 5) and a precision (expressed as coefficient of variance (CV) in percentage) within 20%. To obtain sensitivity, the response of the analyte at LLOQ was compared to the response obtained by the zero calibrator (matrix blank prepared with IS) and was expected to be at least 5 times higher (acceptance factor ≤ 1): 1 [12pt]{minimal}
$$ \ =\ \ ()\ \ \ 5}{\ \ ()\ } $$ Acceptance Factor = Peak Area analyte at zero calibrator × 5 Peak Area analyte LLOQ In addition, signal-to-noise (S/N) ratios were calculated by the Analyst Software for the lowest calibration level for each analyte on nine different days (= nine calibration runs). A 10:1 ratio was considered to be appropriate. Selectivity To differentiate the analytes and the IS from endogenous and other components in the matrix, selectivity was demonstrated by analyzing blank plasma samples from six different healthy donors. The impact of hemolytic, icteric, and lipemic serum was also tested as part of the selectivity assessment and matrix effect. Spiked QC samples in serum as matrix and blank serum were also measured to ensure the absence of interference in serum and matrix effects of serum. All MRM traces were monitored and investigated for the absence of interference by matrix or crosstalk between MRM transitions. Selectivity of the method was accepted if the response of blank samples was free of interference at the retention times of analytes and IS. The absence of interfering peaks was characterized by blank responses < 5% for IS and < 20% of LLOQ, respectively. Carryover Carryover was investigated by analyzing blank samples (prepared without IS) injected directly after ULOQ samples (1500 ng/mL or 500 ng/mL, n = 5). A mean signal ≤ 20% of LLOQ and less than 5% for IS was defined as absence of carryover effects. Five replicates were measured. Peak area was used for calculation. Recovery and matrix effects To investigate matrix effects, KI-free plasma from six healthy donors were used to prepare QC-H and QC-L samples ( n = 6 per level). These samples in biological matrix were compared to QC-H and QC-L samples in ACN as matrix. Both batches were prepared by serial dilution as described in the “ .” For each analyte and IS, matrix factor (MF) was calculated. 2 [12pt]{minimal}
$$ =\ \ \ \ \ \ \ }{\ \ \ \ \ } $$ MF = Peak Area in the presence of matrix Peak Area in absence of matrix In addition, the IS-normalized MF was calculated by dividing the MF of analyte by the MF of IS . Matrix effects were considered absent if the CV of the IS-normalized MF calculated from the six lots of matrix was ≤ 15%. To confirm efficient recovery, analytical results of blank KI-free plasma from six different healthy donors spiked with analyte after extraction (A.E.) (representing 100% recovery) were compared to samples from six different healthy donors spiked with analyte before extraction (B.E.) at QC-H, QC-M, and QC-L ( n = 6 per level). The following equation was used to calculate recovery: 3 [12pt]{minimal}
$$ \%=\ \ ()..$}\!/ \!\ \ (\ )..$}.}{\ \ ()..$}\!/ \!\ \ (\ )..$}.} 100 $$ Recovery % = Peak Area analyte B . E . Peak Area internal standard B . E . Peak Area analyte A . E . Peak Area internal standard A . E . × 100 Dilution integrity In case patient samples have a concentration higher than ULOQ, dilution integrity was tested by preparing samples above ULOQ (3000 ng/mL and 1000 ng/mL, respectively) using blank human plasma or physiological saline solution as diluents (1:2). Six replicates were measured. Accuracy and precision Accuracy and precision were evaluated using QC-H, QC-M, QC-L, and QC-LLOQ. Intra-day accuracy and precision were determined by measuring five replicates of every QC in a single run. Inter-day accuracy and precision were assessed by performing single runs with five replicates of each QC on three different days. The limits for accuracy and precision were ± 15% and CV ≤ 15%, respectively. According to guidelines, the limits for accuracy and precision (CV) of LLOQ were ± 20% and ≤ 20%, respectively. Total within-day and between-day imprecisions were calculated according to the method of Krouwer et al. . 4 [12pt]{minimal}
$$ \ (\%)=\ \ ()\ \ \ \ }{\ \ \ \ } 100 $$ Precision % = standard deviation SD per QC level mean concentration per QC Level × 100 Stability of samples and stock solutions Pre-preparative stability (bench-top stability) of KI in human plasma was investigated by comparing freshly prepared QC samples (QC-H and QC-L) to unprocessed QC samples kept at room temperature for 24 h without daylight and at room temperature for 48 h with or without daylight, in the fridge at 4 °C for 6 weeks and after incubation at 56 °C for 1 h. Post-preparative stability was tested by measuring processed samples after being kept in the autosampler for 24 h (autosampler stability). Long-term stability of samples was tested by comparing freshly prepared QC in plasma to QC samples in plasma stored at − 20 °C or − 80 °C for 3 months. Freeze-thaw stability was investigated by measuring QC samples after three cycles of freezing at − 20 °C and thawing at ambient temperature for 2 h. Stability for OSI at different storage conditions and long-term stability were tested separately since preparation of calibration standards and QC samples for OSI had to be changed prior to validation. Long-term stability of stock solutions in DMSO was tested by comparing freshly prepared solutions in DMSO with 4-month-old solutions kept at − 80 °C. Stability of working solutions in methanol was tested by comparing a freshly prepared working solution with a 4-month-old working solution which underwent several freeze (− 20 °C) and thaw cycles. Stability was accepted, if the measured concentrations met the nominal concentration ± 15%. Incurred sample reanalysis (ISR) The accuracy of incurred samples was evaluated by reanalysis of study samples in separate runs at different days. About 10% of the samples from each run were analyzed again. The following equation was used for calculation according to EMA guidelines: 5 [12pt]{minimal}
$$ \%=\ \ )\ }{\ } 100 $$ % difference = repeat value – initial value mean value × 100 Clinical application The method was applied to determine AFA and OSI concentrations in daily clinical routine. One sample per visit was collected from adult patients receiving AFA and OSI for the treatment of EGFR-mutant metastatic lung adenocarcinoma who had a predicted prognosis of > 2 months. Data regarding the patients’ additional condition and co-medication were accessed by questionnaires and documented history (ESM Table ). Twenty-eight steady-state serum samples of three patients receiving AFA 20 mg ( n = 3) or OSI 80 mg ( n = 25) on a once daily schedule were collected during routine patient visits in an outpatient setting. Steady state was assumed 8 days after initiation of AFA and 15 days after initiation of OSI therapy according to the information obtained from the summary of product characteristics of Giotrif and Tagrisso, respectively . For both compounds, trough and non-trough levels were analyzed. The study was approved by the Ethics Commission of the University of Wuerzburg (ref199/18-am) and was conducted in accordance with the declaration of Helsinki. All patients gave written informed consent.
Axitinib and lenvatinib were purchased from Alsachim (Illkirch-Graffenstaden, France), and afatinib, bosutinib monohydrate, cabozantinib malate, dabrafenib mesylate, osimertinib mesylate, and ruxolitinib were acquired from Carbosynth Ltd. (Berkshire, UK). Nilotinib was bought from LC Laboratories (Woburn, MA, USA) and trametinib from Ark Pharm (Arlington Heights, IL, USA). 2 H 6 -Afatinib, 2 H 9 -bosutinib, 2 H 9 -dabrafenib, 2 H 5 -lenvatinib, 13 C 2 H 3 -osimertinib, and 13 C 2 H 6 -trametinib were obtained from Alsachim (Illkirch-Graffenstaden, France), and 13 C 2 H 3 -axitnib, 2 H 4 -cabozantinib, 2 H 6 -nilotinib, and 2 H 4 -ruxolitinib were purchased from Biozol Diagnostica GmbH (Eching, Germany). Ammonium bicarbonate was bought from Sigma-Aldrich Inc. (Steinheim, Germany). Acetonitrile, methanol, water (all LC-MS-grade), and dimethyl sulfoxide were purchased from Merck KGaA (Darmstadt, Germany). Blank human plasma was provided by the Institute for Transfusion Medicine and Haemotherapy of the University of Wuerzburg Medical Center.
Stock solutions of all analytes were separately prepared at a concentration of 1 mg/mL (calculated as free base) in dimethyl sulfoxide (DMSO). Two independent stock solutions were used for the preparation of calibration standards and quality control (QC) samples. The stock solutions were combined and diluted with methanol to obtain working solutions. Stock solutions for all internal standards (IS) were separately prepared at a concentration of 1 mg/mL in DMSO and were diluted with DMSO to prepare an IS working stock solution at a concentration of 20 μg/mL. Subsequently, the mixture was diluted with acetonitrile (ACN) to yield the precipitating agent at an IS concentration of 50 ng/mL (for each IS). Stock solutions were stored at − 80 °C in 2.0 mL polypropylene tubes and working solutions were stored at − 20 °C in glass tubes (0.5 mL). AXI and DAB were stored in amber-colored tubes (2.0 mL) due to their light sensitivity .
Nine hundred fifty microliters of blank human plasma was spiked with 50 μL calibration standard working solution to obtain the highest calibration standard, containing all KI but OSI. The highest QC sample was established by spiking 50 μL of the separately prepared working solution to 950 μL of blank human plasma. QC low (QC-L), QC middle (QC-M), QC high (QC-H), and the remaining target calibrators were obtained by serial dilution with human plasma. To determine the lower limit of quantification (LLOQ), QC-LLOQ was prepared equivalently. Aliquots of 50 μL for each concentration level were stored at − 20 °C. Both validation and analytical runs contained nine calibration levels over the range (one replicate per level). All validation runs, except for stability assessment runs, consisted of five replicates of each QC level (in total n = 20 per run). Stability assessment runs and analytical runs included QC-H, QC-M, and QC-L in triplicates (in total n = 9 per run). OSI calibration standards and QC samples were first prepared in methanol in a 20-fold higher concentration compared to the final concentrations in human plasma. Nine hundred fifty microliters of blank human plasma was then spiked with 50 μL of each concentration level to obtain final calibrators and QC samples. The calibration range for CAB, DAB, NIL, and OSI was 6–1500 ng/mL. The concentrations for QC-LLOQ, QC-L, QC-M, and QC-H were 6, 15, 600, and 1200 ng/mL, respectively. AFA, AXI, BOS, LEN, RUX, and TRA calibration was applied in the range of 2–500 ng/mL. QC samples for these analytes were prepared at concentrations of 2, 5, 200, and 400 ng/mL for QC-LLOQ, QC-L, QC-M, and QC-H, respectively. Dilution of QC-H yielded QC-M (1:2), which was used to prepare an intermediate (level 1) (1:4). Another intermediate (level 2) was prepared from this solution (1:3 for CAB, DAB, NIL OSI, or 2:5 for AFA, AXI, BOS, LEN, RUX, TRA, respectively). Dilution of intermediate level 2 was used to prepare QC-L (1:4) which then again was diluted to QC-LLOQ (2:5).
Immediately after sample collection by venipuncture, patient blood samples were centrifuged for 10 min at 4500 rcf at 18 °C. Serum was subsequently isolated and aliquots of 300 μL were stored at − 80 °C. Light protection of AXI samples was implemented by packaging samples in opaque plastic bags for transportation. Samples were thawed at room temperature prior to processing while being protected from daylight. OSI samples were thawed in the fridge (8 °C).
For each validation and analytical run, freshly prepared QC samples and calibrators were used. Samples were prepared by protein precipitation with ACN as precipitation agent. One hundred fifty microliters of ice-cooled ACN containing IS was added to 50 μL sample, calibrator, or QC sample. The samples were vortexed for 15 s and centrifuged for 5 min at 4 °C and 11,000 rcf. Fifty microliters of supernatant was subsequently transferred to another tube containing 450 μL mobile phase A and was vortexed for an additional 10 s. Three hundred microliters was transferred to an autosampler vial with polypropylene insert. Forty microliters was injected into the chromatographic system.
Chromatographic separation was carried out using an Agilent 1290 Infinity LC System equipped with binary pump, autosampler, and thermostatted column compartment (Waldbronn, Germany). Chromatographic separation was achieved using a Waters XBridge® Phenyl 3.5 μm (2.1 × 50 mm) column and an eluent consisting of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A and methanol-water (9:1, v/v) containing 10 mM ammonium bicarbonate as phase B. To degas mobile phases and for further purification, vacuum filtration with a Millipore filtering system consisting of a ground joint flask, glass funnel, glass frit base, and clamp (Millipore Corporation, Billerica, MA, USA) using Sartorius™ SARTOLON polyamide membrane filter, 0.45 μm (Sartorius Stedim Biotech GmbH, Goettingen, Germany), was used. Gradient elution was applied at a flow rate of 400 μL/min using the following time program: 0–0.5 min 60% B, 0.5–2.00 min linear increase to 80% B, 2.00–5.00 min 80% B, 5.00–5.25 min linear decline to 60% B and remained at 60% B for the last 2 min. ACN-water (9:1, v/v) was used as needle wash solution. The temperature of the autosampler was kept at 10 °C. The column temperature was set to ambient temperature (controlled at 18 °C by air conditioning). Acquisition time was 7.0 min per run.
For detection, a Sciex QTRAP 4500 MD mass spectrometer (Framingham, MA, USA) meeting the legal requirements for medical devices (MD) was used. Pneumatically assisted electrospray ionization (ESI) in positive mode and multiple reaction monitoring (MRM) were configured for ionization and fragmentation, respectively. The linear ion trap was not used in this analysis. Source parameters were set as follows: ion spray gas 3000 Volt, nebulizer gas 30 AU, collision gas medium, curtain gas 30 AU, temperature 400 °C, heater gas 30 AU. The mass spectrometer settings for each compound are illustrated in Electronic Supplementary Material (ESM) Table .
Analytical results were recorded and processed using Analyst Software 1.6.3 MD (Sciex, Framingham, MA, USA). Validation results were processed using Microsoft Excel Version 13.36 (Microsoft Corporation, Redmond, WA, USA). Patient data were analyzed using R Version 3.6.0 (R Foundation, Austria). Plots were generated using the “ggplot2” package in R. Chemical structures were illustrated with ChemDraw Version 19.0.1.332 (PerkinElmer Informatics, Waltham, MA, USA).
The assay was validated according to the European Medical Agency (EMA) (2011) and US Food and Drug Administration (2018) regulatory guidelines on bioanalytical method validation . Method robustness The method’s robustness was monitored daily by comparing the intensity of the analytes’ and IS’ signal for each concentration and compound. Retention times of each analyte were compared to previous runs. The system’s pressure during analysis and prior to analysis, variations in pressure, and its possible sources were also monitored. Another lot of column and solvents were used to check for differences in intensity and retention times. The pressure during a run was also randomly compared between samples and to previous runs for the same concentration. To ensure the accuracy of freshly prepared calibrators and QC samples, they were measured against previous calibrators or QC samples. Robustness of the method was also evaluated by the guidelines’ requirements of intra-day and inter-day accuracy and precision. Imprecision was calculated to monitor daily variations and was not expected to be < 15%. Calibration curves, linearity, and sensitivity The ratio of analyte peak area and isotopically labeled IS was defined as response and the concentration as the independent variable. The upper limit of quantitation (ULOQ) was defined as the concentration of the highest calibration level with accuracy between 85 and 115% ( n = 5) and a precision within 15%. LLOQ was defined as the concentration of the lowest calibrator with an accuracy between 80 and 120% ( n = 5) and a precision (expressed as coefficient of variance (CV) in percentage) within 20%. To obtain sensitivity, the response of the analyte at LLOQ was compared to the response obtained by the zero calibrator (matrix blank prepared with IS) and was expected to be at least 5 times higher (acceptance factor ≤ 1): 1 [12pt]{minimal}
$$ \ =\ \ ()\ \ \ 5}{\ \ ()\ } $$ Acceptance Factor = Peak Area analyte at zero calibrator × 5 Peak Area analyte LLOQ In addition, signal-to-noise (S/N) ratios were calculated by the Analyst Software for the lowest calibration level for each analyte on nine different days (= nine calibration runs). A 10:1 ratio was considered to be appropriate. Selectivity To differentiate the analytes and the IS from endogenous and other components in the matrix, selectivity was demonstrated by analyzing blank plasma samples from six different healthy donors. The impact of hemolytic, icteric, and lipemic serum was also tested as part of the selectivity assessment and matrix effect. Spiked QC samples in serum as matrix and blank serum were also measured to ensure the absence of interference in serum and matrix effects of serum. All MRM traces were monitored and investigated for the absence of interference by matrix or crosstalk between MRM transitions. Selectivity of the method was accepted if the response of blank samples was free of interference at the retention times of analytes and IS. The absence of interfering peaks was characterized by blank responses < 5% for IS and < 20% of LLOQ, respectively. Carryover Carryover was investigated by analyzing blank samples (prepared without IS) injected directly after ULOQ samples (1500 ng/mL or 500 ng/mL, n = 5). A mean signal ≤ 20% of LLOQ and less than 5% for IS was defined as absence of carryover effects. Five replicates were measured. Peak area was used for calculation. Recovery and matrix effects To investigate matrix effects, KI-free plasma from six healthy donors were used to prepare QC-H and QC-L samples ( n = 6 per level). These samples in biological matrix were compared to QC-H and QC-L samples in ACN as matrix. Both batches were prepared by serial dilution as described in the “ .” For each analyte and IS, matrix factor (MF) was calculated. 2 [12pt]{minimal}
$$ =\ \ \ \ \ \ \ }{\ \ \ \ \ } $$ MF = Peak Area in the presence of matrix Peak Area in absence of matrix In addition, the IS-normalized MF was calculated by dividing the MF of analyte by the MF of IS . Matrix effects were considered absent if the CV of the IS-normalized MF calculated from the six lots of matrix was ≤ 15%. To confirm efficient recovery, analytical results of blank KI-free plasma from six different healthy donors spiked with analyte after extraction (A.E.) (representing 100% recovery) were compared to samples from six different healthy donors spiked with analyte before extraction (B.E.) at QC-H, QC-M, and QC-L ( n = 6 per level). The following equation was used to calculate recovery: 3 [12pt]{minimal}
$$ \%=\ \ ()..$}\!/ \!\ \ (\ )..$}.}{\ \ ()..$}\!/ \!\ \ (\ )..$}.} 100 $$ Recovery % = Peak Area analyte B . E . Peak Area internal standard B . E . Peak Area analyte A . E . Peak Area internal standard A . E . × 100 Dilution integrity In case patient samples have a concentration higher than ULOQ, dilution integrity was tested by preparing samples above ULOQ (3000 ng/mL and 1000 ng/mL, respectively) using blank human plasma or physiological saline solution as diluents (1:2). Six replicates were measured. Accuracy and precision Accuracy and precision were evaluated using QC-H, QC-M, QC-L, and QC-LLOQ. Intra-day accuracy and precision were determined by measuring five replicates of every QC in a single run. Inter-day accuracy and precision were assessed by performing single runs with five replicates of each QC on three different days. The limits for accuracy and precision were ± 15% and CV ≤ 15%, respectively. According to guidelines, the limits for accuracy and precision (CV) of LLOQ were ± 20% and ≤ 20%, respectively. Total within-day and between-day imprecisions were calculated according to the method of Krouwer et al. . 4 [12pt]{minimal}
$$ \ (\%)=\ \ ()\ \ \ \ }{\ \ \ \ } 100 $$ Precision % = standard deviation SD per QC level mean concentration per QC Level × 100 Stability of samples and stock solutions Pre-preparative stability (bench-top stability) of KI in human plasma was investigated by comparing freshly prepared QC samples (QC-H and QC-L) to unprocessed QC samples kept at room temperature for 24 h without daylight and at room temperature for 48 h with or without daylight, in the fridge at 4 °C for 6 weeks and after incubation at 56 °C for 1 h. Post-preparative stability was tested by measuring processed samples after being kept in the autosampler for 24 h (autosampler stability). Long-term stability of samples was tested by comparing freshly prepared QC in plasma to QC samples in plasma stored at − 20 °C or − 80 °C for 3 months. Freeze-thaw stability was investigated by measuring QC samples after three cycles of freezing at − 20 °C and thawing at ambient temperature for 2 h. Stability for OSI at different storage conditions and long-term stability were tested separately since preparation of calibration standards and QC samples for OSI had to be changed prior to validation. Long-term stability of stock solutions in DMSO was tested by comparing freshly prepared solutions in DMSO with 4-month-old solutions kept at − 80 °C. Stability of working solutions in methanol was tested by comparing a freshly prepared working solution with a 4-month-old working solution which underwent several freeze (− 20 °C) and thaw cycles. Stability was accepted, if the measured concentrations met the nominal concentration ± 15%. Incurred sample reanalysis (ISR) The accuracy of incurred samples was evaluated by reanalysis of study samples in separate runs at different days. About 10% of the samples from each run were analyzed again. The following equation was used for calculation according to EMA guidelines: 5 [12pt]{minimal}
$$ \%=\ \ )\ }{\ } 100 $$ % difference = repeat value – initial value mean value × 100
The method’s robustness was monitored daily by comparing the intensity of the analytes’ and IS’ signal for each concentration and compound. Retention times of each analyte were compared to previous runs. The system’s pressure during analysis and prior to analysis, variations in pressure, and its possible sources were also monitored. Another lot of column and solvents were used to check for differences in intensity and retention times. The pressure during a run was also randomly compared between samples and to previous runs for the same concentration. To ensure the accuracy of freshly prepared calibrators and QC samples, they were measured against previous calibrators or QC samples. Robustness of the method was also evaluated by the guidelines’ requirements of intra-day and inter-day accuracy and precision. Imprecision was calculated to monitor daily variations and was not expected to be < 15%.
The ratio of analyte peak area and isotopically labeled IS was defined as response and the concentration as the independent variable. The upper limit of quantitation (ULOQ) was defined as the concentration of the highest calibration level with accuracy between 85 and 115% ( n = 5) and a precision within 15%. LLOQ was defined as the concentration of the lowest calibrator with an accuracy between 80 and 120% ( n = 5) and a precision (expressed as coefficient of variance (CV) in percentage) within 20%. To obtain sensitivity, the response of the analyte at LLOQ was compared to the response obtained by the zero calibrator (matrix blank prepared with IS) and was expected to be at least 5 times higher (acceptance factor ≤ 1): 1 [12pt]{minimal}
$$ \ =\ \ ()\ \ \ 5}{\ \ ()\ } $$ Acceptance Factor = Peak Area analyte at zero calibrator × 5 Peak Area analyte LLOQ In addition, signal-to-noise (S/N) ratios were calculated by the Analyst Software for the lowest calibration level for each analyte on nine different days (= nine calibration runs). A 10:1 ratio was considered to be appropriate.
To differentiate the analytes and the IS from endogenous and other components in the matrix, selectivity was demonstrated by analyzing blank plasma samples from six different healthy donors. The impact of hemolytic, icteric, and lipemic serum was also tested as part of the selectivity assessment and matrix effect. Spiked QC samples in serum as matrix and blank serum were also measured to ensure the absence of interference in serum and matrix effects of serum. All MRM traces were monitored and investigated for the absence of interference by matrix or crosstalk between MRM transitions. Selectivity of the method was accepted if the response of blank samples was free of interference at the retention times of analytes and IS. The absence of interfering peaks was characterized by blank responses < 5% for IS and < 20% of LLOQ, respectively.
Carryover was investigated by analyzing blank samples (prepared without IS) injected directly after ULOQ samples (1500 ng/mL or 500 ng/mL, n = 5). A mean signal ≤ 20% of LLOQ and less than 5% for IS was defined as absence of carryover effects. Five replicates were measured. Peak area was used for calculation.
To investigate matrix effects, KI-free plasma from six healthy donors were used to prepare QC-H and QC-L samples ( n = 6 per level). These samples in biological matrix were compared to QC-H and QC-L samples in ACN as matrix. Both batches were prepared by serial dilution as described in the “ .” For each analyte and IS, matrix factor (MF) was calculated. 2 [12pt]{minimal}
$$ =\ \ \ \ \ \ \ }{\ \ \ \ \ } $$ MF = Peak Area in the presence of matrix Peak Area in absence of matrix In addition, the IS-normalized MF was calculated by dividing the MF of analyte by the MF of IS . Matrix effects were considered absent if the CV of the IS-normalized MF calculated from the six lots of matrix was ≤ 15%. To confirm efficient recovery, analytical results of blank KI-free plasma from six different healthy donors spiked with analyte after extraction (A.E.) (representing 100% recovery) were compared to samples from six different healthy donors spiked with analyte before extraction (B.E.) at QC-H, QC-M, and QC-L ( n = 6 per level). The following equation was used to calculate recovery: 3 [12pt]{minimal}
$$ \%=\ \ ()..$}\!/ \!\ \ (\ )..$}.}{\ \ ()..$}\!/ \!\ \ (\ )..$}.} 100 $$ Recovery % = Peak Area analyte B . E . Peak Area internal standard B . E . Peak Area analyte A . E . Peak Area internal standard A . E . × 100
In case patient samples have a concentration higher than ULOQ, dilution integrity was tested by preparing samples above ULOQ (3000 ng/mL and 1000 ng/mL, respectively) using blank human plasma or physiological saline solution as diluents (1:2). Six replicates were measured.
Accuracy and precision were evaluated using QC-H, QC-M, QC-L, and QC-LLOQ. Intra-day accuracy and precision were determined by measuring five replicates of every QC in a single run. Inter-day accuracy and precision were assessed by performing single runs with five replicates of each QC on three different days. The limits for accuracy and precision were ± 15% and CV ≤ 15%, respectively. According to guidelines, the limits for accuracy and precision (CV) of LLOQ were ± 20% and ≤ 20%, respectively. Total within-day and between-day imprecisions were calculated according to the method of Krouwer et al. . 4 [12pt]{minimal}
$$ \ (\%)=\ \ ()\ \ \ \ }{\ \ \ \ } 100 $$ Precision % = standard deviation SD per QC level mean concentration per QC Level × 100
Pre-preparative stability (bench-top stability) of KI in human plasma was investigated by comparing freshly prepared QC samples (QC-H and QC-L) to unprocessed QC samples kept at room temperature for 24 h without daylight and at room temperature for 48 h with or without daylight, in the fridge at 4 °C for 6 weeks and after incubation at 56 °C for 1 h. Post-preparative stability was tested by measuring processed samples after being kept in the autosampler for 24 h (autosampler stability). Long-term stability of samples was tested by comparing freshly prepared QC in plasma to QC samples in plasma stored at − 20 °C or − 80 °C for 3 months. Freeze-thaw stability was investigated by measuring QC samples after three cycles of freezing at − 20 °C and thawing at ambient temperature for 2 h. Stability for OSI at different storage conditions and long-term stability were tested separately since preparation of calibration standards and QC samples for OSI had to be changed prior to validation. Long-term stability of stock solutions in DMSO was tested by comparing freshly prepared solutions in DMSO with 4-month-old solutions kept at − 80 °C. Stability of working solutions in methanol was tested by comparing a freshly prepared working solution with a 4-month-old working solution which underwent several freeze (− 20 °C) and thaw cycles. Stability was accepted, if the measured concentrations met the nominal concentration ± 15%.
The accuracy of incurred samples was evaluated by reanalysis of study samples in separate runs at different days. About 10% of the samples from each run were analyzed again. The following equation was used for calculation according to EMA guidelines: 5 [12pt]{minimal}
$$ \%=\ \ )\ }{\ } 100 $$ % difference = repeat value – initial value mean value × 100
The method was applied to determine AFA and OSI concentrations in daily clinical routine. One sample per visit was collected from adult patients receiving AFA and OSI for the treatment of EGFR-mutant metastatic lung adenocarcinoma who had a predicted prognosis of > 2 months. Data regarding the patients’ additional condition and co-medication were accessed by questionnaires and documented history (ESM Table ). Twenty-eight steady-state serum samples of three patients receiving AFA 20 mg ( n = 3) or OSI 80 mg ( n = 25) on a once daily schedule were collected during routine patient visits in an outpatient setting. Steady state was assumed 8 days after initiation of AFA and 15 days after initiation of OSI therapy according to the information obtained from the summary of product characteristics of Giotrif and Tagrisso, respectively . For both compounds, trough and non-trough levels were analyzed. The study was approved by the Ethics Commission of the University of Wuerzburg (ref199/18-am) and was conducted in accordance with the declaration of Helsinki. All patients gave written informed consent.
Method characterization Method robustness The system’s pressure prior to analysis and during analysis was reproducible on a daily basis and compared to prior runs. Using a differing lot of column and solvents showed neither significant changes in the intensity of signals for the same concentration nor in the retention times of the analytes. Freshly prepared calibrators and QC samples were first measured against an older set of calibrator and QC samples and were only used for validation or analytical runs if their accuracy met the accuracy and precision criteria of ± 15% and ≤ 15%, respectively. Hence, reproducibility of the method was guaranteed. Finally, results of the inter-day and intra-day accuracy and precision according to the abovementioned validation guidelines (2.9.7) ensured the method’s robustness on a daily basis. Calibration curves, linearity, and sensitivity The mathematical model which fitted best was a weighted ( [12pt]{minimal}
$$ $$ 1 conc 2 ) linear regression model for all analytes. The calibration curves were linear ( R 2 ≥ 0.995–0.999) and reproducible for all analytes. Calculation of the acceptance factor for each KI showed the method’s sensitivity at LLOQ (ESM Table ). In addition, S/N ratios were calculated for the lowest calibration level and met the acceptance criteria 10:1 (ESM Table ). Selectivity The response in the blanks were less than 20% of LLOQ and ≤ 5% for IS (ESM Table ). When injecting IS of AXI, a small degree of crosstalk or contamination was detected in the MRM trace of the analyte at the retention time for AXI (Fig. and ESM Figs. ). However, it did not exceed the required limit of 20% of LLOQ. Carryover Carryover of the analytes did not exceed 20% of LLOQ and carryover of IS were below 5% (ESM Table ). Recovery and matrix effects Recovery results were reproducible and consistent for concentrations near the lower (91.1–110%) and upper limits of quantification (89.5–105%). Hemolytic, icteric, and lipemic serum did not have any impact on accuracy and precision (ESM Table ). Values were in the range of ± 15% (accuracy) and ≤ 15% (precision). Neither accuracy nor precision were affected when using serum as matrix instead of plasma (ESM Table ). The CV of the IS-normalized MF calculated for the 6 lots of matrix was less than 15% for all QC levels (range 2.86–10.1%). IS-normalized MF ranged from 0.74–1.02. Dilution integrity To dilute patient samples above ULOQ, blank human plasma and physiological saline solution were adequate and did not affect accuracy and precision (ESM Table ). Accuracy and (im)precision Intra-day accuracy and precision (expressed as CV in %) met the acceptance criteria (Table ). Between-day imprecision was also found adequate (Table ). Stability Pre-preparative stability (bench-top stability) investigations of AXI, BOS, CAB, DAB, LEN, NIL, RUX, and TRA showed that they can be stored in the fridge (4 °C) for 6 weeks and for 24 h at room temperature (Table ). Prolonged storage at room temperature and light exposure did not show any impact on accuracy and precision either (Table ). Incubation at 56 °C for 1 h also did not affect accuracy and precision (Table ). Post-preparative stability values (autosampler stability) were within acceptance criteria (Table ). Long-term stability investigations confirmed stability at − 20 °C (≥ 86.2%, CV ≤ 5.40%) and − 80 °C (≥ 96.1%, CV ≤ 5.92%) for at least 3 months. Samples in plasma were stable during three freeze-thaw cycles (≥ 96.3% ± 2.35, CV ≤ 7.79%). Stability results are illustrated in ESM Table . AFA and OSI stability differed from the remaining KI. QC-H and QC-L of AFA did not meet the acceptance criteria at 4 °C for 6 weeks (QC-H 66.1% ± 1.38, CV 2.08% and QC-L 38.9% ± 0.42, CV 1.08%) and QC-L at room temperature for 24 h (72.8% ± 3.5, CV 4.80%). Consequently, storage at room temperature for 48 h confirmed the instability of AFA at room temperature without showing any light dependency (QC-H 81.3% ± 3.41–83.5% ± 0.46, CV 4.19–0.55% and QC-L 63.0% ± 0.78 to 63.7% ± 5.05, CV 1.94–7.93%). All results are shown in Table . Instability of OSI under different conditions was investigated: after 24 h at room temperature, only 6.24% ± 0.04, CV 0.58% (with daylight) and 3.51% ± 0.27, CV 7.58% (without daylight), and after incubation at 56 °C for 1 h, only 26.6% ± 0.72, CV 2.70%, of the spiked analyte were detectable in QC-H. Quantitation of OSI in QC-L resulted in values below LLOQ under these conditions. In contrast, storage at 4 °C for 24 h met the accuracy and precision criteria (87.9% ± 0.00–90.3% ± 3.00, CV ≤ 3.00%). Bench-top and post-preparative stability results are shown in Table . Freeze-thaw stability of OSI missed the acceptance criteria in the third cycle for QC-L (81.5% ± 2.46, CV 3.02%). Long-term stability of OSI in human plasma was demonstrated at − 80 °C (QC-H 89.5% ± 0.49, CV 0.55%, QC-L 91.9% ± 4.82, CV 5.25%) and − 20 °C (QC-H 89.8% ± 1.47, CV 1.63%, QC-L 92.4% ± 3.31, CV 3.58%) for 4 weeks (ESM Table ). Stock solution stability results show that all KI, but AFA, were stable in DMSO for 4 months at − 80 °C (ESM Table ). Working solutions in methanol were stable for 4 months (ESM Table ). Incurred sample reanalysis (ISR) AFA sample reanalysis showed no difference. OSI difference ranged from − 5.4 to + 12.3% for three samples (= 10% of all samples). Analysis of patient samples AFA concentrations were analyzed in a 74-year-old Caucasian female patient receiving 20 mg/day for treatment of EGFR-mutant metastatic lung adenocarcinoma (study inclusion 16 months after initial dose). Each of the three samples was collected 4–5 h post dose (AFA taken at home) at the patient’s routine visit every 4 months (three visits in total). The average AFA serum concentration was 11.5 ng/mL ± 4.6 ng/mL (median 11.8 ng/mL, IQR 4.60 ng/mL, range 6.81–11.8 ng/mL). OSI concentrations were analyzed in samples collected every 4 to 6 weeks in two Caucasian female patients (58 and 40 years, study inclusion 4 weeks after initial dose) receiving 80 mg/day OSI for treatment of EGFR-mutant metastatic lung adenocarcinoma (Fig. ). The average OSI serum concentration of all levels in patient 1 ( n = 11) was 323 ng/mL ± 158 ng/mL (median 246 ng/mL, IQR 234 ng/mL, range 146–582 ng/mL). The analysis of six trough levels (24.5–25 h post dose) showed an average OSI concentration of 433 ng/mL ± 131 ng/mL (median 446 ng/mL, IQR 179 ng/mL, range 246–582 ng/mL). Five samples were obtained 1–4 h post dose. Analysis of untimed OSI samples in patient 2 ( n = 14, including 13 trough levels (23–27.5 h post dose)) revealed an average OSI serum concentration of 265 ± 260 ng/mL (median 180 ng/mL, IQR 243 ng/mL, range 61.3–1030 ng/mL).
Method robustness The system’s pressure prior to analysis and during analysis was reproducible on a daily basis and compared to prior runs. Using a differing lot of column and solvents showed neither significant changes in the intensity of signals for the same concentration nor in the retention times of the analytes. Freshly prepared calibrators and QC samples were first measured against an older set of calibrator and QC samples and were only used for validation or analytical runs if their accuracy met the accuracy and precision criteria of ± 15% and ≤ 15%, respectively. Hence, reproducibility of the method was guaranteed. Finally, results of the inter-day and intra-day accuracy and precision according to the abovementioned validation guidelines (2.9.7) ensured the method’s robustness on a daily basis. Calibration curves, linearity, and sensitivity The mathematical model which fitted best was a weighted ( [12pt]{minimal}
$$ $$ 1 conc 2 ) linear regression model for all analytes. The calibration curves were linear ( R 2 ≥ 0.995–0.999) and reproducible for all analytes. Calculation of the acceptance factor for each KI showed the method’s sensitivity at LLOQ (ESM Table ). In addition, S/N ratios were calculated for the lowest calibration level and met the acceptance criteria 10:1 (ESM Table ). Selectivity The response in the blanks were less than 20% of LLOQ and ≤ 5% for IS (ESM Table ). When injecting IS of AXI, a small degree of crosstalk or contamination was detected in the MRM trace of the analyte at the retention time for AXI (Fig. and ESM Figs. ). However, it did not exceed the required limit of 20% of LLOQ. Carryover Carryover of the analytes did not exceed 20% of LLOQ and carryover of IS were below 5% (ESM Table ). Recovery and matrix effects Recovery results were reproducible and consistent for concentrations near the lower (91.1–110%) and upper limits of quantification (89.5–105%). Hemolytic, icteric, and lipemic serum did not have any impact on accuracy and precision (ESM Table ). Values were in the range of ± 15% (accuracy) and ≤ 15% (precision). Neither accuracy nor precision were affected when using serum as matrix instead of plasma (ESM Table ). The CV of the IS-normalized MF calculated for the 6 lots of matrix was less than 15% for all QC levels (range 2.86–10.1%). IS-normalized MF ranged from 0.74–1.02. Dilution integrity To dilute patient samples above ULOQ, blank human plasma and physiological saline solution were adequate and did not affect accuracy and precision (ESM Table ). Accuracy and (im)precision Intra-day accuracy and precision (expressed as CV in %) met the acceptance criteria (Table ). Between-day imprecision was also found adequate (Table ). Stability Pre-preparative stability (bench-top stability) investigations of AXI, BOS, CAB, DAB, LEN, NIL, RUX, and TRA showed that they can be stored in the fridge (4 °C) for 6 weeks and for 24 h at room temperature (Table ). Prolonged storage at room temperature and light exposure did not show any impact on accuracy and precision either (Table ). Incubation at 56 °C for 1 h also did not affect accuracy and precision (Table ). Post-preparative stability values (autosampler stability) were within acceptance criteria (Table ). Long-term stability investigations confirmed stability at − 20 °C (≥ 86.2%, CV ≤ 5.40%) and − 80 °C (≥ 96.1%, CV ≤ 5.92%) for at least 3 months. Samples in plasma were stable during three freeze-thaw cycles (≥ 96.3% ± 2.35, CV ≤ 7.79%). Stability results are illustrated in ESM Table . AFA and OSI stability differed from the remaining KI. QC-H and QC-L of AFA did not meet the acceptance criteria at 4 °C for 6 weeks (QC-H 66.1% ± 1.38, CV 2.08% and QC-L 38.9% ± 0.42, CV 1.08%) and QC-L at room temperature for 24 h (72.8% ± 3.5, CV 4.80%). Consequently, storage at room temperature for 48 h confirmed the instability of AFA at room temperature without showing any light dependency (QC-H 81.3% ± 3.41–83.5% ± 0.46, CV 4.19–0.55% and QC-L 63.0% ± 0.78 to 63.7% ± 5.05, CV 1.94–7.93%). All results are shown in Table . Instability of OSI under different conditions was investigated: after 24 h at room temperature, only 6.24% ± 0.04, CV 0.58% (with daylight) and 3.51% ± 0.27, CV 7.58% (without daylight), and after incubation at 56 °C for 1 h, only 26.6% ± 0.72, CV 2.70%, of the spiked analyte were detectable in QC-H. Quantitation of OSI in QC-L resulted in values below LLOQ under these conditions. In contrast, storage at 4 °C for 24 h met the accuracy and precision criteria (87.9% ± 0.00–90.3% ± 3.00, CV ≤ 3.00%). Bench-top and post-preparative stability results are shown in Table . Freeze-thaw stability of OSI missed the acceptance criteria in the third cycle for QC-L (81.5% ± 2.46, CV 3.02%). Long-term stability of OSI in human plasma was demonstrated at − 80 °C (QC-H 89.5% ± 0.49, CV 0.55%, QC-L 91.9% ± 4.82, CV 5.25%) and − 20 °C (QC-H 89.8% ± 1.47, CV 1.63%, QC-L 92.4% ± 3.31, CV 3.58%) for 4 weeks (ESM Table ). Stock solution stability results show that all KI, but AFA, were stable in DMSO for 4 months at − 80 °C (ESM Table ). Working solutions in methanol were stable for 4 months (ESM Table ). Incurred sample reanalysis (ISR) AFA sample reanalysis showed no difference. OSI difference ranged from − 5.4 to + 12.3% for three samples (= 10% of all samples).
The system’s pressure prior to analysis and during analysis was reproducible on a daily basis and compared to prior runs. Using a differing lot of column and solvents showed neither significant changes in the intensity of signals for the same concentration nor in the retention times of the analytes. Freshly prepared calibrators and QC samples were first measured against an older set of calibrator and QC samples and were only used for validation or analytical runs if their accuracy met the accuracy and precision criteria of ± 15% and ≤ 15%, respectively. Hence, reproducibility of the method was guaranteed. Finally, results of the inter-day and intra-day accuracy and precision according to the abovementioned validation guidelines (2.9.7) ensured the method’s robustness on a daily basis.
The mathematical model which fitted best was a weighted ( [12pt]{minimal}
$$ $$ 1 conc 2 ) linear regression model for all analytes. The calibration curves were linear ( R 2 ≥ 0.995–0.999) and reproducible for all analytes. Calculation of the acceptance factor for each KI showed the method’s sensitivity at LLOQ (ESM Table ). In addition, S/N ratios were calculated for the lowest calibration level and met the acceptance criteria 10:1 (ESM Table ).
The response in the blanks were less than 20% of LLOQ and ≤ 5% for IS (ESM Table ). When injecting IS of AXI, a small degree of crosstalk or contamination was detected in the MRM trace of the analyte at the retention time for AXI (Fig. and ESM Figs. ). However, it did not exceed the required limit of 20% of LLOQ.
Carryover of the analytes did not exceed 20% of LLOQ and carryover of IS were below 5% (ESM Table ).
Recovery results were reproducible and consistent for concentrations near the lower (91.1–110%) and upper limits of quantification (89.5–105%). Hemolytic, icteric, and lipemic serum did not have any impact on accuracy and precision (ESM Table ). Values were in the range of ± 15% (accuracy) and ≤ 15% (precision). Neither accuracy nor precision were affected when using serum as matrix instead of plasma (ESM Table ). The CV of the IS-normalized MF calculated for the 6 lots of matrix was less than 15% for all QC levels (range 2.86–10.1%). IS-normalized MF ranged from 0.74–1.02.
To dilute patient samples above ULOQ, blank human plasma and physiological saline solution were adequate and did not affect accuracy and precision (ESM Table ).
Intra-day accuracy and precision (expressed as CV in %) met the acceptance criteria (Table ). Between-day imprecision was also found adequate (Table ).
Pre-preparative stability (bench-top stability) investigations of AXI, BOS, CAB, DAB, LEN, NIL, RUX, and TRA showed that they can be stored in the fridge (4 °C) for 6 weeks and for 24 h at room temperature (Table ). Prolonged storage at room temperature and light exposure did not show any impact on accuracy and precision either (Table ). Incubation at 56 °C for 1 h also did not affect accuracy and precision (Table ). Post-preparative stability values (autosampler stability) were within acceptance criteria (Table ). Long-term stability investigations confirmed stability at − 20 °C (≥ 86.2%, CV ≤ 5.40%) and − 80 °C (≥ 96.1%, CV ≤ 5.92%) for at least 3 months. Samples in plasma were stable during three freeze-thaw cycles (≥ 96.3% ± 2.35, CV ≤ 7.79%). Stability results are illustrated in ESM Table . AFA and OSI stability differed from the remaining KI. QC-H and QC-L of AFA did not meet the acceptance criteria at 4 °C for 6 weeks (QC-H 66.1% ± 1.38, CV 2.08% and QC-L 38.9% ± 0.42, CV 1.08%) and QC-L at room temperature for 24 h (72.8% ± 3.5, CV 4.80%). Consequently, storage at room temperature for 48 h confirmed the instability of AFA at room temperature without showing any light dependency (QC-H 81.3% ± 3.41–83.5% ± 0.46, CV 4.19–0.55% and QC-L 63.0% ± 0.78 to 63.7% ± 5.05, CV 1.94–7.93%). All results are shown in Table . Instability of OSI under different conditions was investigated: after 24 h at room temperature, only 6.24% ± 0.04, CV 0.58% (with daylight) and 3.51% ± 0.27, CV 7.58% (without daylight), and after incubation at 56 °C for 1 h, only 26.6% ± 0.72, CV 2.70%, of the spiked analyte were detectable in QC-H. Quantitation of OSI in QC-L resulted in values below LLOQ under these conditions. In contrast, storage at 4 °C for 24 h met the accuracy and precision criteria (87.9% ± 0.00–90.3% ± 3.00, CV ≤ 3.00%). Bench-top and post-preparative stability results are shown in Table . Freeze-thaw stability of OSI missed the acceptance criteria in the third cycle for QC-L (81.5% ± 2.46, CV 3.02%). Long-term stability of OSI in human plasma was demonstrated at − 80 °C (QC-H 89.5% ± 0.49, CV 0.55%, QC-L 91.9% ± 4.82, CV 5.25%) and − 20 °C (QC-H 89.8% ± 1.47, CV 1.63%, QC-L 92.4% ± 3.31, CV 3.58%) for 4 weeks (ESM Table ). Stock solution stability results show that all KI, but AFA, were stable in DMSO for 4 months at − 80 °C (ESM Table ). Working solutions in methanol were stable for 4 months (ESM Table ).
AFA sample reanalysis showed no difference. OSI difference ranged from − 5.4 to + 12.3% for three samples (= 10% of all samples).
AFA concentrations were analyzed in a 74-year-old Caucasian female patient receiving 20 mg/day for treatment of EGFR-mutant metastatic lung adenocarcinoma (study inclusion 16 months after initial dose). Each of the three samples was collected 4–5 h post dose (AFA taken at home) at the patient’s routine visit every 4 months (three visits in total). The average AFA serum concentration was 11.5 ng/mL ± 4.6 ng/mL (median 11.8 ng/mL, IQR 4.60 ng/mL, range 6.81–11.8 ng/mL). OSI concentrations were analyzed in samples collected every 4 to 6 weeks in two Caucasian female patients (58 and 40 years, study inclusion 4 weeks after initial dose) receiving 80 mg/day OSI for treatment of EGFR-mutant metastatic lung adenocarcinoma (Fig. ). The average OSI serum concentration of all levels in patient 1 ( n = 11) was 323 ng/mL ± 158 ng/mL (median 246 ng/mL, IQR 234 ng/mL, range 146–582 ng/mL). The analysis of six trough levels (24.5–25 h post dose) showed an average OSI concentration of 433 ng/mL ± 131 ng/mL (median 446 ng/mL, IQR 179 ng/mL, range 246–582 ng/mL). Five samples were obtained 1–4 h post dose. Analysis of untimed OSI samples in patient 2 ( n = 14, including 13 trough levels (23–27.5 h post dose)) revealed an average OSI serum concentration of 265 ± 260 ng/mL (median 180 ng/mL, IQR 243 ng/mL, range 61.3–1030 ng/mL).
Several LC-MS/MS methods for the determination of KI comprising different analyte panels (range 17 to two different KI) exist. While methods by Merienne et al. Reis et al. , Rousset et al , and Nijenhuis et al. use different sample preparation techniques, acidic mobile phase and C18 columns for separation, we focused on a simple and time-efficient sample preparation and used a phenyl-hexyl end-capped column at alkaline conditions. Moreover, we tried to keep the instrumentation of the LC system simple. Hence, we only used an inline filter in the autosampler and did not use any online extraction techniques or guard columns. However, calibration ranges slightly differ in these methods: As an example, Reis et al. showed a linear range from 5 to 250 ng/mL for AFA, Merienne et al. calibrated AFA in a range from 4 to 800 ng/mL, whereas we found a range from 2 to 500 ng/mL to be adequate for measurements of trough levels in daily clinical setting. In comparison, our lower limit of quantification would allow determination of plasma levels prior to steady state or in PK studies. Sample preparation One of the main advantages of our method is the simple and time-efficient sample preparation as it is developed to be used in daily routine. Existing methods use solid-phase extraction , liquid-liquid extraction , or a mixture of both , which are more time-consuming. Protein precipitation resulted in high analyte recovery, a short turnaround time, and was devoid of strong matrix effects. Chromatographic and mass spectrometric method Since all analytes are organic bases, weak retention and poor peak shape (excessive tailing) were the results of using formic acid, a common modifier in positive ESI, often combined with reversed phase chromatography. In contrast, a basic pH was chosen to shift the equilibrium towards the free base in order to increase analyte retention on reversed phase material. Excellent peak shape (with regard to symmetry and width) and sufficient retention were achieved using ammonium hydrogen carbonate as mobile phase additive together with a phenyl-hexyl–modified stationary phase with extended pH stability at the alkaline condition. The phenyl-hexyl modification was chosen to exploit planar π-π interactions for increased selectivity (Fig. ). Gradient elution enabled the separation of all analytes from hydrophilic matrix compounds, while shortening retention time (elution within 3 min). In order to avoid a gradient in ionic strength, mobile phase A and B both contained ammonium hydrogen carbonate at 10 mM. Methanol was chosen as organic modifier due to the fact that ACN might undergo hydrolysis at alkaline pH (hydrolysis of nitriles to the ammonium salt of a carboxylic acid). Despite of the basic conditions, [M+H] + ions were generated efficiently (comparable to using formic acid) in ESI-positive mode, due to the weakly acidic character of the hydrogen carbonate ion. However, using an alkaline mobile phase which contains hydrogen carbonate ions impedes changing to an acidic mobile phase on the same LC system as additional washing steps are required to prevent formation of carbon dioxide in the system. In comparison to existing methods which use acetic acid or formic acid , this can be seen as a limitation of the method’s feasibility. Operating the column temperature at elevated temperature was not required, ensuring column stability despite high throughput and alkaline conditions, which usually tend to shorten the lifetime of chromatographic columns. Even after more than 1500 injections on the same column, retention time and analytical results remained stable. The high analyte retention, as a result of the chosen mobile phase composition, pH, and stationary phase, enabled using the diverter valve to separate early eluting matrix components to waste, leading to effective reduction of ion suppression, ensuring reproducible and accurate analytical results. Moreover, effects of ion suppression were minimized by using isotopically labeled IS for every analyte. However, mobile phases containing ammonium hydrogen carbonate were not stable for a prolonged period of time and had to be discarded after 1 week at the latest. During method development, the starting composition of the mobile phase for the first 0.5 min and after 5.0 min had to be adjusted to 60% because of major carryover of OSI and AXI (method development started with 40%). Carryover was assumed to be caused by OSI or AXI residues in either autosampler or MS detector valves. Hence, prior to injection, the autosampler loop was switched from inline to waste and the higher strength of the mobile phase helped removing remaining residues. Same applied to remaining residues in the MS valves. Therefore, carryover could be reduced. Furthermore, different needle wash solutions such as 90% methanol, 90% isopropanol, and a mixture of formic acid in methanol/water (0.1:50:49.9, v/v) were tested to reduce carryover. Using 90% acetonitrile minimized carryover significantly, but due to the hydrophobic character of the compounds, carryover could not be avoided completely. Dilution integrity was tested using blank human plasma but also saline solution, since it is easily accessible in daily clinical routine. The saline solution was able to substitute blank human plasma, making it an ethically interesting alternative for this purpose. Recovery To investigate recovery, samples were first prepared in water as matrix. However, solubility issues in water made it impossible to prepare matrix-free samples by serial dilution. ACN was tested as matrix instead and showed reproducible results. Stability Light sensitivity of DAB in organic solvents and plasma and light sensitivity of AXI were reported in the literature. Investigating light exposure effects during our validation showed that DAB in plasma did not show any significant changes in concentrations, whereas concentrations of AXI in plasma decreased by about 7.0% in samples after light exposure (Table ). While Nijenhuis et al. report the instability of DAB in plasma at ambient temperature for 24 h, Herbrinks et al. come to the same conclusion as we did and report stability for both compounds in plasma at room temperature for 48 h. Nijenhuis et al. concluded that DAB in plasma was only stable for a period of 6 h kept at room temperature. Unfortunately, exact conditions for their stress test (e.g., material of container and anticoagulant) were not reported impeding the direct comparison. Sparidans et al. in addition confirm our findings on the stability of AXI in plasma at ambient temperature for 24 h while protected from daylight. Our method successfully separated the cis-/trans isomers of AXI enabling a differentiated quantification by consideration of the photolytic degradation product. Testing stability at 56 °C showed that at this condition, all KI, but OSI, are stable. Therefore, virus inactivation by heat (e.g., hepatitis c virus or human immunodeficiency virus and other enveloped viruses, such as SARS-CoV-2) of probably infectious or infectious samples could be performed. Stock solution stability results show that AFA in DMSO is not stable for 4 months at − 80 °C (82.8% ± 1.23, CV 1.49%). Therefore, we recommend to either prepare stock solutions freshly or investigate further stability conditions either in other solvents or for a shorter period of time. Stability investigations showed that AFA and OSI did not meet the acceptance criteria being kept at room temperature for more than 24 h, at 4 °C for 6 weeks and during incubation at 56 °C. These findings are in accordance with the fact that both compounds bind covalently to plasma proteins and other nucleophiles . The same conclusions were drawn by Veermann et al. and Rood et al. when they tested the stability of OSI at ambient temperature for 3 and 4 h, respectively; Veermann and colleagues suggested working on ice (+ 4 °C). As this was considered inconvenient for daily routine measurements, we chose a different approach. Working quickly through the simple sample preparation while storing and thawing samples at 4 °C produced acceptable results for accuracy and precision. However, we observed that freshly prepared calibration standards and QC samples in human matrix prior to analysis showed a better accuracy for OSI samples. Post-preparative stability investigations of OSI samples indicate that degradation and binding to matrix constituents is compensated by the IS. It is therefore crucial to use the corresponding isotope-labeled compounds for such analytes. For other analytes, CR and QC samples could be prepared in bulk and stored at − 80 °C for up to 3 months until analysis. Rood et al. reported non-linearity for OSI in their method. Having the same difficulties with assay reproducibility and accuracy, we changed the CR and QC preparation technique for OSI. Solubility issues combined with serial dilution in matrix seemed to have an effect on accuracy and precision between and within runs. Preparing the QC and CR samples differently solved the initial problems. The samples were serially diluted in organic solvent at a concentration 20 times higher compared to the intended plasma level. Each level was then individually prepared by dilution with blank matrix (1:20). Reproducibility of this assay is underlined by the ISR results. Patient samples We were able to demonstrate that our assay can easily be applied in a routine clinical setting. In contrast to reported AFA levels by Wind et al. and Merienne et al. (vide infra), the analysis of three AFA samples from our patient during a year revealed low concentrations of 11.5 ng/mL ± 4.6 ng/mL, despite the fact that samples where obtained close to expected time to maximum concentration (t max ) (4–5 h post dose). In PK investigations performed in 15 patients by Wind et al., the mean maximum concentration in steady state (C max,ss ) at once daily oral administration of 20 mg AFA was 24.5 ng/mL (geometric CV 88.5%) . Reis et al. measured 15 AFA trough concentrations in steady state (C ss,min ) originating from an unknown number of patients and reported a mean concentration of 23.0 ± 14.4 ng/mL for a daily dose of 40 mg . Merienne et al. analyzed eight trough levels in six patients receiving 40 mg/day and reported a median of 16.7 ng/mL (range 5–38.8 ng/mL) and proposed a minimum concentration (C ss,min ) of > 10–55 ng/mL as PK target . Considering that our patient was treated with 20 mg daily and given the broad range of concentrations in the above-named studies, our results seem plausible. In our patient, dose reduction from initially 40 to 30 mg and later 20 mg was performed due to severe cutaneous adverse events. On a 20 mg daily schedule, only mild adverse events remained, while the oncological situation is still stable (32 months after initiation of therapy), suggesting that lower concentrations might be necessary to control adverse events while still preventing tumor progression. This is in accordance with the findings of a retrospective study in 254 patients conducted by Lim et al. which demonstrated that a daily dose of 20 mg did not result in lower progression-free survival (PFS) in comparison to daily doses up to 40 mg, whereas significant differences in PFS could be seen when dosing regimens < 20 mg/day were applied . Our results of quantification of OSI trough concentrations in clinical routine samples of 433 ng/mL ± 131 ng/mL (patient 1) and 278 ± 264 ng/mL (patient 2) are in accordance with existing data. Rood et al. reported a mean OSI trough concentration of 301.6 ± 164 ng/mL in 34 patients (2 technical replicates for each patient). Janssen et al. reported a mean plasma level of 331 ng/mL (range 123–798 ng/mL) in 10 samples from an unknown number of patients. As sampling in daily clinical routine was described, we assumed that the reported concentrations also were trough levels. In contrast, Reis et al. reported a slightly lower mean trough concentration of 194 ± 88 ng/mL in 15 samples obtained from an unknown number of patients. Given the broad range of trough levels in patient 2 (61–1030 ng/mL) and the significant standard deviation reported in ours, as well as other published studies, a high degree of inter- and intraindividual variability in drug exposure can be presumed, possibly promoting treatment failure or adverse events. As both compounds are irreversible inhibitors of the EGFR, area under the curve (AUC) might be a more promising target compared to trough plasma concentrations.
One of the main advantages of our method is the simple and time-efficient sample preparation as it is developed to be used in daily routine. Existing methods use solid-phase extraction , liquid-liquid extraction , or a mixture of both , which are more time-consuming. Protein precipitation resulted in high analyte recovery, a short turnaround time, and was devoid of strong matrix effects.
Since all analytes are organic bases, weak retention and poor peak shape (excessive tailing) were the results of using formic acid, a common modifier in positive ESI, often combined with reversed phase chromatography. In contrast, a basic pH was chosen to shift the equilibrium towards the free base in order to increase analyte retention on reversed phase material. Excellent peak shape (with regard to symmetry and width) and sufficient retention were achieved using ammonium hydrogen carbonate as mobile phase additive together with a phenyl-hexyl–modified stationary phase with extended pH stability at the alkaline condition. The phenyl-hexyl modification was chosen to exploit planar π-π interactions for increased selectivity (Fig. ). Gradient elution enabled the separation of all analytes from hydrophilic matrix compounds, while shortening retention time (elution within 3 min). In order to avoid a gradient in ionic strength, mobile phase A and B both contained ammonium hydrogen carbonate at 10 mM. Methanol was chosen as organic modifier due to the fact that ACN might undergo hydrolysis at alkaline pH (hydrolysis of nitriles to the ammonium salt of a carboxylic acid). Despite of the basic conditions, [M+H] + ions were generated efficiently (comparable to using formic acid) in ESI-positive mode, due to the weakly acidic character of the hydrogen carbonate ion. However, using an alkaline mobile phase which contains hydrogen carbonate ions impedes changing to an acidic mobile phase on the same LC system as additional washing steps are required to prevent formation of carbon dioxide in the system. In comparison to existing methods which use acetic acid or formic acid , this can be seen as a limitation of the method’s feasibility. Operating the column temperature at elevated temperature was not required, ensuring column stability despite high throughput and alkaline conditions, which usually tend to shorten the lifetime of chromatographic columns. Even after more than 1500 injections on the same column, retention time and analytical results remained stable. The high analyte retention, as a result of the chosen mobile phase composition, pH, and stationary phase, enabled using the diverter valve to separate early eluting matrix components to waste, leading to effective reduction of ion suppression, ensuring reproducible and accurate analytical results. Moreover, effects of ion suppression were minimized by using isotopically labeled IS for every analyte. However, mobile phases containing ammonium hydrogen carbonate were not stable for a prolonged period of time and had to be discarded after 1 week at the latest. During method development, the starting composition of the mobile phase for the first 0.5 min and after 5.0 min had to be adjusted to 60% because of major carryover of OSI and AXI (method development started with 40%). Carryover was assumed to be caused by OSI or AXI residues in either autosampler or MS detector valves. Hence, prior to injection, the autosampler loop was switched from inline to waste and the higher strength of the mobile phase helped removing remaining residues. Same applied to remaining residues in the MS valves. Therefore, carryover could be reduced. Furthermore, different needle wash solutions such as 90% methanol, 90% isopropanol, and a mixture of formic acid in methanol/water (0.1:50:49.9, v/v) were tested to reduce carryover. Using 90% acetonitrile minimized carryover significantly, but due to the hydrophobic character of the compounds, carryover could not be avoided completely. Dilution integrity was tested using blank human plasma but also saline solution, since it is easily accessible in daily clinical routine. The saline solution was able to substitute blank human plasma, making it an ethically interesting alternative for this purpose.
To investigate recovery, samples were first prepared in water as matrix. However, solubility issues in water made it impossible to prepare matrix-free samples by serial dilution. ACN was tested as matrix instead and showed reproducible results.
Light sensitivity of DAB in organic solvents and plasma and light sensitivity of AXI were reported in the literature. Investigating light exposure effects during our validation showed that DAB in plasma did not show any significant changes in concentrations, whereas concentrations of AXI in plasma decreased by about 7.0% in samples after light exposure (Table ). While Nijenhuis et al. report the instability of DAB in plasma at ambient temperature for 24 h, Herbrinks et al. come to the same conclusion as we did and report stability for both compounds in plasma at room temperature for 48 h. Nijenhuis et al. concluded that DAB in plasma was only stable for a period of 6 h kept at room temperature. Unfortunately, exact conditions for their stress test (e.g., material of container and anticoagulant) were not reported impeding the direct comparison. Sparidans et al. in addition confirm our findings on the stability of AXI in plasma at ambient temperature for 24 h while protected from daylight. Our method successfully separated the cis-/trans isomers of AXI enabling a differentiated quantification by consideration of the photolytic degradation product. Testing stability at 56 °C showed that at this condition, all KI, but OSI, are stable. Therefore, virus inactivation by heat (e.g., hepatitis c virus or human immunodeficiency virus and other enveloped viruses, such as SARS-CoV-2) of probably infectious or infectious samples could be performed. Stock solution stability results show that AFA in DMSO is not stable for 4 months at − 80 °C (82.8% ± 1.23, CV 1.49%). Therefore, we recommend to either prepare stock solutions freshly or investigate further stability conditions either in other solvents or for a shorter period of time. Stability investigations showed that AFA and OSI did not meet the acceptance criteria being kept at room temperature for more than 24 h, at 4 °C for 6 weeks and during incubation at 56 °C. These findings are in accordance with the fact that both compounds bind covalently to plasma proteins and other nucleophiles . The same conclusions were drawn by Veermann et al. and Rood et al. when they tested the stability of OSI at ambient temperature for 3 and 4 h, respectively; Veermann and colleagues suggested working on ice (+ 4 °C). As this was considered inconvenient for daily routine measurements, we chose a different approach. Working quickly through the simple sample preparation while storing and thawing samples at 4 °C produced acceptable results for accuracy and precision. However, we observed that freshly prepared calibration standards and QC samples in human matrix prior to analysis showed a better accuracy for OSI samples. Post-preparative stability investigations of OSI samples indicate that degradation and binding to matrix constituents is compensated by the IS. It is therefore crucial to use the corresponding isotope-labeled compounds for such analytes. For other analytes, CR and QC samples could be prepared in bulk and stored at − 80 °C for up to 3 months until analysis. Rood et al. reported non-linearity for OSI in their method. Having the same difficulties with assay reproducibility and accuracy, we changed the CR and QC preparation technique for OSI. Solubility issues combined with serial dilution in matrix seemed to have an effect on accuracy and precision between and within runs. Preparing the QC and CR samples differently solved the initial problems. The samples were serially diluted in organic solvent at a concentration 20 times higher compared to the intended plasma level. Each level was then individually prepared by dilution with blank matrix (1:20). Reproducibility of this assay is underlined by the ISR results.
We were able to demonstrate that our assay can easily be applied in a routine clinical setting. In contrast to reported AFA levels by Wind et al. and Merienne et al. (vide infra), the analysis of three AFA samples from our patient during a year revealed low concentrations of 11.5 ng/mL ± 4.6 ng/mL, despite the fact that samples where obtained close to expected time to maximum concentration (t max ) (4–5 h post dose). In PK investigations performed in 15 patients by Wind et al., the mean maximum concentration in steady state (C max,ss ) at once daily oral administration of 20 mg AFA was 24.5 ng/mL (geometric CV 88.5%) . Reis et al. measured 15 AFA trough concentrations in steady state (C ss,min ) originating from an unknown number of patients and reported a mean concentration of 23.0 ± 14.4 ng/mL for a daily dose of 40 mg . Merienne et al. analyzed eight trough levels in six patients receiving 40 mg/day and reported a median of 16.7 ng/mL (range 5–38.8 ng/mL) and proposed a minimum concentration (C ss,min ) of > 10–55 ng/mL as PK target . Considering that our patient was treated with 20 mg daily and given the broad range of concentrations in the above-named studies, our results seem plausible. In our patient, dose reduction from initially 40 to 30 mg and later 20 mg was performed due to severe cutaneous adverse events. On a 20 mg daily schedule, only mild adverse events remained, while the oncological situation is still stable (32 months after initiation of therapy), suggesting that lower concentrations might be necessary to control adverse events while still preventing tumor progression. This is in accordance with the findings of a retrospective study in 254 patients conducted by Lim et al. which demonstrated that a daily dose of 20 mg did not result in lower progression-free survival (PFS) in comparison to daily doses up to 40 mg, whereas significant differences in PFS could be seen when dosing regimens < 20 mg/day were applied . Our results of quantification of OSI trough concentrations in clinical routine samples of 433 ng/mL ± 131 ng/mL (patient 1) and 278 ± 264 ng/mL (patient 2) are in accordance with existing data. Rood et al. reported a mean OSI trough concentration of 301.6 ± 164 ng/mL in 34 patients (2 technical replicates for each patient). Janssen et al. reported a mean plasma level of 331 ng/mL (range 123–798 ng/mL) in 10 samples from an unknown number of patients. As sampling in daily clinical routine was described, we assumed that the reported concentrations also were trough levels. In contrast, Reis et al. reported a slightly lower mean trough concentration of 194 ± 88 ng/mL in 15 samples obtained from an unknown number of patients. Given the broad range of trough levels in patient 2 (61–1030 ng/mL) and the significant standard deviation reported in ours, as well as other published studies, a high degree of inter- and intraindividual variability in drug exposure can be presumed, possibly promoting treatment failure or adverse events. As both compounds are irreversible inhibitors of the EGFR, area under the curve (AUC) might be a more promising target compared to trough plasma concentrations.
The developed state-of-the art LC-MS/MS method is a sensitive and rapid method for the quantification of AFA, AXI, BOS, CAB, DAB, LEN, NIL, OSI, RUX, and TRA in human serum and plasma. The application of stable isotope-labeled internal standards has proven necessary to yield acceptable stability, especially for the irreversible inhibitors AFA and OSI. We can also demonstrate that either plasma or serum can be used for the determination of drug concentrations. However, plasma should be the preferred species for the determination of osimertinib and afatinib due to the pronounced instability at ambient temperature. Samples containing these analytes should be kept at 2–8 °C immediately after sample collection and frozen as quickly as possible. Since heat inactivation of the samples was possible in the case of ruxolitinib, the method could also be used for the monitoring of patients treated with this KI for tocilizumab-refractory severe COVID-19. Our method provides the basis for further investigation on correlation between drug exposure, AE, efficiency, and other patient characteristics. Additional data from clinical routine settings need to be collected to define PK targets for an effective and safe treatment with KI.
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Robotics and cybersurgery in ophthalmology: a current perspective | 2ec437d2-b515-453f-80da-6c515d87e550 | 9838251 | Ophthalmology[mh] | Ophthalmology is a field with rapid progression. This field includes medical and surgical specialties with distinct demands. Ocular procedures can be divided into an extraocular, intraocular anterior segment, or intraocular posterior segment surgery. Surgical microscopes are needed in intraocular surgeries. In addition, ocular surgery necessitates visualization systems and specific parameters, which make integrating robotics in ocular surgery difficult. Artificial intelligence (A.I.) has emerged recently in medical and surgical fields. Ophthalmology is one of the most enriched fields that allowed the A.I. domain to be part of its point of interest in scientific research . Many applications with the aid of A.I. helped diagnose many pathologies through image recognition and deep learning (DL)1. A.I., Machine Learning (ML), and DL have been used in an ophthalmic setting to validate the diagnosis of diseases, read images, and perform corneal topographic mapping and intraocular lens calculations. Diabetic retinopathy (D.R.), age-related macular degeneration (AMD), and glaucoma are the three most common causes of irreversible blindness on a global scale . COVID-19 has affected healthcare systems. A.I. applications have emerged in ophthalmology and will be used more in clinical research, education, and patient healthcare . When it comes to A.I., the surgical field in ophthalmology is in its infancy. Ophthalmic surgery requires high precision and high degrees of magnification. Surgical microscopes are the main tools used. Assistance facilitated by surgical robots improves movement control, cancels tremors, and enhances visualization and distance sensing. Robotic technology is only in its initial stages in ocular surgery . Cybersurgery, also referred to as Telesurgery, is most commonly defined as a surgical technique that allows a surgeon to operate on a patient remotely, either from a different location or nearby, through a telecommunications channel attached to a robotic operating machine . This technology not only benefits the shortage of surgeons and the sanitary crisis of COVID-19, but it also eliminates geographical barriers that prevent timely and high-quality surgical intervention, financial burden, complications, and often risky long-distance travel. This study aimed to focus on the current perspectives on the development of Robotic and Cybersurgery in Ophthalmology, evolution, innovation, and reasons for the delay.
A review of the literature with the aid of Google Scholar, Pubmed, CINAHL, MEDLINE (N.H.S. Evidence), Cochrane, AMed, EMBASE, PsychINFO, SCOPUS, and Web of Science was performed to gather information from articles. Keywords used: Cybersurgery, Telesurgery, ophthalmology robotics, Da Vinci robotic system, artificial intelligence in ophthalmology, training on robotic surgery, ethics of the use of robots in medicine, legal aspects, and economics of cybersurgery and robotics. 150 abstracts were reviewed for inclusion, and 68 articles focusing on ophthalmology were included for full-text review. (Flowchart Fig. ). Inclusion criteria Articles or case series containing the application of robotic surgery with a particular focus on using robotics in ophthalmology, cybersurgery, Telesurgery, and ethical and legal aspects of their service were included. Exclusion criteria Inaccessible articles or articles published in bulletins without an impact factor were excluded. Ethical approval The study is conducted according to the French data protection law. No submission to IRB/ethical committee was needed. The study adheres to the tenets of the Declaration of Helsinki.
Articles or case series containing the application of robotic surgery with a particular focus on using robotics in ophthalmology, cybersurgery, Telesurgery, and ethical and legal aspects of their service were included.
Inaccessible articles or articles published in bulletins without an impact factor were excluded.
The study is conducted according to the French data protection law. No submission to IRB/ethical committee was needed. The study adheres to the tenets of the Declaration of Helsinki.
Robotic surgery in ophthalmology Robotics history The term robotics derives from “robota” Czech word meaning “servant” or “worker” . It is known that the word was coined by Karel Capek in the theatrical spectacle R.U.R. (Rossum’s Universal Robots). However, the term was popularized only years later, through the works of Russian Isaac Asimov, responsible for making the “Three Laws of Robotics” , which, in fiction, standardize the robot’s behavior . The application of robots started in the industry by replacing workers in dangerous functions, such as car assembly lines, to prevent injuries . The use of robots in surgeries could help improve the gesture of tasks, decrease tremors, better visualization, and distance control. Robotics has been used in different medical fields for more than 20 years and assisted physicians in surgical rooms. The first robotic surgery was conducted in 1985 with the help of a robotic arm called Puma 560, which was used for non-laparoscopic neurosurgical biopsies . The first robot, Probot, was designed primarily to aid the medical team in the transurethral resection of the prostate in 1991 . In 1992, the U.S. Food and Drug Administration (F.D.A.) approved the first medical use of a robot . Uses in surgical fields Recent publications proved superior functional outcomes with equal oncologic safety compared to conventional open surgery. Its field of application may extend to nasopharynx and skull base surgery. The preliminary results encourage the role of trans-oral robotic surgery in head and neck cancer . Other surgical fields use robotic surgery for minimally invasive surgery, such as cardiac, digestive, gynecology, plastic reconstructive surgery, throat surgery, neurosurgery, vascular surgery, hand surgery, and peripheral nerve surgery . Role of robotic surgery in ophthalmology Analysis of previous ocular robotic assisted surgery studies summerized in (Table ). Definition of main ocular surgical procedures : Phacoemulsification: Removal of the intraocular lens with an ultrasound machine and a manual arm. Keratoplasty: Performing corneal grafts with donor corneas to be sutured or implemented to a host recipient. Vitrectomy: The procedure of removing the vitreous from the posterior chamber of the eye just before the retina using instruments called vitrectomy attached to specified machines. Intravitreal injection: The instillation of drugs in the intravitreal cavity using needles/syringes. Ocular microsurgery was successfully performed using the Da Vinci surgical robot in the porcine model. The robotic system provided excellent visualization and controlled and delicate placement of the sutures at corneal level . Back in 2009, Bourges et al. performed Robot-assisted Penetrating Keratoplasty . Three arms of the Da Vinci surgical robot were loaded with a dual-channel video and two 360°-rotating, 8 mm, wrested-end effector instruments and placed over porcine eyes or a human cadaver head. Trephination of corneal grafts, cardinal sutures, continuous 10.0 nylon sutures and adjustments on both eyes were performed remotely on both porcine and human eyes facilitated by the wrested-end forceps. No limitation of surgical motion was noted . Micro-hands of 4 mm in length were developed pneumatically with microelectromechanical systems (MEMS) technology to mimic a human hand for small object manipulation needed in retinal manipulation . Robotically assisted pterygium surgeries in non-living biological pterygium models were performed using the DaVinci Si H.D. robotic surgical system. Twelve models were prepared, and 12 pterygium excision and conjunctival autografts were performed . Robot-assisted Penetrating Keratoplasty was also successfully performed on human donor 12 corneas with low endothelial cell count mounter on the artificial anterior chamber. The mean duration of the procedures was 43.4 6 8.9 min (range 28.5–61.1 min). There were no unexpected intraoperative events . Amniotic membrane transplantation on corneal pathologies including (Neurotrophic keratitis, graft failure and post-radiation keratoconjunctivitis sicca) has been successfully performed on three human patients . Robot-assisted cataract phacoemulsification surgery was successfully performed on 25 lens nuclei with a mean operative time of 26.44 min ± 5.15 (S.D.). Intraocular dexterity and operative field visualization are necessary for achieving the main steps of the phacoemulsification procedure . There are current uses and developments of cataract surgeries aided by Femtosecond lasers. It is a partially performed cataract operation where many steps of the procedure are done in another setting. The rest is left for the surgeon’s intervention. Femtosecond lasers are used in corneal and almost all types of refractive surgery, such as laser in situ keratomileusis (LASIK), small incision lenticule extraction (SMILE), penetrating keratoplasty (P.K.P.), insertion of intra-corneal ring segments, anterior and posterior lamellar keratoplasty Deep anterior lamellar keratoplasty (DALK), and Descemet's stripping endothelial Keratoplasty (DSEK). In addition, femtosecond lasers provide more accurate and safe procedures . Robot-assisted strabismus procedures were successfully performed on six eyes. The feasibility of robot-assisted simulated strabismus surgery is confirmed . Classic microsurgery of the eye is performed using an operating microscope. The structures of the eye anterior to the vitreous are operated on under direct vision, whereas posterior regions, such as the retina and vitreous, use a specialized lens and viewing systems. Robotic-assisted uses in the posterior region of the Retina and Vitreous include Retinal surgery, Gene therapy, Retinal implantation, drug therapy, Retinal Vein Cannulation and intravitreal injections . Using devices designed by PRECEYES, a Dutch medical robotics firm, the procedure involved removing a membrane from the back of the eye. Successful human intraocular surgery performed using the Preceyes surgical system . Apart from Preceyes’ B.V. research platform, none of the currently eye-specific systems has reached a commercial stage . The robotic system was used to carry out micro-cannulation experiments on a pig’s eye. As a result, a surgeon was able to perform micro-cannulation successfully. The Gamma Knife, designed by Lars Leksell in the early 1950s gave rise to a new discipline of medicine-stereotactic radiosurgery. The gamma-ray beam concentration can be used to treat uveal melanoma, choroidal hemangioma, orbital tumors or even choroidal neovascularization . Robotics types used in ophthalmology The robotic Da Vinci Surgical System (Intuitive Surgical Inc., Sunnyvale, CA) and the ARES (Auris Surgical Endoscopy System) robot (Auris Surgical Robotics, San Carlos, CA) are the only two surgical robots approved by the U.S. Food and Drug Administration for human surgery not being specifically designed for microsurgical specialties such as ocular surgery. 1. The Da Vinci Robot (Fig. ): It is the most widespread platform used in human surgery. Since 2000, the indications for operations assisted by robotics systems are emerging progressively. They rose from 1500 in 2000 to more than 20,000 in 2004 . It includes three‐dimensional stereoscopic vision with three robotic slave arms that can be equipped with instruments with 7 degrees of freedom and wrist‐like motions. Four models have been launched since they received U.S. Food and Drug Administration approval in 2000: S, Si, Si H.D., and Xi. Surgeons can control the tools and camera from a remote workstation. However, limitations have been documented including the artificial wrist movements that differ from the human range of motion and endoscopic vision, as a result, difficulties in performing microsurgical steps such as sclerotomies could be encountered . 2. Intraocular robotic interventional surgical system (IRISS) : This ophthalmic platform was proposed by Jules Stein Eye Institute and the UCLA Department of Mechanical and Aerospace Engineering. It is composed of master controller with two joysticks and a slave manipulator. The manipulator has two independent arms that each hold surgical instruments. The arms have an independent pivot point and 7 degrees of freedom necessary for surgical maneuvers. This system has been used in anterior and posterior ocular procedures, such as capsulorhexis, lens cortex removal, core vitrectomies, and retinal vein micro-cannulation in porcine eyes . 3. Johns Hopkins steady-hand eye robot (Fig. ): This robot is designed to share the control of surgical instruments, mainly during posterior segment surgeries. It consists of three major components: the X.Y.Z. system, the rolling mechanism, and the tilt mechanism. The X.Y.Z. system allows movement of the surgical tool in all directions. The roll mechanism consists of a rotating table designed to optimize the access of the surgical device to the patient’s eye. The tilt mechanism is attached to the tool holder at one end and the rolling mechanism at the other, allowing the instrument to be at any angle. As a result, the robot improves the effectiveness of each movement. This instrument can be used free-hand or incorporated into the Steady-Hand-Eye robot . The latest model has improved the range of motion, stiffness, and speed of holder release. 4. “Smart” instruments : Additional systems and “smart” instruments have been developed to improve technical performance . For example, sensors detect the force applied to the eye; this could be transferred directly to the surgeon via an auditory feedback system . In addition, they can notice tactile sensations lower than the human threshold, which could minimize the risk of possible surgical complications. Retinal membrane removal was successfully performed with the aid of PRECEYES surgical system robotic assistant, which serves as an instrument holder for over six patients as part of a trial at Oxford's John Radcliffe hospital . The median time was longer (four minutes and 55 s) than the traditional method (one minute and 20 s) . Assistive devices for Intravitreal Injection have been demonstrated through ex vivo experiments with porcine eyes. It used an automatic fine positioning and intravitreal injection through the pars plana. In addition, several safety features, such as continuous eye-tracking and iris recognition, have been implemented . Prototypes development and innovation in ophthalmology Guerrouad and Vidal in 1989 created a robotic ocular system composed of a Stereotaxical Microtelemanipulator (SMOS), a spherical micromanipulator mounted on an x, y, and z stage, which allowed 6 degrees of freedom. No development was made after this stage. Robot-Assisted Microsurgery (RAMS) tele-robotic platform emerged in 1997 (Charles S 1997). It comprises a slave robot arm (2.5 cm in diameter and 25 cm long) and a primary device supported by cables and encoders facilitating the operator's arm movement guided by computers . In the same year, another prototype used the Stewart-based platform (Jensen, Grace et al. 1997). This was developed to measure intraluminal (20–130 microns) retinal vessel pressure and to extract blood from these vessels for research purposes. In 2009, Ueta et al. developed a newer prototype with more accuracy adapted to assist in vitreoretinal surgeries. Cybersurgery History and background Electrocardiogram was first introduced in 1906, the first step in telemedicine. Cybersurgery, also referred to as Telesurgery, from the Greek tele, “far off”, also called “remote surgery”, is defined as a surgical technique which allows for a surgeon to operate on a patient remotely, either from a different location or at proximity, through a telecommunications channel attached to a robotic operating machine. Tele-surgery is a surgical system that utilizes wireless networking and robotic technology to connect surgeons and patients distantly. It can be divided into three main components: Telesurgery, telementoring, and teleconsultation . The telerobotic Zeus and Da Vinci surgical systems allow surgeons to operate remotely. These telerobots hold the camera, replace the surgeon’s two hands with robotic instruments, and serve in a master–slave relationship for the surgeon. They are characterized by their capabilities to simulate the motions of the surgeon’s wrist and different surgeon positions . In 1988: Minimally invasive surgery enabled surgical procedures to be guided by introducing a camera without requiring an opening of the abdomen or thorax. In 1996: Computer-assisted surgery was introduced, which enabled to transmit surgeons’ actions remotely to manipulation devices. September 7, 2001: Telesurgery: The world's first Telesurgery was performed by a surgical team in New York, U.S.A. using the ZEUS robotic system (Intuitive Surgical, Sunnyvale, CA, U.S.A.). This project produced a successful two-hour-long laparoscopic cholecystectomy performed on a female patient at a hospital in Strasbourg, France . The patient had an uneventful recovery . In 2003, a surgical system was set up in Canada between two hospitals 400 kms away . Robotics could be helpful in surgical tele-mentoring by expert surgeons to supervise younger surgeons remotely, given its endoscopic optics and mechanized movement. However, the maturity of these modalities depends on financial factors, legislation and collaboration with cybersecurity experts to ensure safety and cost-effectiveness . Current applications of cybersurgery Current applications of cybersurgery include tele-education, tele-training, telementoring, tele-proctoring, and tele-accreditation. Different projects have been developed; different site videoconferences used images and data transmission at the European Institute of TeleSurgery of Strasbourg via The TESUS project through the realization of international multi-site video conferences between surgeons. The WEBSurg project created the first virtual university by placing surgical techniques at the surgeon’s disposal through the Internet. It is a comprehensive source of knowledge in minimally invasive surgery. It promotes technological advances in its fields, such as general and digestive surgery, urology, gynecology, pediatric surgery, endoscopic surgery, skull base surgery, arthroscopy, and upper limb surgery . The HESSOS project (Hepatic Surgery Simulation and Operative Strategy) uses virtual reality as a surgical simulation system, allowing the development of the concept of distant tele-manipulation. It serves as an operative system available for clinical application in liver surgery. In addition, it allows worldwide surgical teaching . Tele-cystoscopy was tested suitable for diagnosis. The trade-offs between cost and tele-cystoscopy system component quality were compared with efficiency frontiers to elucidate the optimal system . Tele-oncology covers diagnosis, treatment, supportive care of cancers, education, and medical training. Modern strategies were addressed to ensure global access to essential cancer care services (Telemedicine and Telesurgery in Cancer Care (TTCC) conference) . To overcome the shortage of surgeons, the “Virtual Interactive Presence” (V.I.P.) platform allows remote participants to simultaneously view each other’s visual field, creating a shared field of view for real-time surgical tele-collaboration . Video analysis yielded a mean compositing delay of 760 ± 606 ms (when compared with the audio signal). Image resolution adequately visualizes neurosurgery’s complex intracranial anatomy and provides interactive guidance . Based on preclinical work, trans-oral robotic surgery (TORS) was performed in February 2007 on a patient with a para-pharyngeal to infratemporal fossa cystic neoplasm as part of a large prospective human trial. The robotic procedure allowed adequate and safe identification of the internal carotid artery and cranial nerves, and excellent hemostasis was achieved with no complications during or after surgery . Later, the Telelap Alf-x, telesurgical system was introduced. It composed of individual arms, which enabled free access to the patient throughout surgery, an extensive range of reusable surgical instruments, an open console with an eye-tracking system, where the camera followed the eye and head movements of the surgeon. The existing force feedback enables for the first time to feel the consistency of the tissues and avoid tearing the stitches while suturing. The system combines the benefits of open surgery and endoscopy . The first clinical application, which involved 146 operations at the gynecological department of the Gemelli University Hospital in Rome, proved the safety and the surgical team’s quick adaptation to the system . In 1992, the National Aeronautics and Space Administration and the Department of Defense supported Telesurgery to rescue wounded soldiers. The Defense Advanced Research Projects Agency invested in tele-medical technologies to help operate injured soldiers remotely . Applications of cybersurgery or telesurgery in ophthalmology There is no current application of Cybersurgery or Telesurgery in Ophthalmology. However, the feasibility of telerobotic microsurgical repair of corneal lacerations has been evaluated . Five mm central full-thickness corneal wounds were fashioned in five enucleated rabbit eyes and repaired remotely using the telerobotic system . The feasibility of using the Robotic Slave Micromanipulator Unit (RSMU) in photocoagulating the ciliary body remotely to treat glaucoma with the diode laser was tested in fresh un-operated, enucleated human eyes. Histology examination of remote robotic contact trans-scleral cyclophotocoagulation and “by hand” technique produced similar degrees of ciliary body tissue disruption . Various projects have recently been launched at academic and corporate levels to develop lightweight, miniaturized surgical robotic prototypes . This delay could be explained by the delicacy of this field which deals with the sense of vision and the small anatomical size. Advanced virtualization and augmented-reality techniques should help human operators to adapt better to special conditions . To meet safety standards and requirements in space, a three-layered architecture is recommended to provide the highest quality of telepresence technically achievable for provisional exploration missions .
Robotics history The term robotics derives from “robota” Czech word meaning “servant” or “worker” . It is known that the word was coined by Karel Capek in the theatrical spectacle R.U.R. (Rossum’s Universal Robots). However, the term was popularized only years later, through the works of Russian Isaac Asimov, responsible for making the “Three Laws of Robotics” , which, in fiction, standardize the robot’s behavior . The application of robots started in the industry by replacing workers in dangerous functions, such as car assembly lines, to prevent injuries . The use of robots in surgeries could help improve the gesture of tasks, decrease tremors, better visualization, and distance control. Robotics has been used in different medical fields for more than 20 years and assisted physicians in surgical rooms. The first robotic surgery was conducted in 1985 with the help of a robotic arm called Puma 560, which was used for non-laparoscopic neurosurgical biopsies . The first robot, Probot, was designed primarily to aid the medical team in the transurethral resection of the prostate in 1991 . In 1992, the U.S. Food and Drug Administration (F.D.A.) approved the first medical use of a robot . Uses in surgical fields Recent publications proved superior functional outcomes with equal oncologic safety compared to conventional open surgery. Its field of application may extend to nasopharynx and skull base surgery. The preliminary results encourage the role of trans-oral robotic surgery in head and neck cancer . Other surgical fields use robotic surgery for minimally invasive surgery, such as cardiac, digestive, gynecology, plastic reconstructive surgery, throat surgery, neurosurgery, vascular surgery, hand surgery, and peripheral nerve surgery . Role of robotic surgery in ophthalmology Analysis of previous ocular robotic assisted surgery studies summerized in (Table ). Definition of main ocular surgical procedures : Phacoemulsification: Removal of the intraocular lens with an ultrasound machine and a manual arm. Keratoplasty: Performing corneal grafts with donor corneas to be sutured or implemented to a host recipient. Vitrectomy: The procedure of removing the vitreous from the posterior chamber of the eye just before the retina using instruments called vitrectomy attached to specified machines. Intravitreal injection: The instillation of drugs in the intravitreal cavity using needles/syringes. Ocular microsurgery was successfully performed using the Da Vinci surgical robot in the porcine model. The robotic system provided excellent visualization and controlled and delicate placement of the sutures at corneal level . Back in 2009, Bourges et al. performed Robot-assisted Penetrating Keratoplasty . Three arms of the Da Vinci surgical robot were loaded with a dual-channel video and two 360°-rotating, 8 mm, wrested-end effector instruments and placed over porcine eyes or a human cadaver head. Trephination of corneal grafts, cardinal sutures, continuous 10.0 nylon sutures and adjustments on both eyes were performed remotely on both porcine and human eyes facilitated by the wrested-end forceps. No limitation of surgical motion was noted . Micro-hands of 4 mm in length were developed pneumatically with microelectromechanical systems (MEMS) technology to mimic a human hand for small object manipulation needed in retinal manipulation . Robotically assisted pterygium surgeries in non-living biological pterygium models were performed using the DaVinci Si H.D. robotic surgical system. Twelve models were prepared, and 12 pterygium excision and conjunctival autografts were performed . Robot-assisted Penetrating Keratoplasty was also successfully performed on human donor 12 corneas with low endothelial cell count mounter on the artificial anterior chamber. The mean duration of the procedures was 43.4 6 8.9 min (range 28.5–61.1 min). There were no unexpected intraoperative events . Amniotic membrane transplantation on corneal pathologies including (Neurotrophic keratitis, graft failure and post-radiation keratoconjunctivitis sicca) has been successfully performed on three human patients . Robot-assisted cataract phacoemulsification surgery was successfully performed on 25 lens nuclei with a mean operative time of 26.44 min ± 5.15 (S.D.). Intraocular dexterity and operative field visualization are necessary for achieving the main steps of the phacoemulsification procedure . There are current uses and developments of cataract surgeries aided by Femtosecond lasers. It is a partially performed cataract operation where many steps of the procedure are done in another setting. The rest is left for the surgeon’s intervention. Femtosecond lasers are used in corneal and almost all types of refractive surgery, such as laser in situ keratomileusis (LASIK), small incision lenticule extraction (SMILE), penetrating keratoplasty (P.K.P.), insertion of intra-corneal ring segments, anterior and posterior lamellar keratoplasty Deep anterior lamellar keratoplasty (DALK), and Descemet's stripping endothelial Keratoplasty (DSEK). In addition, femtosecond lasers provide more accurate and safe procedures . Robot-assisted strabismus procedures were successfully performed on six eyes. The feasibility of robot-assisted simulated strabismus surgery is confirmed . Classic microsurgery of the eye is performed using an operating microscope. The structures of the eye anterior to the vitreous are operated on under direct vision, whereas posterior regions, such as the retina and vitreous, use a specialized lens and viewing systems. Robotic-assisted uses in the posterior region of the Retina and Vitreous include Retinal surgery, Gene therapy, Retinal implantation, drug therapy, Retinal Vein Cannulation and intravitreal injections . Using devices designed by PRECEYES, a Dutch medical robotics firm, the procedure involved removing a membrane from the back of the eye. Successful human intraocular surgery performed using the Preceyes surgical system . Apart from Preceyes’ B.V. research platform, none of the currently eye-specific systems has reached a commercial stage . The robotic system was used to carry out micro-cannulation experiments on a pig’s eye. As a result, a surgeon was able to perform micro-cannulation successfully. The Gamma Knife, designed by Lars Leksell in the early 1950s gave rise to a new discipline of medicine-stereotactic radiosurgery. The gamma-ray beam concentration can be used to treat uveal melanoma, choroidal hemangioma, orbital tumors or even choroidal neovascularization . Robotics types used in ophthalmology The robotic Da Vinci Surgical System (Intuitive Surgical Inc., Sunnyvale, CA) and the ARES (Auris Surgical Endoscopy System) robot (Auris Surgical Robotics, San Carlos, CA) are the only two surgical robots approved by the U.S. Food and Drug Administration for human surgery not being specifically designed for microsurgical specialties such as ocular surgery. 1. The Da Vinci Robot (Fig. ): It is the most widespread platform used in human surgery. Since 2000, the indications for operations assisted by robotics systems are emerging progressively. They rose from 1500 in 2000 to more than 20,000 in 2004 . It includes three‐dimensional stereoscopic vision with three robotic slave arms that can be equipped with instruments with 7 degrees of freedom and wrist‐like motions. Four models have been launched since they received U.S. Food and Drug Administration approval in 2000: S, Si, Si H.D., and Xi. Surgeons can control the tools and camera from a remote workstation. However, limitations have been documented including the artificial wrist movements that differ from the human range of motion and endoscopic vision, as a result, difficulties in performing microsurgical steps such as sclerotomies could be encountered . 2. Intraocular robotic interventional surgical system (IRISS) : This ophthalmic platform was proposed by Jules Stein Eye Institute and the UCLA Department of Mechanical and Aerospace Engineering. It is composed of master controller with two joysticks and a slave manipulator. The manipulator has two independent arms that each hold surgical instruments. The arms have an independent pivot point and 7 degrees of freedom necessary for surgical maneuvers. This system has been used in anterior and posterior ocular procedures, such as capsulorhexis, lens cortex removal, core vitrectomies, and retinal vein micro-cannulation in porcine eyes . 3. Johns Hopkins steady-hand eye robot (Fig. ): This robot is designed to share the control of surgical instruments, mainly during posterior segment surgeries. It consists of three major components: the X.Y.Z. system, the rolling mechanism, and the tilt mechanism. The X.Y.Z. system allows movement of the surgical tool in all directions. The roll mechanism consists of a rotating table designed to optimize the access of the surgical device to the patient’s eye. The tilt mechanism is attached to the tool holder at one end and the rolling mechanism at the other, allowing the instrument to be at any angle. As a result, the robot improves the effectiveness of each movement. This instrument can be used free-hand or incorporated into the Steady-Hand-Eye robot . The latest model has improved the range of motion, stiffness, and speed of holder release. 4. “Smart” instruments : Additional systems and “smart” instruments have been developed to improve technical performance . For example, sensors detect the force applied to the eye; this could be transferred directly to the surgeon via an auditory feedback system . In addition, they can notice tactile sensations lower than the human threshold, which could minimize the risk of possible surgical complications. Retinal membrane removal was successfully performed with the aid of PRECEYES surgical system robotic assistant, which serves as an instrument holder for over six patients as part of a trial at Oxford's John Radcliffe hospital . The median time was longer (four minutes and 55 s) than the traditional method (one minute and 20 s) . Assistive devices for Intravitreal Injection have been demonstrated through ex vivo experiments with porcine eyes. It used an automatic fine positioning and intravitreal injection through the pars plana. In addition, several safety features, such as continuous eye-tracking and iris recognition, have been implemented . Prototypes development and innovation in ophthalmology Guerrouad and Vidal in 1989 created a robotic ocular system composed of a Stereotaxical Microtelemanipulator (SMOS), a spherical micromanipulator mounted on an x, y, and z stage, which allowed 6 degrees of freedom. No development was made after this stage. Robot-Assisted Microsurgery (RAMS) tele-robotic platform emerged in 1997 (Charles S 1997). It comprises a slave robot arm (2.5 cm in diameter and 25 cm long) and a primary device supported by cables and encoders facilitating the operator's arm movement guided by computers . In the same year, another prototype used the Stewart-based platform (Jensen, Grace et al. 1997). This was developed to measure intraluminal (20–130 microns) retinal vessel pressure and to extract blood from these vessels for research purposes. In 2009, Ueta et al. developed a newer prototype with more accuracy adapted to assist in vitreoretinal surgeries.
The term robotics derives from “robota” Czech word meaning “servant” or “worker” . It is known that the word was coined by Karel Capek in the theatrical spectacle R.U.R. (Rossum’s Universal Robots). However, the term was popularized only years later, through the works of Russian Isaac Asimov, responsible for making the “Three Laws of Robotics” , which, in fiction, standardize the robot’s behavior . The application of robots started in the industry by replacing workers in dangerous functions, such as car assembly lines, to prevent injuries . The use of robots in surgeries could help improve the gesture of tasks, decrease tremors, better visualization, and distance control. Robotics has been used in different medical fields for more than 20 years and assisted physicians in surgical rooms. The first robotic surgery was conducted in 1985 with the help of a robotic arm called Puma 560, which was used for non-laparoscopic neurosurgical biopsies . The first robot, Probot, was designed primarily to aid the medical team in the transurethral resection of the prostate in 1991 . In 1992, the U.S. Food and Drug Administration (F.D.A.) approved the first medical use of a robot . Uses in surgical fields Recent publications proved superior functional outcomes with equal oncologic safety compared to conventional open surgery. Its field of application may extend to nasopharynx and skull base surgery. The preliminary results encourage the role of trans-oral robotic surgery in head and neck cancer . Other surgical fields use robotic surgery for minimally invasive surgery, such as cardiac, digestive, gynecology, plastic reconstructive surgery, throat surgery, neurosurgery, vascular surgery, hand surgery, and peripheral nerve surgery . Role of robotic surgery in ophthalmology Analysis of previous ocular robotic assisted surgery studies summerized in (Table ). Definition of main ocular surgical procedures : Phacoemulsification: Removal of the intraocular lens with an ultrasound machine and a manual arm. Keratoplasty: Performing corneal grafts with donor corneas to be sutured or implemented to a host recipient. Vitrectomy: The procedure of removing the vitreous from the posterior chamber of the eye just before the retina using instruments called vitrectomy attached to specified machines. Intravitreal injection: The instillation of drugs in the intravitreal cavity using needles/syringes. Ocular microsurgery was successfully performed using the Da Vinci surgical robot in the porcine model. The robotic system provided excellent visualization and controlled and delicate placement of the sutures at corneal level . Back in 2009, Bourges et al. performed Robot-assisted Penetrating Keratoplasty . Three arms of the Da Vinci surgical robot were loaded with a dual-channel video and two 360°-rotating, 8 mm, wrested-end effector instruments and placed over porcine eyes or a human cadaver head. Trephination of corneal grafts, cardinal sutures, continuous 10.0 nylon sutures and adjustments on both eyes were performed remotely on both porcine and human eyes facilitated by the wrested-end forceps. No limitation of surgical motion was noted . Micro-hands of 4 mm in length were developed pneumatically with microelectromechanical systems (MEMS) technology to mimic a human hand for small object manipulation needed in retinal manipulation . Robotically assisted pterygium surgeries in non-living biological pterygium models were performed using the DaVinci Si H.D. robotic surgical system. Twelve models were prepared, and 12 pterygium excision and conjunctival autografts were performed . Robot-assisted Penetrating Keratoplasty was also successfully performed on human donor 12 corneas with low endothelial cell count mounter on the artificial anterior chamber. The mean duration of the procedures was 43.4 6 8.9 min (range 28.5–61.1 min). There were no unexpected intraoperative events . Amniotic membrane transplantation on corneal pathologies including (Neurotrophic keratitis, graft failure and post-radiation keratoconjunctivitis sicca) has been successfully performed on three human patients . Robot-assisted cataract phacoemulsification surgery was successfully performed on 25 lens nuclei with a mean operative time of 26.44 min ± 5.15 (S.D.). Intraocular dexterity and operative field visualization are necessary for achieving the main steps of the phacoemulsification procedure . There are current uses and developments of cataract surgeries aided by Femtosecond lasers. It is a partially performed cataract operation where many steps of the procedure are done in another setting. The rest is left for the surgeon’s intervention. Femtosecond lasers are used in corneal and almost all types of refractive surgery, such as laser in situ keratomileusis (LASIK), small incision lenticule extraction (SMILE), penetrating keratoplasty (P.K.P.), insertion of intra-corneal ring segments, anterior and posterior lamellar keratoplasty Deep anterior lamellar keratoplasty (DALK), and Descemet's stripping endothelial Keratoplasty (DSEK). In addition, femtosecond lasers provide more accurate and safe procedures . Robot-assisted strabismus procedures were successfully performed on six eyes. The feasibility of robot-assisted simulated strabismus surgery is confirmed . Classic microsurgery of the eye is performed using an operating microscope. The structures of the eye anterior to the vitreous are operated on under direct vision, whereas posterior regions, such as the retina and vitreous, use a specialized lens and viewing systems. Robotic-assisted uses in the posterior region of the Retina and Vitreous include Retinal surgery, Gene therapy, Retinal implantation, drug therapy, Retinal Vein Cannulation and intravitreal injections . Using devices designed by PRECEYES, a Dutch medical robotics firm, the procedure involved removing a membrane from the back of the eye. Successful human intraocular surgery performed using the Preceyes surgical system . Apart from Preceyes’ B.V. research platform, none of the currently eye-specific systems has reached a commercial stage . The robotic system was used to carry out micro-cannulation experiments on a pig’s eye. As a result, a surgeon was able to perform micro-cannulation successfully. The Gamma Knife, designed by Lars Leksell in the early 1950s gave rise to a new discipline of medicine-stereotactic radiosurgery. The gamma-ray beam concentration can be used to treat uveal melanoma, choroidal hemangioma, orbital tumors or even choroidal neovascularization . Robotics types used in ophthalmology The robotic Da Vinci Surgical System (Intuitive Surgical Inc., Sunnyvale, CA) and the ARES (Auris Surgical Endoscopy System) robot (Auris Surgical Robotics, San Carlos, CA) are the only two surgical robots approved by the U.S. Food and Drug Administration for human surgery not being specifically designed for microsurgical specialties such as ocular surgery. 1. The Da Vinci Robot (Fig. ): It is the most widespread platform used in human surgery. Since 2000, the indications for operations assisted by robotics systems are emerging progressively. They rose from 1500 in 2000 to more than 20,000 in 2004 . It includes three‐dimensional stereoscopic vision with three robotic slave arms that can be equipped with instruments with 7 degrees of freedom and wrist‐like motions. Four models have been launched since they received U.S. Food and Drug Administration approval in 2000: S, Si, Si H.D., and Xi. Surgeons can control the tools and camera from a remote workstation. However, limitations have been documented including the artificial wrist movements that differ from the human range of motion and endoscopic vision, as a result, difficulties in performing microsurgical steps such as sclerotomies could be encountered . 2. Intraocular robotic interventional surgical system (IRISS) : This ophthalmic platform was proposed by Jules Stein Eye Institute and the UCLA Department of Mechanical and Aerospace Engineering. It is composed of master controller with two joysticks and a slave manipulator. The manipulator has two independent arms that each hold surgical instruments. The arms have an independent pivot point and 7 degrees of freedom necessary for surgical maneuvers. This system has been used in anterior and posterior ocular procedures, such as capsulorhexis, lens cortex removal, core vitrectomies, and retinal vein micro-cannulation in porcine eyes . 3. Johns Hopkins steady-hand eye robot (Fig. ): This robot is designed to share the control of surgical instruments, mainly during posterior segment surgeries. It consists of three major components: the X.Y.Z. system, the rolling mechanism, and the tilt mechanism. The X.Y.Z. system allows movement of the surgical tool in all directions. The roll mechanism consists of a rotating table designed to optimize the access of the surgical device to the patient’s eye. The tilt mechanism is attached to the tool holder at one end and the rolling mechanism at the other, allowing the instrument to be at any angle. As a result, the robot improves the effectiveness of each movement. This instrument can be used free-hand or incorporated into the Steady-Hand-Eye robot . The latest model has improved the range of motion, stiffness, and speed of holder release. 4. “Smart” instruments : Additional systems and “smart” instruments have been developed to improve technical performance . For example, sensors detect the force applied to the eye; this could be transferred directly to the surgeon via an auditory feedback system . In addition, they can notice tactile sensations lower than the human threshold, which could minimize the risk of possible surgical complications. Retinal membrane removal was successfully performed with the aid of PRECEYES surgical system robotic assistant, which serves as an instrument holder for over six patients as part of a trial at Oxford's John Radcliffe hospital . The median time was longer (four minutes and 55 s) than the traditional method (one minute and 20 s) . Assistive devices for Intravitreal Injection have been demonstrated through ex vivo experiments with porcine eyes. It used an automatic fine positioning and intravitreal injection through the pars plana. In addition, several safety features, such as continuous eye-tracking and iris recognition, have been implemented . Prototypes development and innovation in ophthalmology Guerrouad and Vidal in 1989 created a robotic ocular system composed of a Stereotaxical Microtelemanipulator (SMOS), a spherical micromanipulator mounted on an x, y, and z stage, which allowed 6 degrees of freedom. No development was made after this stage. Robot-Assisted Microsurgery (RAMS) tele-robotic platform emerged in 1997 (Charles S 1997). It comprises a slave robot arm (2.5 cm in diameter and 25 cm long) and a primary device supported by cables and encoders facilitating the operator's arm movement guided by computers . In the same year, another prototype used the Stewart-based platform (Jensen, Grace et al. 1997). This was developed to measure intraluminal (20–130 microns) retinal vessel pressure and to extract blood from these vessels for research purposes. In 2009, Ueta et al. developed a newer prototype with more accuracy adapted to assist in vitreoretinal surgeries.
Recent publications proved superior functional outcomes with equal oncologic safety compared to conventional open surgery. Its field of application may extend to nasopharynx and skull base surgery. The preliminary results encourage the role of trans-oral robotic surgery in head and neck cancer . Other surgical fields use robotic surgery for minimally invasive surgery, such as cardiac, digestive, gynecology, plastic reconstructive surgery, throat surgery, neurosurgery, vascular surgery, hand surgery, and peripheral nerve surgery .
Analysis of previous ocular robotic assisted surgery studies summerized in (Table ). Definition of main ocular surgical procedures : Phacoemulsification: Removal of the intraocular lens with an ultrasound machine and a manual arm. Keratoplasty: Performing corneal grafts with donor corneas to be sutured or implemented to a host recipient. Vitrectomy: The procedure of removing the vitreous from the posterior chamber of the eye just before the retina using instruments called vitrectomy attached to specified machines. Intravitreal injection: The instillation of drugs in the intravitreal cavity using needles/syringes. Ocular microsurgery was successfully performed using the Da Vinci surgical robot in the porcine model. The robotic system provided excellent visualization and controlled and delicate placement of the sutures at corneal level . Back in 2009, Bourges et al. performed Robot-assisted Penetrating Keratoplasty . Three arms of the Da Vinci surgical robot were loaded with a dual-channel video and two 360°-rotating, 8 mm, wrested-end effector instruments and placed over porcine eyes or a human cadaver head. Trephination of corneal grafts, cardinal sutures, continuous 10.0 nylon sutures and adjustments on both eyes were performed remotely on both porcine and human eyes facilitated by the wrested-end forceps. No limitation of surgical motion was noted . Micro-hands of 4 mm in length were developed pneumatically with microelectromechanical systems (MEMS) technology to mimic a human hand for small object manipulation needed in retinal manipulation . Robotically assisted pterygium surgeries in non-living biological pterygium models were performed using the DaVinci Si H.D. robotic surgical system. Twelve models were prepared, and 12 pterygium excision and conjunctival autografts were performed . Robot-assisted Penetrating Keratoplasty was also successfully performed on human donor 12 corneas with low endothelial cell count mounter on the artificial anterior chamber. The mean duration of the procedures was 43.4 6 8.9 min (range 28.5–61.1 min). There were no unexpected intraoperative events . Amniotic membrane transplantation on corneal pathologies including (Neurotrophic keratitis, graft failure and post-radiation keratoconjunctivitis sicca) has been successfully performed on three human patients . Robot-assisted cataract phacoemulsification surgery was successfully performed on 25 lens nuclei with a mean operative time of 26.44 min ± 5.15 (S.D.). Intraocular dexterity and operative field visualization are necessary for achieving the main steps of the phacoemulsification procedure . There are current uses and developments of cataract surgeries aided by Femtosecond lasers. It is a partially performed cataract operation where many steps of the procedure are done in another setting. The rest is left for the surgeon’s intervention. Femtosecond lasers are used in corneal and almost all types of refractive surgery, such as laser in situ keratomileusis (LASIK), small incision lenticule extraction (SMILE), penetrating keratoplasty (P.K.P.), insertion of intra-corneal ring segments, anterior and posterior lamellar keratoplasty Deep anterior lamellar keratoplasty (DALK), and Descemet's stripping endothelial Keratoplasty (DSEK). In addition, femtosecond lasers provide more accurate and safe procedures . Robot-assisted strabismus procedures were successfully performed on six eyes. The feasibility of robot-assisted simulated strabismus surgery is confirmed . Classic microsurgery of the eye is performed using an operating microscope. The structures of the eye anterior to the vitreous are operated on under direct vision, whereas posterior regions, such as the retina and vitreous, use a specialized lens and viewing systems. Robotic-assisted uses in the posterior region of the Retina and Vitreous include Retinal surgery, Gene therapy, Retinal implantation, drug therapy, Retinal Vein Cannulation and intravitreal injections . Using devices designed by PRECEYES, a Dutch medical robotics firm, the procedure involved removing a membrane from the back of the eye. Successful human intraocular surgery performed using the Preceyes surgical system . Apart from Preceyes’ B.V. research platform, none of the currently eye-specific systems has reached a commercial stage . The robotic system was used to carry out micro-cannulation experiments on a pig’s eye. As a result, a surgeon was able to perform micro-cannulation successfully. The Gamma Knife, designed by Lars Leksell in the early 1950s gave rise to a new discipline of medicine-stereotactic radiosurgery. The gamma-ray beam concentration can be used to treat uveal melanoma, choroidal hemangioma, orbital tumors or even choroidal neovascularization .
The robotic Da Vinci Surgical System (Intuitive Surgical Inc., Sunnyvale, CA) and the ARES (Auris Surgical Endoscopy System) robot (Auris Surgical Robotics, San Carlos, CA) are the only two surgical robots approved by the U.S. Food and Drug Administration for human surgery not being specifically designed for microsurgical specialties such as ocular surgery. 1. The Da Vinci Robot (Fig. ): It is the most widespread platform used in human surgery. Since 2000, the indications for operations assisted by robotics systems are emerging progressively. They rose from 1500 in 2000 to more than 20,000 in 2004 . It includes three‐dimensional stereoscopic vision with three robotic slave arms that can be equipped with instruments with 7 degrees of freedom and wrist‐like motions. Four models have been launched since they received U.S. Food and Drug Administration approval in 2000: S, Si, Si H.D., and Xi. Surgeons can control the tools and camera from a remote workstation. However, limitations have been documented including the artificial wrist movements that differ from the human range of motion and endoscopic vision, as a result, difficulties in performing microsurgical steps such as sclerotomies could be encountered . 2. Intraocular robotic interventional surgical system (IRISS) : This ophthalmic platform was proposed by Jules Stein Eye Institute and the UCLA Department of Mechanical and Aerospace Engineering. It is composed of master controller with two joysticks and a slave manipulator. The manipulator has two independent arms that each hold surgical instruments. The arms have an independent pivot point and 7 degrees of freedom necessary for surgical maneuvers. This system has been used in anterior and posterior ocular procedures, such as capsulorhexis, lens cortex removal, core vitrectomies, and retinal vein micro-cannulation in porcine eyes . 3. Johns Hopkins steady-hand eye robot (Fig. ): This robot is designed to share the control of surgical instruments, mainly during posterior segment surgeries. It consists of three major components: the X.Y.Z. system, the rolling mechanism, and the tilt mechanism. The X.Y.Z. system allows movement of the surgical tool in all directions. The roll mechanism consists of a rotating table designed to optimize the access of the surgical device to the patient’s eye. The tilt mechanism is attached to the tool holder at one end and the rolling mechanism at the other, allowing the instrument to be at any angle. As a result, the robot improves the effectiveness of each movement. This instrument can be used free-hand or incorporated into the Steady-Hand-Eye robot . The latest model has improved the range of motion, stiffness, and speed of holder release. 4. “Smart” instruments : Additional systems and “smart” instruments have been developed to improve technical performance . For example, sensors detect the force applied to the eye; this could be transferred directly to the surgeon via an auditory feedback system . In addition, they can notice tactile sensations lower than the human threshold, which could minimize the risk of possible surgical complications. Retinal membrane removal was successfully performed with the aid of PRECEYES surgical system robotic assistant, which serves as an instrument holder for over six patients as part of a trial at Oxford's John Radcliffe hospital . The median time was longer (four minutes and 55 s) than the traditional method (one minute and 20 s) . Assistive devices for Intravitreal Injection have been demonstrated through ex vivo experiments with porcine eyes. It used an automatic fine positioning and intravitreal injection through the pars plana. In addition, several safety features, such as continuous eye-tracking and iris recognition, have been implemented .
Guerrouad and Vidal in 1989 created a robotic ocular system composed of a Stereotaxical Microtelemanipulator (SMOS), a spherical micromanipulator mounted on an x, y, and z stage, which allowed 6 degrees of freedom. No development was made after this stage. Robot-Assisted Microsurgery (RAMS) tele-robotic platform emerged in 1997 (Charles S 1997). It comprises a slave robot arm (2.5 cm in diameter and 25 cm long) and a primary device supported by cables and encoders facilitating the operator's arm movement guided by computers . In the same year, another prototype used the Stewart-based platform (Jensen, Grace et al. 1997). This was developed to measure intraluminal (20–130 microns) retinal vessel pressure and to extract blood from these vessels for research purposes. In 2009, Ueta et al. developed a newer prototype with more accuracy adapted to assist in vitreoretinal surgeries.
History and background Electrocardiogram was first introduced in 1906, the first step in telemedicine. Cybersurgery, also referred to as Telesurgery, from the Greek tele, “far off”, also called “remote surgery”, is defined as a surgical technique which allows for a surgeon to operate on a patient remotely, either from a different location or at proximity, through a telecommunications channel attached to a robotic operating machine. Tele-surgery is a surgical system that utilizes wireless networking and robotic technology to connect surgeons and patients distantly. It can be divided into three main components: Telesurgery, telementoring, and teleconsultation . The telerobotic Zeus and Da Vinci surgical systems allow surgeons to operate remotely. These telerobots hold the camera, replace the surgeon’s two hands with robotic instruments, and serve in a master–slave relationship for the surgeon. They are characterized by their capabilities to simulate the motions of the surgeon’s wrist and different surgeon positions . In 1988: Minimally invasive surgery enabled surgical procedures to be guided by introducing a camera without requiring an opening of the abdomen or thorax. In 1996: Computer-assisted surgery was introduced, which enabled to transmit surgeons’ actions remotely to manipulation devices. September 7, 2001: Telesurgery: The world's first Telesurgery was performed by a surgical team in New York, U.S.A. using the ZEUS robotic system (Intuitive Surgical, Sunnyvale, CA, U.S.A.). This project produced a successful two-hour-long laparoscopic cholecystectomy performed on a female patient at a hospital in Strasbourg, France . The patient had an uneventful recovery . In 2003, a surgical system was set up in Canada between two hospitals 400 kms away . Robotics could be helpful in surgical tele-mentoring by expert surgeons to supervise younger surgeons remotely, given its endoscopic optics and mechanized movement. However, the maturity of these modalities depends on financial factors, legislation and collaboration with cybersecurity experts to ensure safety and cost-effectiveness . Current applications of cybersurgery Current applications of cybersurgery include tele-education, tele-training, telementoring, tele-proctoring, and tele-accreditation. Different projects have been developed; different site videoconferences used images and data transmission at the European Institute of TeleSurgery of Strasbourg via The TESUS project through the realization of international multi-site video conferences between surgeons. The WEBSurg project created the first virtual university by placing surgical techniques at the surgeon’s disposal through the Internet. It is a comprehensive source of knowledge in minimally invasive surgery. It promotes technological advances in its fields, such as general and digestive surgery, urology, gynecology, pediatric surgery, endoscopic surgery, skull base surgery, arthroscopy, and upper limb surgery . The HESSOS project (Hepatic Surgery Simulation and Operative Strategy) uses virtual reality as a surgical simulation system, allowing the development of the concept of distant tele-manipulation. It serves as an operative system available for clinical application in liver surgery. In addition, it allows worldwide surgical teaching . Tele-cystoscopy was tested suitable for diagnosis. The trade-offs between cost and tele-cystoscopy system component quality were compared with efficiency frontiers to elucidate the optimal system . Tele-oncology covers diagnosis, treatment, supportive care of cancers, education, and medical training. Modern strategies were addressed to ensure global access to essential cancer care services (Telemedicine and Telesurgery in Cancer Care (TTCC) conference) . To overcome the shortage of surgeons, the “Virtual Interactive Presence” (V.I.P.) platform allows remote participants to simultaneously view each other’s visual field, creating a shared field of view for real-time surgical tele-collaboration . Video analysis yielded a mean compositing delay of 760 ± 606 ms (when compared with the audio signal). Image resolution adequately visualizes neurosurgery’s complex intracranial anatomy and provides interactive guidance . Based on preclinical work, trans-oral robotic surgery (TORS) was performed in February 2007 on a patient with a para-pharyngeal to infratemporal fossa cystic neoplasm as part of a large prospective human trial. The robotic procedure allowed adequate and safe identification of the internal carotid artery and cranial nerves, and excellent hemostasis was achieved with no complications during or after surgery . Later, the Telelap Alf-x, telesurgical system was introduced. It composed of individual arms, which enabled free access to the patient throughout surgery, an extensive range of reusable surgical instruments, an open console with an eye-tracking system, where the camera followed the eye and head movements of the surgeon. The existing force feedback enables for the first time to feel the consistency of the tissues and avoid tearing the stitches while suturing. The system combines the benefits of open surgery and endoscopy . The first clinical application, which involved 146 operations at the gynecological department of the Gemelli University Hospital in Rome, proved the safety and the surgical team’s quick adaptation to the system . In 1992, the National Aeronautics and Space Administration and the Department of Defense supported Telesurgery to rescue wounded soldiers. The Defense Advanced Research Projects Agency invested in tele-medical technologies to help operate injured soldiers remotely . Applications of cybersurgery or telesurgery in ophthalmology There is no current application of Cybersurgery or Telesurgery in Ophthalmology. However, the feasibility of telerobotic microsurgical repair of corneal lacerations has been evaluated . Five mm central full-thickness corneal wounds were fashioned in five enucleated rabbit eyes and repaired remotely using the telerobotic system . The feasibility of using the Robotic Slave Micromanipulator Unit (RSMU) in photocoagulating the ciliary body remotely to treat glaucoma with the diode laser was tested in fresh un-operated, enucleated human eyes. Histology examination of remote robotic contact trans-scleral cyclophotocoagulation and “by hand” technique produced similar degrees of ciliary body tissue disruption . Various projects have recently been launched at academic and corporate levels to develop lightweight, miniaturized surgical robotic prototypes . This delay could be explained by the delicacy of this field which deals with the sense of vision and the small anatomical size. Advanced virtualization and augmented-reality techniques should help human operators to adapt better to special conditions . To meet safety standards and requirements in space, a three-layered architecture is recommended to provide the highest quality of telepresence technically achievable for provisional exploration missions .
Electrocardiogram was first introduced in 1906, the first step in telemedicine. Cybersurgery, also referred to as Telesurgery, from the Greek tele, “far off”, also called “remote surgery”, is defined as a surgical technique which allows for a surgeon to operate on a patient remotely, either from a different location or at proximity, through a telecommunications channel attached to a robotic operating machine. Tele-surgery is a surgical system that utilizes wireless networking and robotic technology to connect surgeons and patients distantly. It can be divided into three main components: Telesurgery, telementoring, and teleconsultation . The telerobotic Zeus and Da Vinci surgical systems allow surgeons to operate remotely. These telerobots hold the camera, replace the surgeon’s two hands with robotic instruments, and serve in a master–slave relationship for the surgeon. They are characterized by their capabilities to simulate the motions of the surgeon’s wrist and different surgeon positions . In 1988: Minimally invasive surgery enabled surgical procedures to be guided by introducing a camera without requiring an opening of the abdomen or thorax. In 1996: Computer-assisted surgery was introduced, which enabled to transmit surgeons’ actions remotely to manipulation devices. September 7, 2001: Telesurgery: The world's first Telesurgery was performed by a surgical team in New York, U.S.A. using the ZEUS robotic system (Intuitive Surgical, Sunnyvale, CA, U.S.A.). This project produced a successful two-hour-long laparoscopic cholecystectomy performed on a female patient at a hospital in Strasbourg, France . The patient had an uneventful recovery . In 2003, a surgical system was set up in Canada between two hospitals 400 kms away . Robotics could be helpful in surgical tele-mentoring by expert surgeons to supervise younger surgeons remotely, given its endoscopic optics and mechanized movement. However, the maturity of these modalities depends on financial factors, legislation and collaboration with cybersecurity experts to ensure safety and cost-effectiveness .
Current applications of cybersurgery include tele-education, tele-training, telementoring, tele-proctoring, and tele-accreditation. Different projects have been developed; different site videoconferences used images and data transmission at the European Institute of TeleSurgery of Strasbourg via The TESUS project through the realization of international multi-site video conferences between surgeons. The WEBSurg project created the first virtual university by placing surgical techniques at the surgeon’s disposal through the Internet. It is a comprehensive source of knowledge in minimally invasive surgery. It promotes technological advances in its fields, such as general and digestive surgery, urology, gynecology, pediatric surgery, endoscopic surgery, skull base surgery, arthroscopy, and upper limb surgery . The HESSOS project (Hepatic Surgery Simulation and Operative Strategy) uses virtual reality as a surgical simulation system, allowing the development of the concept of distant tele-manipulation. It serves as an operative system available for clinical application in liver surgery. In addition, it allows worldwide surgical teaching . Tele-cystoscopy was tested suitable for diagnosis. The trade-offs between cost and tele-cystoscopy system component quality were compared with efficiency frontiers to elucidate the optimal system . Tele-oncology covers diagnosis, treatment, supportive care of cancers, education, and medical training. Modern strategies were addressed to ensure global access to essential cancer care services (Telemedicine and Telesurgery in Cancer Care (TTCC) conference) . To overcome the shortage of surgeons, the “Virtual Interactive Presence” (V.I.P.) platform allows remote participants to simultaneously view each other’s visual field, creating a shared field of view for real-time surgical tele-collaboration . Video analysis yielded a mean compositing delay of 760 ± 606 ms (when compared with the audio signal). Image resolution adequately visualizes neurosurgery’s complex intracranial anatomy and provides interactive guidance . Based on preclinical work, trans-oral robotic surgery (TORS) was performed in February 2007 on a patient with a para-pharyngeal to infratemporal fossa cystic neoplasm as part of a large prospective human trial. The robotic procedure allowed adequate and safe identification of the internal carotid artery and cranial nerves, and excellent hemostasis was achieved with no complications during or after surgery . Later, the Telelap Alf-x, telesurgical system was introduced. It composed of individual arms, which enabled free access to the patient throughout surgery, an extensive range of reusable surgical instruments, an open console with an eye-tracking system, where the camera followed the eye and head movements of the surgeon. The existing force feedback enables for the first time to feel the consistency of the tissues and avoid tearing the stitches while suturing. The system combines the benefits of open surgery and endoscopy . The first clinical application, which involved 146 operations at the gynecological department of the Gemelli University Hospital in Rome, proved the safety and the surgical team’s quick adaptation to the system . In 1992, the National Aeronautics and Space Administration and the Department of Defense supported Telesurgery to rescue wounded soldiers. The Defense Advanced Research Projects Agency invested in tele-medical technologies to help operate injured soldiers remotely .
There is no current application of Cybersurgery or Telesurgery in Ophthalmology. However, the feasibility of telerobotic microsurgical repair of corneal lacerations has been evaluated . Five mm central full-thickness corneal wounds were fashioned in five enucleated rabbit eyes and repaired remotely using the telerobotic system . The feasibility of using the Robotic Slave Micromanipulator Unit (RSMU) in photocoagulating the ciliary body remotely to treat glaucoma with the diode laser was tested in fresh un-operated, enucleated human eyes. Histology examination of remote robotic contact trans-scleral cyclophotocoagulation and “by hand” technique produced similar degrees of ciliary body tissue disruption . Various projects have recently been launched at academic and corporate levels to develop lightweight, miniaturized surgical robotic prototypes . This delay could be explained by the delicacy of this field which deals with the sense of vision and the small anatomical size. Advanced virtualization and augmented-reality techniques should help human operators to adapt better to special conditions . To meet safety standards and requirements in space, a three-layered architecture is recommended to provide the highest quality of telepresence technically achievable for provisional exploration missions .
Ophthalmology is one of the most enriched fields that allowed artificial intelligence to be part of its point of interest in scientific research. Robotic surgery in ocular surgeries is not well established and still in the experimental process in ophthalmology . Although the current study is limited by the number of published studies discussing robotic and cybersurgeries in ophthalmology, being mainly in experimental stages, a review of current aspects of robotic and cybersurgery in ophthalmology could help in the progression of these disciplines. The advantages of robots in surgery originate from the need to achieve two goals: telepresence and the performance of repetitive and accurate tasks which are the gold standards of ocular surgery. An accelerometer can cancel the operator's physiologic tremor in real-time. Robotic arms minimize the natural limits of human wrists, favoring more precise and efficient movements. The risk of human errors combined with mechanical failure as electrical current and misapplication to surrounding tissues coupled with a longer duration of surgery are disadvantages of this technology. Robotics platforms and prototypes specializing in ophthalmology surgeries are not yet met. Speed and velocity are required during ocular surgery. The time delay threshold must be acceptable by adopting strategies that preserve path-tracking accuracy. The latest model of Johns Hopkins Steady-Hand Eye Robot has improved the range of motion, stiffness, and speed of holder release. These criteria would be helpful in emergencies requiring rapid actions. In addition, smart instruments coupled with robotics could minimize the risk of possible surgical complications. However, apart from Preceyes’ B.V. research platform, none of the currently eye-specific systems has reached a commercial stage. Anterior segment surgeries, such as cataract, strabismus, pterygium, keratoplasty, and amniotic membrane suturing, were successfully performed on porcine and human donors. Subretinal drug delivery to treat submacular hemorrhage aided by robots, demonstrated its feasibility and safety; this could be useful in gene or cell therapy . Further development in the instruments used in intravitreal injections could enhance the technique . Cybersurgery benefits today’s shortage of surgeons and eliminates geographical barriers, financial burdens, complications, and often risky long-distance travel. Fibreoptic A.T.M. lines to minimize latency and optimize connectivity and computer motion are elements to consider when planning Telesurgery. In addition, Telesurgery allows for international surgical collaboration and helps in improving surgical education. Different models have been used, such as virtual simulators (DV-Trainer ® , Robotic Mentor ® , DVSS ® ), mechanical simulators, microsurgery and wet lab using ex vivo animal organs, anaesthetized animals, and cadavers . There was no significant difference between the lengths of the learning curves for robot-assisted vitreoretinal surgery compared to manual surgery. However, robot-assisted vitreoretinal surgery was more precise, associated with less tissue damage, and slower . Surgical robots are rarely found in healthcare systems and are provided for other surgical specialties, where evidence-based medicine confirms their feasibility. Ophthalmology specialized hospitals are more commonly separated, making access to surgical robots difficult and time-consuming. This factor could contribute to the low number of studies and trials on ocular surgery after the high cost of these robots. Data regarding costs and litigation of robotics and cyber-surgery versus conventional techniques in ophthalmology are limited. This may be responsible for the delay in this field . Another drawback is the low number of qualified, trained surgeons in robotic surgery. The technical specifications of robotics used in microsurgery are highly challenging . Future perspectives include technologies, such as 5G, Tactile Internet, and A.I., to help reduce resource scheduling problems in cybersurgery. In addition, specific prototypes to be implemented for robotic ocular surgery to increase the accuracy, as seen in previous studies are crucial for developing this field. Increasing the number of short- and long-term clinical training programs in robotic surgery could facilitate this field's progression.
The robotic Da Vinci Surgical System and the ARES (Auris Surgical Endoscopy System) robot are the only two surgical robots approved for human surgery; however, it is not designed for microsurgical specialties. Robotic technology has only recently been integrated into ophthalmology; hence, the progression is only in its initial stages. The cybernetic revolution in surgery supported by artificial intelligence could enable surgeons to perform surgeries remotely. Tele-surgery can provide urgent medical services and allows highly skilled doctors to operate globally. Technologies, such as 5G and Tactile Internet, are required to help reduce resource scheduling problems in cybersurgery. In addition, prototype development and the integration of artificial intelligence applications could further enhance the safety and precision of ocular surgery. Surgeons must embrace these technologies to render these technologies available; however, further studies to overcome these challenges limiting the progression of these fields in terms of cost, availability, legislation, and ethics are crucial.
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Dental evidence for extended growth in early | 05f2b935-553c-41f0-952d-d2104bf95606 | 11602720 | Dentistry[mh] | Humans exhibit several aspects of life history different from great apes, such as helpless newborns, early weaning and allomaternal infant care, a prolonged postnatal brain growth period, delayed somatic maturation, late onset of reproduction, transgenerational co-operation and a long post-reproductive life . In particular, humans have an extended period of immaturity called childhood—the time after weaning and before the attainment of nutritional independence—that is characterized by (allo)parental care and ample opportunities for social learning . These traits do not fossilize, however, so inferences on the evolution of human life history must be drawn from developmental proxies preserved in hominin fossils , , , . Teeth are of special relevance because their crowns and roots grow by the daily incremental deposition of enamel and dentin layers, respectively, thus conserving detailed information about the maturation schedule of an individual’s masticatory system , . Humans, compared to great apes, show a marked slow-down in dental maturation, especially of the permanent molars, which correlates well with the pace of cerebral development and somatic maturation across primates , . Dental incremental growth data from fossil hominins have yielded reliable estimates of the age at death of immature specimens , permitting researchers to use dental growth chronologies to calibrate cerebral, cranial and somatic development. For example, microstructural dental evidence from the immature Australopithecus afarensis specimen from Dikika, Ethiopia , obtained using propagation phase-contrast synchrotron microtomography (PPC-SRµCT), suggests that this individual essentially followed an ape-like dental ontogenetic schedule. However, its endocranial volume (ECV) at the time of death (2.4 years) had reached a smaller proportion of adult ECV than in great apes, which has been interpreted as the first evidence of prolonged postnatal brain growth. There is still relatively little information available on the exact timing of dental ontogeny in early Homo . The Nariokotome individual (KNM-WT 15 000), dated to approximately 1.6–1.5 million years ago (Ma), is thought to have followed a great-ape-like rather than human-like dental ontogenetic schedule – , but these inferences are based on estimates rather than direct measurements of its dental microstructural growth. When exactly the characteristic life-history slow-down occurred in the evolution of our genus Homo is therefore still an open question. Here we present a comprehensive and non-destructive microstructural analysis of dental development in an early Homo individual from Dmanisi and draw inferences on the life-history evolution of early Homo . The Dmanisi site, dated to 1.85–1.77 Ma (ref. ), has yielded five well-preserved crania, associated mandibles and various postcranial elements – . Together with a rich record of faunal remains and lithic artefacts, the Dmanisi ensemble has provided unique insights into the palaeobiology of an early Homo population outside Africa, its life history, social organization and cognitive capabilities , . Here we focus on a subadult individual that is represented by cranium D2700, mandible D2735 and various postcranial elements , . The individual’s permanent dentition is well preserved, and each tooth type is represented by at least one antimere, except for the lower first incisors (Fig. ). The first molar teeth (M1s) exhibit extensive occlusal wear, resulting in cuspal dentin exposure. The second molars (M2s) only exhibit moderate wear. The lower right third molar (LRM3) was found in isolation, but fits well in its alveolar cavity. The corresponding alveolus for the lower left M3 (LLM3) is not present, indicating LLM3 agenesis , . The upper right M3 (URM3) exhibits a supernumerary cusp (Extended Data Fig. ) and was excluded from further microstructural analyses to ensure data consistency, its antimere having a normal morphology. All M3s exhibit well-developed, but not fully formed, roots, while already exhibiting some wear facets. The individual therefore died before reaching dental maturity, which allows an accurate determination of the individual’s age at death through quantification of the incremental growth structures preserved in the tooth enamel and dentin. We used PPC-SRµCT virtual histology , to track the development of the individual’s best-preserved maxillary and mandibular teeth (Fig. , , Supplementary Data and Extended Data Fig. ). To obtain a complete record of dental development, the worn crown cusps were reconstructed using comparative data from better-preserved teeth (Extended Data Fig. ; ). The cusps of the M1 typically comprise the neonatal line (a perinatal stress marker) in their apical-most region. Because this information was lost through dental wear, all following analyses are based on the assumption that the timing of M1 crown initiation was similar to that in living humans and great apes, that is, shortly before, to around, birth . Following this preparatory work, we reconstructed the developmental profiles of all teeth, as follows . (1) We identified major stress markers (indicating discrete systemic stress events during development), which are visible as hypoplastic discontinuities in the dental growth line sequences (Fig. and Extended Data Figs. – ). Stress lines were matched across all teeth, resulting in a relative chronology of dental development (Fig. ). (2) High-resolution (0.72 μm voxel size) virtual cross-sections through the best-preserved enamel and dentin regions revealed a long-period (Retzius/Andresen) growth line timing of 6 days (which is at the fast end of variation in fossil hominins, living humans and great apes ) (Supplementary Data ), and an average dentin secretion rate of 4.28 μm per day (Supplementary Data ). (3) We counted the well-preserved long-period growth lines of the dentin (Andresen lines), and combined this information with the long-period line spacing and the dentin secretion rate data to establish the absolute timing of the major stress events, and of root formation from initiation to completion. (4) These data were combined with stress marker information from the enamel and calibrated with the Andresen line counts in the dentin to reconstruct crown formation from initiation to completion (Extended Data Figs. – ). (5) Finally, the crown and root formation schedules of all the teeth were aggregated into a composite record of dental ontogeny from birth to death (Fig. ). Our data indicate that individual D2700/D2735 died at an age of 11.36 ± 0.65 years (the error margin results from interdental variation in the distances between the stress markers, variation in the reconstruction of worn cusps, variation in the assumed M1 crown initiation and variation in the daily dentin secretion rate) (Supplementary Data – ). The time to root apex closure of the third molars was estimated to be 1.3–1.5 years . Thus, D2700/D2735 died shortly before reaching full dental maturity (12.0–13.5 years). The almost complete incremental growth record of this individual’s permanent teeth allowed us to reconstruct the longitudinal ontogenetic trajectory of its dentition from birth to dental maturity (Figs. and and Supplementary Videos and ). We recorded the ontogeny of D2700/D2735’s dentition at 6-month intervals from birth to death and compared this with corresponding data from living humans, great apes and fossil hominins ( and Supplementary Data ). To characterize taxon-specific modes of dental ontogeny, we distinguished between the pattern and rate of permanent dentition formation. The pattern describes how the eight tooth types (I1, I2, C, P3, P4, M1, M2, M3) matured relative to each other (Fig. ), while the rate describes how the dentition, as a whole, matures with increasing age (Fig. ). In quantitative terms, the pattern is given by the dental maturation scores (DMSs) of all tooth types of an individual’s dentition (with the tooth types indicated by the subscript labels DMS I1 , DMS I2 , DMS C , DMS P3 , DMS P4 , DMS M1 , DMS M2 and DMS M3 ), while the rate is given by the DMS of the entire dentition as a function of individual age . Figure summarizes the timing of the key dental ontogeny events in humans, chimpanzees and Dmanisi. The intraspecific variation in crown initiation and completion, tooth emergence and root completion is substantial – . However, a principal component analysis (PCA) of the DMS I1…M3 data (DMS-PCA (ref. )) showed that humans have a distinctly different pattern of dental ontogeny compared to all great apes (chimpanzees, bonobos, gorillas and orangutans) (Fig. ). Notably, the Dmanisi pattern is within the human range of ontogenetic variation and outside the great ape range of variation (Fig. ). Figure shows taxon-specific maturation trajectories of the entire dentition. Chimpanzee dentition matures rapidly during the first few years of postnatal life, reaching full maturity at around 11–13 years of age . Data from gorilla and orangutan specimens of known age suggest trajectories in the upper and lower ranges of the chimpanzee variation, respectively , . By contrast, human dentition is characterized by a slow early postnatal maturation, reaching full maturity around 18–22 years of age. The dentition of the Dmanisi individual initially followed a slow human-like rate trajectory until approximately 4 years of age, then catching up to reach a chimpanzee-like trajectory around 8 years, and finally reaching dental maturity at 12.0–13.5 years. Figure shows the inferred instantaneous maturation rate (total DMS per year) along the trajectories. In the chimpanzee dentition, the rate is highest in the first 2 years of postnatal life and then declines towards adulthood. By contrast, in living humans, the rate is comparatively low in the early postnatal period, but then increases to a peak at around 7.0 years of age, after which it declines steadily towards maturity. We refer to the peak rate as the dentition growth spurt (DGS). Notably, the Dmanisi individual also exhibits a DGS, but this occurred at approximately 5.3 years of age, which is significantly later than in chimpanzees (shortly after birth) and close to the lower range of the modern human DGS (in the range 6.0–8.5 years) (Fig. and Extended Data Table ).
Dmanisi’s dental ontogeny appears as a combination of derived human-like and primitive great-ape-like features, on both relative and absolute time scales. Its pattern of ontogeny is within the range of human variation (Fig. ), with a delayed initiation of the posterior teeth (P3, P4, M2, M3, except M1) compared to the anterior teeth (I1, I2, C), and a wide spacing between the M1, M2 and M3 initiation times compared to great apes (Fig. ). As an effect, Dmanisi’s dentition, as a whole, matured slowly during the first approximately 5 years of life and exhibited a late DGS (Fig. ). However, Dmanisi’s tooth-specific maturation rates differed from those of humans in several ways. Once the crowns of the premolars, M2 and M3, were initiated, their formation rates were exceptionally high, resulting in Dmanisi catching up to a chimpanzee-like maturation trajectory (Fig. ), and the estimated eruption times of the molars (M1: around 3.8 years; M2: around 7.6 years; M3: around 10.6 years) being more similar to those observed in wild chimpanzees , than in humans . Also, the predicted age of dental maturity (around 12.0–13.5 years) is more similar to chimpanzees and other great apes (11–13 years) than to modern humans (18–22 years). How did Dmanisi’s mode of dental ontogeny evolve? Our data suggest a diversity of dental ontogenetic patterns and rates among both fossil hominin and extant great ape taxa. Great apes show a common pattern of ontogeny (Fig. ), but taxon-specific differences in rate , (Fig. ). The data for A. afarensis (specimens AL 333-105, Dikika; geological age around 3.3 Ma) and Australopithecus africanus (Taung, Sts 24; around 2.8 Ma) suggest that the dental ontogenies of these species were probably within the range of ontogenetic pattern and rate variation seen in extant great apes, whereas Paranthropus (DNH 107, SK 63; around 2.0–1.8 Ma) demonstrates a mode of dental ontogeny not found in extant taxa , (Figs. and ). Fossils representing African early Homo (KNM-WT 15 000 Nariokotome, KNM-ER 1590; 1.8–1.6 Ma) conform with the Dmanisi/human pattern of ontogeny (Figs. and ), supporting the notion that this pattern is an evolutionary hallmark of the genus Homo . What are the implications for the evolution of hominin life history? Because teeth are central to masticatory function, evolutionary changes in their maturation schedules tend to reflect changes in masticatory biomechanics and in the timing of dietary transitions during an individual’s life, from breastfeeding to solid foods and from nutritional dependence to independence. These changes, in turn, are thought to reflect evolutionary changes in the allocation of metabolic resources to cerebral, somatic and reproductive maturation, and thus changes in life history. In Dmanisi, compared to great apes, the characteristically delayed maturation of the posterior permanent dentition (note the relatively late eruption of P3 and P4 in Fig. ) and the late DGS imply a prolonged use of the deciduous molars. This probably indicates an extended period of nutritional dependence. The long childhood period in humans is characterized by delayed dental and somatic maturation relative to brain growth and maturation , , , . This pattern is typically thought to have evolved because the large human brain requires considerably more metabolic energy to grow to adult size than in great apes , , . The resulting delay in somatic maturation, in turn, would have provided new opportunities for immature individuals to acquire advanced cognitive skills in a complex social environment while the brain was still growing and maturing . However, this evolutionary scenario is not applicable to early Homo , as the advanced dental and skeletal maturity of Dmanisi and Nariokotome at their respective ages of death are more similar to those of great apes than living humans , – (Extended Data Table ). Furthermore, the brain size of early Homo at Dmanisi (ECV range: 546–730 cm 3 ) was only moderately larger than that of great apes, and therefore probably had a moderate impact on the metabolic trade-off between brain and body growth. We therefore propose an alternative scenario for early Homo that assumes a diversity of life-history strategies among Plio-Pleistocene hominins, as reflected by the diversity of dental ontogenies . Australopithecus probably exhibited a primitive ape-like mode of dental ontogeny (Figs. and ). However, immature Australopithecus individuals show evidence of a prolonged phase of postnatal brain growth and maturation compared to great apes , , suggesting that this pattern emerged before a marked evolutionary expansion of the brain. In early Homo , according to this scenario, the extended growth phase did not evolve as a consequence of brain expansion and the associated trade-offs in resource allocation , . Rather, the selective pressures underlying the observed changes must be sought elsewhere. The palaeoanthropological and archaeological context of Dmanisi provides valuable information in this regard. The presence of Mode I stone tools and cut marks on animal bones indicates tool-assisted meat consumption at Dmanisi , . Therefore, it remains to be investigated whether the evolution of the early Homo pattern of dental ontogeny reflects a shift towards tool-assisted meat consumption, implying a reduced overall masticatory load . Furthermore, the sociocultural context and its potential effects on life history and reproduction must be considered , . The four mature individuals found at Dmanisi , – span an age range from early to late adulthood, including one senile individual who had survived several years without teeth , . The possible presence of post-reproductive individuals in the Dmanisi palaeopopulation can be viewed in the context of biocultural reproduction , which implies that intergenerational co-operation facilitated an extended period of growth sustained by alloparental provisioning , and ultimately contributed to increased reproductive success. This raises the question of whether the inferred life-history characteristics of early Homo reflect an evolutionary change in reproduction strategies , —before substantial brain expansion and reorganization , and before a general slow-down in life history. To test these hypotheses, further dental microstructural analyses of key early Homo fossils, such as the Nariokotome individual, and of small-brained fossil hominin taxa, such as Australopithecus sediba and Homo naledi , are required. Overall, our analyses add to the growing body of evidence that the evolution of hominin life histories did not follow a single linear trajectory, but was a branched and modular process, involving diverse selective pressures and adaptive responses.
Propagation phase-contrast synchrotron microtomography Cranium D2700 and associated mandible D2735 were scanned at the ID19 beamline of the European Synchrotron Radiation Facility, Grenoble, France. Volume scans were performed using PPC-SRµCT. For general dental morphological assessment, as well as for the counting of Andresen lines, two configurations were used in two successive series of experiments. The first used voxel sizes of 4.89 µm with a monochromatic beam set at 51 keV to take advantage of the gadolinium K-edge of the 5-μm-thick gadox scintillator. The second series took place a few years later, after substantial technical evolution of the ID19 beamline, and was performed using a 5.06-µm voxel size using a polychromatic beam whose spectrum was shaped with a tungsten filter to isolate a peak between the K-edge of the lutecium from the lutecium–aluminium–garnet–(cerium) scintillator (63.3 keV) and the K-edge of the tungsten filter (69.5 keV). This combination enabled much faster scans than with the monochromatic setup due to the higher flux and higher energy, and with perfect beam stability and improved coherence, resulting in much better phase-contrast imaging. In both cases, the propagation distance was 5,000 mm and the scans were performed in half-acquisition to almost double the field of view of the detector. In addition, scans for daily periodicity assessment were performed at 0.75 μm at 65 KeV, also in half-acquisition. All scanning parameters are listed in Supplementary Data , and a comparison of the monochromatic and polychromatic results is presented in Extended Data Fig. . Acquisition of dental developmental data Determination of developmental planes The dental development was tracked based on the PPC-SRµCT data of at least one tooth of each type (Supplementary Data ). Using the software VGStudio Max v.3.4 and later (Volume Graphics GmbH), virtual cross-sections through the teeth were generated to optimally visualize the microstructural growth lines in the enamel (Retzius lines) and dentin (Andresen lines). The optimum cross-sectional plane, named the developmental plane , was defined as the plane passing through a given dentin horn and the top of the underlying pulp cavity. The plane’s x axis was oriented in the bucco-lingual direction and its y axis along the tooth’s longitudinal axis. The plane’s thickness ( z -axis depth) was set to values between 100 and 150 µm. The use of these ‘thick sections’ enhanced the visibility of the developmental microstructures in the tooth . Reconstruction of worn cusp tips Several teeth of the Dmanisi individual had undergone substantial wear since eruption, as evidenced by missing enamel cusp tips and dentin horn tips (Extended Data Fig. ). Because the most relevant microstructural developmental information in the teeth of this specimen came from the dentin (see ‘Daily secretion rate’), we focused on reconstructing the relatively small missing apical volumes of the dentin horns (Extended Data Fig. ), using better-preserved teeth as guides. This process was repeated in several independent sessions. The reconstruction uncertainty inherent in this inference procedure is reflected in the calculations of the dental developmental timing and the age at death (Supplementary Data ). Matching of stress markers across the entire dentition The general sequence of tooth development is defined by the order in which teeth initiate, emerge and complete their growth. Episodes of major stress during an individual’s development tend to affect the dentition as a whole, resulting in growth discontinuities, which become manifest as stress lines on the outer crown surface and in the microscopic record of enamel and dentin growth lines. A given stress event leaves its mark on all teeth that are still in the enamel and/or dentin deposition phase at this moment. This property is an important prerequisite for placing the development of the individual teeth in a common time frame (Extended Data Fig. ). In the Dmanisi dentition, six major stress events could be identified (numbered 1 to 6 in Supplementary Data and assigned different colours in Fig. ). The matching of these stress lines across all teeth was ensured by cross-correlation between the Retzius and Andresen lines, and was further verified by comparison with the three-dimensional topology of the enamelo-dentin junction as well as the hypoplasias of the root surface (Extended Data Fig. ) and lateral enamel surface (Extended Data Figs. and ), following the methodology proposed in ref. . Long-period duration The deposition of dental enamel and dentin exhibit both short-term (daily) and long-term (several days) periodicities. In virtual PPC-SRµCT cross-sections, the daily incremental growth structures are visible as cross-striations (or laminations) in the enamel (with a size of about 1–3 μm) . Typically, the cross-striations are most visible in the relatively flat areas of the mid-lateral cuspal enamel. In enamel, the long-period structures are visible as Retzius lines (size of about 30–40 μm, measured along the enamel prisms). The number of daily increments between consecutive Retzius lines gives the value of the long-period line periodicity. This is a property that varies between individuals of a given species (for example, in humans, between 5 and 12 days , ), but can also sometimes vary within a given individual, despite general consensus to the contrary. For example, in a modern human sample of n = 40, 18 individuals (45%) showed variation in long periodicity, with longer duration in the anterior than the posterior dentition . To determine the actual long-periodicity of the D2700/D2735 dentition, scans of the enamel at a voxel resolution of 0.72 µm were performed on the URP4, URC, ULM3 and LRM3, and the cross-striations were directly traced and counted between the long-period lines (Supplementary Data and and Extended Data Fig. ). Each tooth was evaluated by two independent observers, and 178 independent measurements were taken all over the teeth. Owing to strong artefacts in the data and the non-perfect preservation of the specimens, periodicities of between 3 and 9 days were observed. The 6-day periodicity represented 41% of the measurements, the 5-day periodicity 31% and the other periodicities 28%. Given the non-normal distribution of these values (Supplementary Data ) (Kolmogorov–Smirnov test: F = 0.2465, P < 9.1693 × 10 −10 ), a Wilcoxon test was performed for comparison with expected periodicities of 5, 6 and 7 days, respectively. The only match between the expected and actual data was observed for the 6-day periodicity ( P < 0.23), while the results for 5 (8.51 × 10 −9 ) and 7 days (7.31 × 10 −10 ) suggest significant differences between the expected and observed distributions. Our data show no evidence of an antero-posterior gradient in the long-period duration . We thus used the 6-day periodicity to reconstruct the developmental profiles of all teeth by counting the Retzius and/or Andresen lines, and multiplying the total number of lines by the long-period duration. Daily secretion rate In the D2700/D2735 specimen, the long-period lines in the cuspal enamel (Retzius lines) were barely visible at the available voxel resolution (4.89 and 5.06 µm), which prevented direct measurements of the daily enamel secretion rate. However, the D2700/D2735 teeth exhibit exceptionally well-preserved dentin microstructures, which permitted an inference of the daily secretion rates by counting the Andresen lines in those areas where they were clearly visible. Specifically, the daily secretion rate of dentin was evaluated as follows. The length ( L , in microns) of a given well-preserved primary dentin tubule was measured (secondary dentin tends to show compression of the Andresen line patterns), and the number, A , of the Andresen line intervals along the tubule was counted. The daily secretion rate ( S , in microns per day) was then evaluated as S = L /( A × P ), where P is the periodicity in days (Extended Data Fig. ). The resulting value of 4.28 μm was high compared to humans and chimpanzees , , but was cross-validated by two observers, and consistent results were obtained when the different sources of information were combined. Finally, this information was combined with the Andresen line counts and the cross-matched stress markers—visible in both the enamel and the dentin—to reconstruct the absolute developmental chronology of the whole dentition (Supplementary Data ). Dental eruption time The time of dental eruption was estimated from the maximum extension rate of the root, termed the root growth spurt , (Extended Data Fig. ). The method used to determine the root extension rates was adapted from previous studies , and proceeded as follows. Along the cemento-dentin junction, a first 200-μm-diameter circle was placed at the cervix and a second was aligned at the junction between the cemento-dentin junction and the Andresen line, passing tangentially on the side of the first circle (Extended Data Fig. ). This procedure was repeated all along the length of the root. The greatest distance along the external surface of the root between the two circles marked the maximum extension rate, that is, the root growth spurt. In great apes, the root growth spurt coincides with the period of dental eruption, whereas in modern humans, the growth spurt precedes eruption , (Supplementary Data ). In the Dmanisi individual, therefore, the maximum rates of root growth should be regarded as minimum estimates of tooth eruption times. Furthermore, tooth eruption is a process that takes place over several months. We therefore represent the estimated eruption period as the time point of the maximum root extension rate ± 3 months (Fig. and Extended Data Fig. ). It should be noted that such a root growth spurt is visible in the third molars on the last third of the developing roots, while these teeth erupted quite some time before the death of the individual, as indicated by the light wear facets. This supports our basic assumption that the root growth spurt in this individual tends to coincide with the dental eruption. Age at death and dental ontogenetic trajectory Synopsis of dental development The time-calibrated microstructural growth data of the tooth crowns and roots permitted reconstruction of the complete sequence of dental development throughout the life of D2700/D2735, from the initiation to completion of each tooth. The resulting diagram is visualized in Fig. and the underlying data are presented in Supplementary Data . These data were used to determine the age at death of the individual, as well as its longitudinal dental ontogenetic trajectory. Age at death The age at death corresponds to the total time of dental development, from the crown initiation of the first molars (estimated from reconstructions of their worn cusp tips) to the last deposited dentin surface in the roots of the third molars. Because the M1 neonatal line was lost through dental wear, Dmanisi’s M1 crown initiation age had to be estimated from comparative data. In humans, the M1 initiation range is from approximately 0.15 years before to approximately 0.03 years after birth, that is, −0.06 ± 0.09 years (refs. , ). With the M1 initiation set to birth (0 years), two independent analyses, performed by P.T. and V.B., yielded an age at death of 11.42 ± 0.56 years (Supplementary Data ). Adding to this number the M1 initiation range yielded the following upper and lower bounds for the age at death of Dmanisi: −0.15 + 11.42 − 0.56 = 10.71 and 0.03 + 11.42 + 0.56 = 12.01 years, which together yield 11.36 ± 0.65 years. Estimating the age at dental maturity Dental maturity is reached with the root closure of the third molars. The time to root closure in the D2700/D2735 M3s was estimated by comparison with the homologous complete M2 roots. The M3 roots were scaled down to match the size of the M2 roots. Following the M2 dentin tubules, the distance from the open end of the M3 root to the closed M2 root tip was measured. Scaling this distance up to the original M3 size, and assuming an average dentin secretion rate of 4.28 μm, gives 469–561 days (about 1.3–1.5 years) to M3 root completion. The lower and upper bounds for the age at dental maturity are 10.71 + 1.3 = 12.0 years and 12.01 + 1.5 = 13.5 years. Visualization of the ontogenetic trajectory The ontogenetic trajectory was visualized using a video capturing the reconstructed developmental stages of each tooth at 6-month intervals, from birth to death, in the form of developmental plane sections (Supplementary Video ). A second video (Supplementary Video ) shows the same data in the form of virtual pseudo-orthopantomograms, which can be compared, to some extent, with clinical radiographic orthopantomograms. The videos were constructed from consecutive virtual snapshots taken at specific time points along the ontogenetic trajectory as follows. Each tooth’s developmental plane (as defined above) was used as a base image. On each image, we first annotated the stress lines (Fig. and Extended Data Figs. – ), which served as absolute time marks. Using the dentin and enamel extension rates as relative measurements of time, we then interpolated the locations of the Andresen and Retzius lines every 6 months, from birth to death. Then, working backwards from the complete developmental plane, dental material was removed virtually, and the resulting images were saved as snapshots for each 6-month step. The videos were recomposed from the single-tooth image sequences as follows. For each series (upper and lower dentition), two reference lines were defined to determine the position of the teeth before and after emergence, respectively. Pre-emergence teeth were positioned with their distal-most part at the pre-emergence line, while post-emergence teeth were positioned with their proximal-most part (crown cusps) at the post-emergence line. Finally, the images of all the teeth for each time step were merged together, and the timed snapshots were combined to form a video. The effects of tooth wear were not simulated in these videos because it would have been too difficult to make accurate interpolations of this process, so the crowns are shown with reconstructed cusp tips (recognizable by them having higher density than the preserved enamel), and the actual tooth wear is only shown at the time of death. Ontogenetic trajectory of the dentition The long dental development record of D2700/D2735 up to the subadult stage (well-developed, but open, M3 roots) allowed an almost complete dental ontogenetic trajectory to be reconstructed. The recovery of an individual longitudinal trajectory of 11.4 years is unique for fossil hominins, and even for individuals of living species, where trajectories are typically obtained by combining data from ontogenetic series rather than longitudinal ontogenetic samples. A scoring system developed previously that makes it possible to compare virtual sections with radiographic surveys was used to determine the overall maturation state of the entire dentition at 6-month intervals, from birth to death. Specifically, the maturation state of each tooth (I1, …, M3) was scored at each time step, following the procedures described in ref. . The resulting DMSs (DMS I1 , …, DMS M3 ) obtained from the virtual histology sections were then converted into equivalent clinical radiographic scores to permit direct comparisons with the radiographic data from the literature. Finally, at each time step, the DMSs of all the teeth were summed to obtain a total DMS (standardized to 100%). The resulting longitudinal ontogenetic trajectories of D2700/D2735 are visualized in Fig. (DMS I1 , …, DMS M3 ) and Fig. (total DMS). Comparative data Comparative data on the developing dentition of known-age chimpanzees and humans were collected from the literature (Supplementary Data ). The data for chimpanzees were from refs. , , and comprise all eight permanent tooth types (I1–M3). The known-age human dataset was combined from studies examining all eight teeth (I1–M3) (refs. , – ) or seven teeth (I1–M2) (refs. , – ). Comparative data for Pan paniscus came from ref. . The data for Gorilla and Pongo were acquired from medical CT scans of collection specimens (Supplementary Data and ref. ). Data on the dental ontogeny of fossil hominin specimens representing Australopithecus , Paranthropus and early Homo were collected from the literature , , , , – . Comparative data on M1 eruption were from refs. , , , . The primary data used in our comparative analyses were the DMSs of each tooth type (DMS I1 , …, DMS M3 ), and of the dentition as a whole (total DMS = sum of DMS I1 , …, DMS M3 ). The included studies were based on different dental-imaging methods (dental radiography, CT, synchrotron-based tomography) and used different DMS systems, which required prior calibration and standardization , . As a common reference, we used the Demirjian DMS system , which subdivides dental maturation into eight stages (four stages each for crown and root formation). The calibration schemes used to convert the 10-, 12- and 14-stage DMS systems to the 8-stage Demirjian system are listed in Supplementary Data . All calibrated data were standardized to total DMS = 100% for a fully formed dentition. Consistency checks to ensure the comparability of the data are described in detail further below. These checks show that, after calibration, the potential residual bias due to the use of different imaging methods and different scoring systems did not exceed the natural variation found in 1-year age bins of chimpanzees and/or humans. Data analysis We analysed the ontogeny of the dentition as a whole (hereafter referred to as the dental ontogeny) and focused on two aspects, the ontogenetic pattern and rate. The ontogenetic pattern of an individual’s dentition was given by the set of DMSs of the eight tooth types (DMS I1 , …, DMS M3 ). For a sample of n specimens, which resulted in an n × 8 DMS matrix. To analyse these multivariate data, we used the approach proposed in ref. . Using the software JMP v.15.2 (SAS Institute Inc.), a PCA was performed on the n × 8 DMS matrix, resulting in eight PCs that accounted for the largest to smallest proportions of the total variance in the sample. Typically, the first few components of a PCA comprise a large proportion of the total variance in the sample, which permits visualization of the relevant patterns of variation in the sample in a low-dimensional subspace. The DMS-PCA method, as we call it here, made it possible to document variation along and across ontogenetic trajectories and to identify taxon-specific differences between the maturation states of the different tooth types relative to each other (Fig. ). Ontogenetic trajectories start with no teeth (0,0,0,0,0,0,0,0) and end with all eight fully formed teeth (1,1,1,1,1,1,1,1), so that, in multivariate space, all trajectories have the same starting point, then diverge, finally converging again to a single end point. Therefore, taxon discrimination is best in the middle of the trajectory. In our analysis of Fig. , PC1 accounted for variation along the ontogenetic trajectories and was essentially equivalent to the total DMS (PC1 = −16.105 + 0.284 × DMS; R 2 = 0.9999; P < 0.0001), as already noted in ref. , with PC2 accounting for taxon-specific differences between trajectories. It is important to note that individual age was not part of the DMS-PCA, but served as an external variable to time-stamp taxon-specific trajectories through multivariate space. Therefore, specimens of unknown individual age, such as wild-lived great apes and many hominin fossils, can also be included in the analysis. While the DMS-PCA method provided a detailed and comprehensive picture of dental ontogenetic patterns, we noted that alternative statistical methods have been used to reach similar conclusions regarding the differences between great ape, human and fossil hominin patterns of dental ontogeny , . Ontogenetic rate To quantify the rate at which the dentition of a given individual or taxon developed, the total DMS (sum of DMS I1 , …, DMS M3 ) was plotted against age. The resulting total DMS trajectories are visualized in Fig. . Furthermore, to summarize the differences between taxon-specific trajectories, we evaluated instantaneous ontogenetic rates = (DMS( t i ) − DMS( t i −Δ t ))/Δ t , with Δ t = 0.5 years (Fig. ). Using MS Excel v.16.16.27 software (Microsoft Office), a moving-average function, comprising time steps t i − Δ t , t i and t i +Δ t , with Δ t = 0.5 years, was then applied to level out, to some extent, the discontinuities resulting from the discrete nature of the scoring procedure (Supplementary Data ). The instantaneous ontogenetic rates trajectory of chimpanzees was evaluated from the data in refs. , , . The human instantaneous ontogenetic rate trajectories were evaluated per population from the maturation score data for eight teeth , – , or seven teeth , , – , , . The human DMS trajectories show a DGS, the timing of which varies between populations (6.3–8.5 years) (see main text and Extended Data Table ). The upper range (8.5 years) largely coincides with M3 crown initiation, such that seven-teeth scoring schemes are unlikely to bias DGS estimates compared to eight-teeth scoring schemes. Consistency checks Influence of imaging methods and scoring schemes on dental maturation trajectories To assess how different imaging methods and different scoring schemes affected the evaluation of the dental ontogenetic rate and pattern, we used data from two independent studies of dental ontogeny in Finnish children, here named Haavikko70 (ref. ) and Nyström07 (ref. ). The Haavikko70 data were based on a ten-stage DMS scheme, whereas the Nyström07 data used the eight-stage Demirjian scheme . To assess the effects of imaging methodology (traditional dental radiography versus tomography-based virtual histology), we recoded the Haavikko70 data from the original radiographic to histological (tomographic) scores, using the conversion scheme presented in ref. . To assess the effects of data standardization, we recoded the Haavikko70 data with the ten-to-eight-stage recoding scheme presented in Supplementary Data . The analyses presented in Extended Data Fig. thus compare four datasets: Nyström07 eight-stage, Haavikko70 ten-stage, Haavikko70 eight-stage radiography and Haavikko70 eight-stage virtual histology. We evaluated the DMS-versus-age profiles (Extended Data Fig. ), and used a DMS-PCA to visualize the ontogenetic trajectories through multivariate space (Extended Data Fig. ). Then results show that the different scoring schemes yielded different DMS-versus-age profiles, but that standardization led to good correspondence between the two datasets. As noted earlier , , , the uncalibrated data from the virtual histology yielded total DMS-versus-age profiles that were consistently more advanced than the radiography-based profiles. Interestingly, however, in the DMS-PCA space, all four datasets (calibrated and uncalibrated) showed dental maturation trajectories that largely coincided, indicating that the DMS-PCA was robust to differences in imaging methods and scoring systems. Dental maturation profiles and evaluation of the dentition growth spurt To assess the influence of different DMS schemes on the evaluation of the DGS, we compared dental maturation data from three independent studies on Finnish children , , . These studies used different scoring schemes (ten-stage versus eight-stage), and different tooth arrays (including/excluding M3) (Extended Data Fig. and Supplementary Data ). Nevertheless, the DMS rate peaks were at similar locations along the age axis (Extended Data Fig. ), indicating that the evaluation of DGS was robust to differences in DMS scoring schemes. Assessment of age-related variation in dental maturation patterns along taxon-specific ontogenetic trajectories DMS-PCA was performed on known-age humans and chimpanzees. As shown in Extended Data Fig. , there is intra-taxon overlap between the 1 s.d.-density ellipses around consecutive 1-year age groups. The natural variation within 1-year age groups along the trajectories tended to be greater than the potential bias remaining after calibration for different data acquisition and scoring methods (Extended Data Fig. ). Reporting summary Further information on research design is available in the linked to this article.
Cranium D2700 and associated mandible D2735 were scanned at the ID19 beamline of the European Synchrotron Radiation Facility, Grenoble, France. Volume scans were performed using PPC-SRµCT. For general dental morphological assessment, as well as for the counting of Andresen lines, two configurations were used in two successive series of experiments. The first used voxel sizes of 4.89 µm with a monochromatic beam set at 51 keV to take advantage of the gadolinium K-edge of the 5-μm-thick gadox scintillator. The second series took place a few years later, after substantial technical evolution of the ID19 beamline, and was performed using a 5.06-µm voxel size using a polychromatic beam whose spectrum was shaped with a tungsten filter to isolate a peak between the K-edge of the lutecium from the lutecium–aluminium–garnet–(cerium) scintillator (63.3 keV) and the K-edge of the tungsten filter (69.5 keV). This combination enabled much faster scans than with the monochromatic setup due to the higher flux and higher energy, and with perfect beam stability and improved coherence, resulting in much better phase-contrast imaging. In both cases, the propagation distance was 5,000 mm and the scans were performed in half-acquisition to almost double the field of view of the detector. In addition, scans for daily periodicity assessment were performed at 0.75 μm at 65 KeV, also in half-acquisition. All scanning parameters are listed in Supplementary Data , and a comparison of the monochromatic and polychromatic results is presented in Extended Data Fig. .
Determination of developmental planes The dental development was tracked based on the PPC-SRµCT data of at least one tooth of each type (Supplementary Data ). Using the software VGStudio Max v.3.4 and later (Volume Graphics GmbH), virtual cross-sections through the teeth were generated to optimally visualize the microstructural growth lines in the enamel (Retzius lines) and dentin (Andresen lines). The optimum cross-sectional plane, named the developmental plane , was defined as the plane passing through a given dentin horn and the top of the underlying pulp cavity. The plane’s x axis was oriented in the bucco-lingual direction and its y axis along the tooth’s longitudinal axis. The plane’s thickness ( z -axis depth) was set to values between 100 and 150 µm. The use of these ‘thick sections’ enhanced the visibility of the developmental microstructures in the tooth . Reconstruction of worn cusp tips Several teeth of the Dmanisi individual had undergone substantial wear since eruption, as evidenced by missing enamel cusp tips and dentin horn tips (Extended Data Fig. ). Because the most relevant microstructural developmental information in the teeth of this specimen came from the dentin (see ‘Daily secretion rate’), we focused on reconstructing the relatively small missing apical volumes of the dentin horns (Extended Data Fig. ), using better-preserved teeth as guides. This process was repeated in several independent sessions. The reconstruction uncertainty inherent in this inference procedure is reflected in the calculations of the dental developmental timing and the age at death (Supplementary Data ). Matching of stress markers across the entire dentition The general sequence of tooth development is defined by the order in which teeth initiate, emerge and complete their growth. Episodes of major stress during an individual’s development tend to affect the dentition as a whole, resulting in growth discontinuities, which become manifest as stress lines on the outer crown surface and in the microscopic record of enamel and dentin growth lines. A given stress event leaves its mark on all teeth that are still in the enamel and/or dentin deposition phase at this moment. This property is an important prerequisite for placing the development of the individual teeth in a common time frame (Extended Data Fig. ). In the Dmanisi dentition, six major stress events could be identified (numbered 1 to 6 in Supplementary Data and assigned different colours in Fig. ). The matching of these stress lines across all teeth was ensured by cross-correlation between the Retzius and Andresen lines, and was further verified by comparison with the three-dimensional topology of the enamelo-dentin junction as well as the hypoplasias of the root surface (Extended Data Fig. ) and lateral enamel surface (Extended Data Figs. and ), following the methodology proposed in ref. . Long-period duration The deposition of dental enamel and dentin exhibit both short-term (daily) and long-term (several days) periodicities. In virtual PPC-SRµCT cross-sections, the daily incremental growth structures are visible as cross-striations (or laminations) in the enamel (with a size of about 1–3 μm) . Typically, the cross-striations are most visible in the relatively flat areas of the mid-lateral cuspal enamel. In enamel, the long-period structures are visible as Retzius lines (size of about 30–40 μm, measured along the enamel prisms). The number of daily increments between consecutive Retzius lines gives the value of the long-period line periodicity. This is a property that varies between individuals of a given species (for example, in humans, between 5 and 12 days , ), but can also sometimes vary within a given individual, despite general consensus to the contrary. For example, in a modern human sample of n = 40, 18 individuals (45%) showed variation in long periodicity, with longer duration in the anterior than the posterior dentition . To determine the actual long-periodicity of the D2700/D2735 dentition, scans of the enamel at a voxel resolution of 0.72 µm were performed on the URP4, URC, ULM3 and LRM3, and the cross-striations were directly traced and counted between the long-period lines (Supplementary Data and and Extended Data Fig. ). Each tooth was evaluated by two independent observers, and 178 independent measurements were taken all over the teeth. Owing to strong artefacts in the data and the non-perfect preservation of the specimens, periodicities of between 3 and 9 days were observed. The 6-day periodicity represented 41% of the measurements, the 5-day periodicity 31% and the other periodicities 28%. Given the non-normal distribution of these values (Supplementary Data ) (Kolmogorov–Smirnov test: F = 0.2465, P < 9.1693 × 10 −10 ), a Wilcoxon test was performed for comparison with expected periodicities of 5, 6 and 7 days, respectively. The only match between the expected and actual data was observed for the 6-day periodicity ( P < 0.23), while the results for 5 (8.51 × 10 −9 ) and 7 days (7.31 × 10 −10 ) suggest significant differences between the expected and observed distributions. Our data show no evidence of an antero-posterior gradient in the long-period duration . We thus used the 6-day periodicity to reconstruct the developmental profiles of all teeth by counting the Retzius and/or Andresen lines, and multiplying the total number of lines by the long-period duration. Daily secretion rate In the D2700/D2735 specimen, the long-period lines in the cuspal enamel (Retzius lines) were barely visible at the available voxel resolution (4.89 and 5.06 µm), which prevented direct measurements of the daily enamel secretion rate. However, the D2700/D2735 teeth exhibit exceptionally well-preserved dentin microstructures, which permitted an inference of the daily secretion rates by counting the Andresen lines in those areas where they were clearly visible. Specifically, the daily secretion rate of dentin was evaluated as follows. The length ( L , in microns) of a given well-preserved primary dentin tubule was measured (secondary dentin tends to show compression of the Andresen line patterns), and the number, A , of the Andresen line intervals along the tubule was counted. The daily secretion rate ( S , in microns per day) was then evaluated as S = L /( A × P ), where P is the periodicity in days (Extended Data Fig. ). The resulting value of 4.28 μm was high compared to humans and chimpanzees , , but was cross-validated by two observers, and consistent results were obtained when the different sources of information were combined. Finally, this information was combined with the Andresen line counts and the cross-matched stress markers—visible in both the enamel and the dentin—to reconstruct the absolute developmental chronology of the whole dentition (Supplementary Data ). Dental eruption time The time of dental eruption was estimated from the maximum extension rate of the root, termed the root growth spurt , (Extended Data Fig. ). The method used to determine the root extension rates was adapted from previous studies , and proceeded as follows. Along the cemento-dentin junction, a first 200-μm-diameter circle was placed at the cervix and a second was aligned at the junction between the cemento-dentin junction and the Andresen line, passing tangentially on the side of the first circle (Extended Data Fig. ). This procedure was repeated all along the length of the root. The greatest distance along the external surface of the root between the two circles marked the maximum extension rate, that is, the root growth spurt. In great apes, the root growth spurt coincides with the period of dental eruption, whereas in modern humans, the growth spurt precedes eruption , (Supplementary Data ). In the Dmanisi individual, therefore, the maximum rates of root growth should be regarded as minimum estimates of tooth eruption times. Furthermore, tooth eruption is a process that takes place over several months. We therefore represent the estimated eruption period as the time point of the maximum root extension rate ± 3 months (Fig. and Extended Data Fig. ). It should be noted that such a root growth spurt is visible in the third molars on the last third of the developing roots, while these teeth erupted quite some time before the death of the individual, as indicated by the light wear facets. This supports our basic assumption that the root growth spurt in this individual tends to coincide with the dental eruption.
The dental development was tracked based on the PPC-SRµCT data of at least one tooth of each type (Supplementary Data ). Using the software VGStudio Max v.3.4 and later (Volume Graphics GmbH), virtual cross-sections through the teeth were generated to optimally visualize the microstructural growth lines in the enamel (Retzius lines) and dentin (Andresen lines). The optimum cross-sectional plane, named the developmental plane , was defined as the plane passing through a given dentin horn and the top of the underlying pulp cavity. The plane’s x axis was oriented in the bucco-lingual direction and its y axis along the tooth’s longitudinal axis. The plane’s thickness ( z -axis depth) was set to values between 100 and 150 µm. The use of these ‘thick sections’ enhanced the visibility of the developmental microstructures in the tooth .
Several teeth of the Dmanisi individual had undergone substantial wear since eruption, as evidenced by missing enamel cusp tips and dentin horn tips (Extended Data Fig. ). Because the most relevant microstructural developmental information in the teeth of this specimen came from the dentin (see ‘Daily secretion rate’), we focused on reconstructing the relatively small missing apical volumes of the dentin horns (Extended Data Fig. ), using better-preserved teeth as guides. This process was repeated in several independent sessions. The reconstruction uncertainty inherent in this inference procedure is reflected in the calculations of the dental developmental timing and the age at death (Supplementary Data ).
The general sequence of tooth development is defined by the order in which teeth initiate, emerge and complete their growth. Episodes of major stress during an individual’s development tend to affect the dentition as a whole, resulting in growth discontinuities, which become manifest as stress lines on the outer crown surface and in the microscopic record of enamel and dentin growth lines. A given stress event leaves its mark on all teeth that are still in the enamel and/or dentin deposition phase at this moment. This property is an important prerequisite for placing the development of the individual teeth in a common time frame (Extended Data Fig. ). In the Dmanisi dentition, six major stress events could be identified (numbered 1 to 6 in Supplementary Data and assigned different colours in Fig. ). The matching of these stress lines across all teeth was ensured by cross-correlation between the Retzius and Andresen lines, and was further verified by comparison with the three-dimensional topology of the enamelo-dentin junction as well as the hypoplasias of the root surface (Extended Data Fig. ) and lateral enamel surface (Extended Data Figs. and ), following the methodology proposed in ref. .
The deposition of dental enamel and dentin exhibit both short-term (daily) and long-term (several days) periodicities. In virtual PPC-SRµCT cross-sections, the daily incremental growth structures are visible as cross-striations (or laminations) in the enamel (with a size of about 1–3 μm) . Typically, the cross-striations are most visible in the relatively flat areas of the mid-lateral cuspal enamel. In enamel, the long-period structures are visible as Retzius lines (size of about 30–40 μm, measured along the enamel prisms). The number of daily increments between consecutive Retzius lines gives the value of the long-period line periodicity. This is a property that varies between individuals of a given species (for example, in humans, between 5 and 12 days , ), but can also sometimes vary within a given individual, despite general consensus to the contrary. For example, in a modern human sample of n = 40, 18 individuals (45%) showed variation in long periodicity, with longer duration in the anterior than the posterior dentition . To determine the actual long-periodicity of the D2700/D2735 dentition, scans of the enamel at a voxel resolution of 0.72 µm were performed on the URP4, URC, ULM3 and LRM3, and the cross-striations were directly traced and counted between the long-period lines (Supplementary Data and and Extended Data Fig. ). Each tooth was evaluated by two independent observers, and 178 independent measurements were taken all over the teeth. Owing to strong artefacts in the data and the non-perfect preservation of the specimens, periodicities of between 3 and 9 days were observed. The 6-day periodicity represented 41% of the measurements, the 5-day periodicity 31% and the other periodicities 28%. Given the non-normal distribution of these values (Supplementary Data ) (Kolmogorov–Smirnov test: F = 0.2465, P < 9.1693 × 10 −10 ), a Wilcoxon test was performed for comparison with expected periodicities of 5, 6 and 7 days, respectively. The only match between the expected and actual data was observed for the 6-day periodicity ( P < 0.23), while the results for 5 (8.51 × 10 −9 ) and 7 days (7.31 × 10 −10 ) suggest significant differences between the expected and observed distributions. Our data show no evidence of an antero-posterior gradient in the long-period duration . We thus used the 6-day periodicity to reconstruct the developmental profiles of all teeth by counting the Retzius and/or Andresen lines, and multiplying the total number of lines by the long-period duration.
In the D2700/D2735 specimen, the long-period lines in the cuspal enamel (Retzius lines) were barely visible at the available voxel resolution (4.89 and 5.06 µm), which prevented direct measurements of the daily enamel secretion rate. However, the D2700/D2735 teeth exhibit exceptionally well-preserved dentin microstructures, which permitted an inference of the daily secretion rates by counting the Andresen lines in those areas where they were clearly visible. Specifically, the daily secretion rate of dentin was evaluated as follows. The length ( L , in microns) of a given well-preserved primary dentin tubule was measured (secondary dentin tends to show compression of the Andresen line patterns), and the number, A , of the Andresen line intervals along the tubule was counted. The daily secretion rate ( S , in microns per day) was then evaluated as S = L /( A × P ), where P is the periodicity in days (Extended Data Fig. ). The resulting value of 4.28 μm was high compared to humans and chimpanzees , , but was cross-validated by two observers, and consistent results were obtained when the different sources of information were combined. Finally, this information was combined with the Andresen line counts and the cross-matched stress markers—visible in both the enamel and the dentin—to reconstruct the absolute developmental chronology of the whole dentition (Supplementary Data ).
The time of dental eruption was estimated from the maximum extension rate of the root, termed the root growth spurt , (Extended Data Fig. ). The method used to determine the root extension rates was adapted from previous studies , and proceeded as follows. Along the cemento-dentin junction, a first 200-μm-diameter circle was placed at the cervix and a second was aligned at the junction between the cemento-dentin junction and the Andresen line, passing tangentially on the side of the first circle (Extended Data Fig. ). This procedure was repeated all along the length of the root. The greatest distance along the external surface of the root between the two circles marked the maximum extension rate, that is, the root growth spurt. In great apes, the root growth spurt coincides with the period of dental eruption, whereas in modern humans, the growth spurt precedes eruption , (Supplementary Data ). In the Dmanisi individual, therefore, the maximum rates of root growth should be regarded as minimum estimates of tooth eruption times. Furthermore, tooth eruption is a process that takes place over several months. We therefore represent the estimated eruption period as the time point of the maximum root extension rate ± 3 months (Fig. and Extended Data Fig. ). It should be noted that such a root growth spurt is visible in the third molars on the last third of the developing roots, while these teeth erupted quite some time before the death of the individual, as indicated by the light wear facets. This supports our basic assumption that the root growth spurt in this individual tends to coincide with the dental eruption.
Synopsis of dental development The time-calibrated microstructural growth data of the tooth crowns and roots permitted reconstruction of the complete sequence of dental development throughout the life of D2700/D2735, from the initiation to completion of each tooth. The resulting diagram is visualized in Fig. and the underlying data are presented in Supplementary Data . These data were used to determine the age at death of the individual, as well as its longitudinal dental ontogenetic trajectory. Age at death The age at death corresponds to the total time of dental development, from the crown initiation of the first molars (estimated from reconstructions of their worn cusp tips) to the last deposited dentin surface in the roots of the third molars. Because the M1 neonatal line was lost through dental wear, Dmanisi’s M1 crown initiation age had to be estimated from comparative data. In humans, the M1 initiation range is from approximately 0.15 years before to approximately 0.03 years after birth, that is, −0.06 ± 0.09 years (refs. , ). With the M1 initiation set to birth (0 years), two independent analyses, performed by P.T. and V.B., yielded an age at death of 11.42 ± 0.56 years (Supplementary Data ). Adding to this number the M1 initiation range yielded the following upper and lower bounds for the age at death of Dmanisi: −0.15 + 11.42 − 0.56 = 10.71 and 0.03 + 11.42 + 0.56 = 12.01 years, which together yield 11.36 ± 0.65 years. Estimating the age at dental maturity Dental maturity is reached with the root closure of the third molars. The time to root closure in the D2700/D2735 M3s was estimated by comparison with the homologous complete M2 roots. The M3 roots were scaled down to match the size of the M2 roots. Following the M2 dentin tubules, the distance from the open end of the M3 root to the closed M2 root tip was measured. Scaling this distance up to the original M3 size, and assuming an average dentin secretion rate of 4.28 μm, gives 469–561 days (about 1.3–1.5 years) to M3 root completion. The lower and upper bounds for the age at dental maturity are 10.71 + 1.3 = 12.0 years and 12.01 + 1.5 = 13.5 years. Visualization of the ontogenetic trajectory The ontogenetic trajectory was visualized using a video capturing the reconstructed developmental stages of each tooth at 6-month intervals, from birth to death, in the form of developmental plane sections (Supplementary Video ). A second video (Supplementary Video ) shows the same data in the form of virtual pseudo-orthopantomograms, which can be compared, to some extent, with clinical radiographic orthopantomograms. The videos were constructed from consecutive virtual snapshots taken at specific time points along the ontogenetic trajectory as follows. Each tooth’s developmental plane (as defined above) was used as a base image. On each image, we first annotated the stress lines (Fig. and Extended Data Figs. – ), which served as absolute time marks. Using the dentin and enamel extension rates as relative measurements of time, we then interpolated the locations of the Andresen and Retzius lines every 6 months, from birth to death. Then, working backwards from the complete developmental plane, dental material was removed virtually, and the resulting images were saved as snapshots for each 6-month step. The videos were recomposed from the single-tooth image sequences as follows. For each series (upper and lower dentition), two reference lines were defined to determine the position of the teeth before and after emergence, respectively. Pre-emergence teeth were positioned with their distal-most part at the pre-emergence line, while post-emergence teeth were positioned with their proximal-most part (crown cusps) at the post-emergence line. Finally, the images of all the teeth for each time step were merged together, and the timed snapshots were combined to form a video. The effects of tooth wear were not simulated in these videos because it would have been too difficult to make accurate interpolations of this process, so the crowns are shown with reconstructed cusp tips (recognizable by them having higher density than the preserved enamel), and the actual tooth wear is only shown at the time of death. Ontogenetic trajectory of the dentition The long dental development record of D2700/D2735 up to the subadult stage (well-developed, but open, M3 roots) allowed an almost complete dental ontogenetic trajectory to be reconstructed. The recovery of an individual longitudinal trajectory of 11.4 years is unique for fossil hominins, and even for individuals of living species, where trajectories are typically obtained by combining data from ontogenetic series rather than longitudinal ontogenetic samples. A scoring system developed previously that makes it possible to compare virtual sections with radiographic surveys was used to determine the overall maturation state of the entire dentition at 6-month intervals, from birth to death. Specifically, the maturation state of each tooth (I1, …, M3) was scored at each time step, following the procedures described in ref. . The resulting DMSs (DMS I1 , …, DMS M3 ) obtained from the virtual histology sections were then converted into equivalent clinical radiographic scores to permit direct comparisons with the radiographic data from the literature. Finally, at each time step, the DMSs of all the teeth were summed to obtain a total DMS (standardized to 100%). The resulting longitudinal ontogenetic trajectories of D2700/D2735 are visualized in Fig. (DMS I1 , …, DMS M3 ) and Fig. (total DMS).
The time-calibrated microstructural growth data of the tooth crowns and roots permitted reconstruction of the complete sequence of dental development throughout the life of D2700/D2735, from the initiation to completion of each tooth. The resulting diagram is visualized in Fig. and the underlying data are presented in Supplementary Data . These data were used to determine the age at death of the individual, as well as its longitudinal dental ontogenetic trajectory.
The age at death corresponds to the total time of dental development, from the crown initiation of the first molars (estimated from reconstructions of their worn cusp tips) to the last deposited dentin surface in the roots of the third molars. Because the M1 neonatal line was lost through dental wear, Dmanisi’s M1 crown initiation age had to be estimated from comparative data. In humans, the M1 initiation range is from approximately 0.15 years before to approximately 0.03 years after birth, that is, −0.06 ± 0.09 years (refs. , ). With the M1 initiation set to birth (0 years), two independent analyses, performed by P.T. and V.B., yielded an age at death of 11.42 ± 0.56 years (Supplementary Data ). Adding to this number the M1 initiation range yielded the following upper and lower bounds for the age at death of Dmanisi: −0.15 + 11.42 − 0.56 = 10.71 and 0.03 + 11.42 + 0.56 = 12.01 years, which together yield 11.36 ± 0.65 years.
Dental maturity is reached with the root closure of the third molars. The time to root closure in the D2700/D2735 M3s was estimated by comparison with the homologous complete M2 roots. The M3 roots were scaled down to match the size of the M2 roots. Following the M2 dentin tubules, the distance from the open end of the M3 root to the closed M2 root tip was measured. Scaling this distance up to the original M3 size, and assuming an average dentin secretion rate of 4.28 μm, gives 469–561 days (about 1.3–1.5 years) to M3 root completion. The lower and upper bounds for the age at dental maturity are 10.71 + 1.3 = 12.0 years and 12.01 + 1.5 = 13.5 years.
The ontogenetic trajectory was visualized using a video capturing the reconstructed developmental stages of each tooth at 6-month intervals, from birth to death, in the form of developmental plane sections (Supplementary Video ). A second video (Supplementary Video ) shows the same data in the form of virtual pseudo-orthopantomograms, which can be compared, to some extent, with clinical radiographic orthopantomograms. The videos were constructed from consecutive virtual snapshots taken at specific time points along the ontogenetic trajectory as follows. Each tooth’s developmental plane (as defined above) was used as a base image. On each image, we first annotated the stress lines (Fig. and Extended Data Figs. – ), which served as absolute time marks. Using the dentin and enamel extension rates as relative measurements of time, we then interpolated the locations of the Andresen and Retzius lines every 6 months, from birth to death. Then, working backwards from the complete developmental plane, dental material was removed virtually, and the resulting images were saved as snapshots for each 6-month step. The videos were recomposed from the single-tooth image sequences as follows. For each series (upper and lower dentition), two reference lines were defined to determine the position of the teeth before and after emergence, respectively. Pre-emergence teeth were positioned with their distal-most part at the pre-emergence line, while post-emergence teeth were positioned with their proximal-most part (crown cusps) at the post-emergence line. Finally, the images of all the teeth for each time step were merged together, and the timed snapshots were combined to form a video. The effects of tooth wear were not simulated in these videos because it would have been too difficult to make accurate interpolations of this process, so the crowns are shown with reconstructed cusp tips (recognizable by them having higher density than the preserved enamel), and the actual tooth wear is only shown at the time of death.
The long dental development record of D2700/D2735 up to the subadult stage (well-developed, but open, M3 roots) allowed an almost complete dental ontogenetic trajectory to be reconstructed. The recovery of an individual longitudinal trajectory of 11.4 years is unique for fossil hominins, and even for individuals of living species, where trajectories are typically obtained by combining data from ontogenetic series rather than longitudinal ontogenetic samples. A scoring system developed previously that makes it possible to compare virtual sections with radiographic surveys was used to determine the overall maturation state of the entire dentition at 6-month intervals, from birth to death. Specifically, the maturation state of each tooth (I1, …, M3) was scored at each time step, following the procedures described in ref. . The resulting DMSs (DMS I1 , …, DMS M3 ) obtained from the virtual histology sections were then converted into equivalent clinical radiographic scores to permit direct comparisons with the radiographic data from the literature. Finally, at each time step, the DMSs of all the teeth were summed to obtain a total DMS (standardized to 100%). The resulting longitudinal ontogenetic trajectories of D2700/D2735 are visualized in Fig. (DMS I1 , …, DMS M3 ) and Fig. (total DMS).
Comparative data on the developing dentition of known-age chimpanzees and humans were collected from the literature (Supplementary Data ). The data for chimpanzees were from refs. , , and comprise all eight permanent tooth types (I1–M3). The known-age human dataset was combined from studies examining all eight teeth (I1–M3) (refs. , – ) or seven teeth (I1–M2) (refs. , – ). Comparative data for Pan paniscus came from ref. . The data for Gorilla and Pongo were acquired from medical CT scans of collection specimens (Supplementary Data and ref. ). Data on the dental ontogeny of fossil hominin specimens representing Australopithecus , Paranthropus and early Homo were collected from the literature , , , , – . Comparative data on M1 eruption were from refs. , , , . The primary data used in our comparative analyses were the DMSs of each tooth type (DMS I1 , …, DMS M3 ), and of the dentition as a whole (total DMS = sum of DMS I1 , …, DMS M3 ). The included studies were based on different dental-imaging methods (dental radiography, CT, synchrotron-based tomography) and used different DMS systems, which required prior calibration and standardization , . As a common reference, we used the Demirjian DMS system , which subdivides dental maturation into eight stages (four stages each for crown and root formation). The calibration schemes used to convert the 10-, 12- and 14-stage DMS systems to the 8-stage Demirjian system are listed in Supplementary Data . All calibrated data were standardized to total DMS = 100% for a fully formed dentition. Consistency checks to ensure the comparability of the data are described in detail further below. These checks show that, after calibration, the potential residual bias due to the use of different imaging methods and different scoring systems did not exceed the natural variation found in 1-year age bins of chimpanzees and/or humans.
We analysed the ontogeny of the dentition as a whole (hereafter referred to as the dental ontogeny) and focused on two aspects, the ontogenetic pattern and rate. The ontogenetic pattern of an individual’s dentition was given by the set of DMSs of the eight tooth types (DMS I1 , …, DMS M3 ). For a sample of n specimens, which resulted in an n × 8 DMS matrix. To analyse these multivariate data, we used the approach proposed in ref. . Using the software JMP v.15.2 (SAS Institute Inc.), a PCA was performed on the n × 8 DMS matrix, resulting in eight PCs that accounted for the largest to smallest proportions of the total variance in the sample. Typically, the first few components of a PCA comprise a large proportion of the total variance in the sample, which permits visualization of the relevant patterns of variation in the sample in a low-dimensional subspace. The DMS-PCA method, as we call it here, made it possible to document variation along and across ontogenetic trajectories and to identify taxon-specific differences between the maturation states of the different tooth types relative to each other (Fig. ). Ontogenetic trajectories start with no teeth (0,0,0,0,0,0,0,0) and end with all eight fully formed teeth (1,1,1,1,1,1,1,1), so that, in multivariate space, all trajectories have the same starting point, then diverge, finally converging again to a single end point. Therefore, taxon discrimination is best in the middle of the trajectory. In our analysis of Fig. , PC1 accounted for variation along the ontogenetic trajectories and was essentially equivalent to the total DMS (PC1 = −16.105 + 0.284 × DMS; R 2 = 0.9999; P < 0.0001), as already noted in ref. , with PC2 accounting for taxon-specific differences between trajectories. It is important to note that individual age was not part of the DMS-PCA, but served as an external variable to time-stamp taxon-specific trajectories through multivariate space. Therefore, specimens of unknown individual age, such as wild-lived great apes and many hominin fossils, can also be included in the analysis. While the DMS-PCA method provided a detailed and comprehensive picture of dental ontogenetic patterns, we noted that alternative statistical methods have been used to reach similar conclusions regarding the differences between great ape, human and fossil hominin patterns of dental ontogeny , . Ontogenetic rate To quantify the rate at which the dentition of a given individual or taxon developed, the total DMS (sum of DMS I1 , …, DMS M3 ) was plotted against age. The resulting total DMS trajectories are visualized in Fig. . Furthermore, to summarize the differences between taxon-specific trajectories, we evaluated instantaneous ontogenetic rates = (DMS( t i ) − DMS( t i −Δ t ))/Δ t , with Δ t = 0.5 years (Fig. ). Using MS Excel v.16.16.27 software (Microsoft Office), a moving-average function, comprising time steps t i − Δ t , t i and t i +Δ t , with Δ t = 0.5 years, was then applied to level out, to some extent, the discontinuities resulting from the discrete nature of the scoring procedure (Supplementary Data ). The instantaneous ontogenetic rates trajectory of chimpanzees was evaluated from the data in refs. , , . The human instantaneous ontogenetic rate trajectories were evaluated per population from the maturation score data for eight teeth , – , or seven teeth , , – , , . The human DMS trajectories show a DGS, the timing of which varies between populations (6.3–8.5 years) (see main text and Extended Data Table ). The upper range (8.5 years) largely coincides with M3 crown initiation, such that seven-teeth scoring schemes are unlikely to bias DGS estimates compared to eight-teeth scoring schemes.
To quantify the rate at which the dentition of a given individual or taxon developed, the total DMS (sum of DMS I1 , …, DMS M3 ) was plotted against age. The resulting total DMS trajectories are visualized in Fig. . Furthermore, to summarize the differences between taxon-specific trajectories, we evaluated instantaneous ontogenetic rates = (DMS( t i ) − DMS( t i −Δ t ))/Δ t , with Δ t = 0.5 years (Fig. ). Using MS Excel v.16.16.27 software (Microsoft Office), a moving-average function, comprising time steps t i − Δ t , t i and t i +Δ t , with Δ t = 0.5 years, was then applied to level out, to some extent, the discontinuities resulting from the discrete nature of the scoring procedure (Supplementary Data ). The instantaneous ontogenetic rates trajectory of chimpanzees was evaluated from the data in refs. , , . The human instantaneous ontogenetic rate trajectories were evaluated per population from the maturation score data for eight teeth , – , or seven teeth , , – , , . The human DMS trajectories show a DGS, the timing of which varies between populations (6.3–8.5 years) (see main text and Extended Data Table ). The upper range (8.5 years) largely coincides with M3 crown initiation, such that seven-teeth scoring schemes are unlikely to bias DGS estimates compared to eight-teeth scoring schemes.
Influence of imaging methods and scoring schemes on dental maturation trajectories To assess how different imaging methods and different scoring schemes affected the evaluation of the dental ontogenetic rate and pattern, we used data from two independent studies of dental ontogeny in Finnish children, here named Haavikko70 (ref. ) and Nyström07 (ref. ). The Haavikko70 data were based on a ten-stage DMS scheme, whereas the Nyström07 data used the eight-stage Demirjian scheme . To assess the effects of imaging methodology (traditional dental radiography versus tomography-based virtual histology), we recoded the Haavikko70 data from the original radiographic to histological (tomographic) scores, using the conversion scheme presented in ref. . To assess the effects of data standardization, we recoded the Haavikko70 data with the ten-to-eight-stage recoding scheme presented in Supplementary Data . The analyses presented in Extended Data Fig. thus compare four datasets: Nyström07 eight-stage, Haavikko70 ten-stage, Haavikko70 eight-stage radiography and Haavikko70 eight-stage virtual histology. We evaluated the DMS-versus-age profiles (Extended Data Fig. ), and used a DMS-PCA to visualize the ontogenetic trajectories through multivariate space (Extended Data Fig. ). Then results show that the different scoring schemes yielded different DMS-versus-age profiles, but that standardization led to good correspondence between the two datasets. As noted earlier , , , the uncalibrated data from the virtual histology yielded total DMS-versus-age profiles that were consistently more advanced than the radiography-based profiles. Interestingly, however, in the DMS-PCA space, all four datasets (calibrated and uncalibrated) showed dental maturation trajectories that largely coincided, indicating that the DMS-PCA was robust to differences in imaging methods and scoring systems. Dental maturation profiles and evaluation of the dentition growth spurt To assess the influence of different DMS schemes on the evaluation of the DGS, we compared dental maturation data from three independent studies on Finnish children , , . These studies used different scoring schemes (ten-stage versus eight-stage), and different tooth arrays (including/excluding M3) (Extended Data Fig. and Supplementary Data ). Nevertheless, the DMS rate peaks were at similar locations along the age axis (Extended Data Fig. ), indicating that the evaluation of DGS was robust to differences in DMS scoring schemes. Assessment of age-related variation in dental maturation patterns along taxon-specific ontogenetic trajectories DMS-PCA was performed on known-age humans and chimpanzees. As shown in Extended Data Fig. , there is intra-taxon overlap between the 1 s.d.-density ellipses around consecutive 1-year age groups. The natural variation within 1-year age groups along the trajectories tended to be greater than the potential bias remaining after calibration for different data acquisition and scoring methods (Extended Data Fig. ).
To assess how different imaging methods and different scoring schemes affected the evaluation of the dental ontogenetic rate and pattern, we used data from two independent studies of dental ontogeny in Finnish children, here named Haavikko70 (ref. ) and Nyström07 (ref. ). The Haavikko70 data were based on a ten-stage DMS scheme, whereas the Nyström07 data used the eight-stage Demirjian scheme . To assess the effects of imaging methodology (traditional dental radiography versus tomography-based virtual histology), we recoded the Haavikko70 data from the original radiographic to histological (tomographic) scores, using the conversion scheme presented in ref. . To assess the effects of data standardization, we recoded the Haavikko70 data with the ten-to-eight-stage recoding scheme presented in Supplementary Data . The analyses presented in Extended Data Fig. thus compare four datasets: Nyström07 eight-stage, Haavikko70 ten-stage, Haavikko70 eight-stage radiography and Haavikko70 eight-stage virtual histology. We evaluated the DMS-versus-age profiles (Extended Data Fig. ), and used a DMS-PCA to visualize the ontogenetic trajectories through multivariate space (Extended Data Fig. ). Then results show that the different scoring schemes yielded different DMS-versus-age profiles, but that standardization led to good correspondence between the two datasets. As noted earlier , , , the uncalibrated data from the virtual histology yielded total DMS-versus-age profiles that were consistently more advanced than the radiography-based profiles. Interestingly, however, in the DMS-PCA space, all four datasets (calibrated and uncalibrated) showed dental maturation trajectories that largely coincided, indicating that the DMS-PCA was robust to differences in imaging methods and scoring systems.
To assess the influence of different DMS schemes on the evaluation of the DGS, we compared dental maturation data from three independent studies on Finnish children , , . These studies used different scoring schemes (ten-stage versus eight-stage), and different tooth arrays (including/excluding M3) (Extended Data Fig. and Supplementary Data ). Nevertheless, the DMS rate peaks were at similar locations along the age axis (Extended Data Fig. ), indicating that the evaluation of DGS was robust to differences in DMS scoring schemes.
DMS-PCA was performed on known-age humans and chimpanzees. As shown in Extended Data Fig. , there is intra-taxon overlap between the 1 s.d.-density ellipses around consecutive 1-year age groups. The natural variation within 1-year age groups along the trajectories tended to be greater than the potential bias remaining after calibration for different data acquisition and scoring methods (Extended Data Fig. ).
Further information on research design is available in the linked to this article.
Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-024-08205-2.
Reporting Summary Peer Review File Supplementary Data 1 Scanning parameters and analysis of dental incremental growth structures of specimen D2700/D2735. Supplementary Data 2 Evaluation of long-periodicity duration in D2700/D2735 teeth. Supplementary Data 3 Evaluation of daily dentin secretion rate in D2700/D2735 teeth. Supplementary Data 4 Comparative data on dental development in humans, great apes and fossil hominins. Supplementary Video 1 Virtual histological reconstruction of dental development in Dmanisi D2700/D2735. Time marks indicate age and major stress events. Tooth wear is only visualized at the time of death. Supplementary Video 2 Virtual orthopantographic reconstruction of dental development in Dmanisi D2700/D2735. Time marks indicate age and major stress events. Red indicates mirror-imaged antimere teeth. Tooth wear is only visualized at the time of death.
Source Data Fig. 3 Source Data Fig. 4
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Cytology of a seminoma in a koi ( | e7806baa-6eea-46c5-b609-54ea2da511ea | 11315794 | Pathology[mh] | Koi ( Cyprinus carpio ) is an ornamental variety of common carp frequently kept as pets in domestic ponds worldwide. Due to their tame behavior and long lifespan, they have high economic and emotional value as pets (Sirri et al. ). To date, only a few studies have reported the prevalence of tumors in koi (Sirri et al. ). Some tumors in fish have been attributed to genetic factors (Meierjohann and Schartl ; Nairn et al. ); others were associated with viral infection (Hanson et al. ; Coffee et al. ) or with environmental contamination (Fabacher and Baumann ; Baumann et al. ; Harshbarger and Clark ). However, since the presence of tumors in koi populations includes just sporadic case reports of tumors worldwide (Knüsel et al. ; Sirri et al. , ; Stegeman et al. ), data about the prevalence or significance of neoplastic lesions in koi are still missing (Ferraro et al. ). Among the tumors, neoplastic lesions of internal organs are particularly represented, with case numbers increasing over the last years (Ott Knüsel et al. ). In cyprinids, a high prevalence of spontaneous gonadal neoplasms has been reported in hybrids of goldfish Carassius auratus L. × common carp Cyprinus carpio L. (Sonstegard ; Leatherland and Sonstegard ; Dickman and Steele ; Granado-Lorencio et al. ; Down and Leatherland ; Sirri et al. ). According to data collected by breeders and examination of various previous documents, ovarian neoplasms in ornamental koi Cyprinus carpio L. are similar to those described in wild goldfish × carp hybrids: they are common in sexually mature females and originate from the ovary, although the cellular origin is often difficult to determine (Groff ). Of the gonadal tumors described in the literature in fish, ovarian tumors are the most reported, while testicular tumors are more rarely described (H. Schmidt-Posthaus & R. Knüsel unpubl. Data; Sirri et al. ). In particular, in 2010 a case of spontaneous testicular tumor was described by Sirri et al. and was classified by histological and immunohistochemical investigation according to the WHO International Histological Classification of the Tumors of the Genital System in use for mammals as diffused and classical seminoma (Kennedy et al. ; Sirri et al. ). Despite what is already reported in the literature, there are no cases in which the application of cytology has been used as a diagnostic tool to obtain an initial diagnosis at the surgical site to be confirmed later by diagnostic methods such as histology or immunohistochemistry. Therefore, the present study is the first case in which cytology is used for this purpose. In the present case, a koi was presented to the referring veterinarian due to coelomic swelling. The carp underwent surgery, which revealed an enlargement of removed testes. Testes measured 19 x 10.5 x 9 cm and 20.5 x 6 x 3.5 cm were cocoonlike and yellow whitish. Some cytological samples were performed. Cytological samples consisted of imprints obtained by placing the mass on the slide and stained with Diff Quick stain. Then, testicular samples were collected, fixed in 10% neutral buffered formalin, and serial sections were obtained and stained with Hematoxylin-Eosin (H&E) for histological examination as previously described (Armando et al. ). The cytological samples were highly cellular, poorly hemodiluted, and composed of a mixed cellular population mainly consisting of atypical cells admixed with occasional lymphocytes and embedded in a moderate amount of bluish fluid (Fig. A). Most cells consisted of round to oval cells with distinct margins, intermediate to high nucleus-cytoplasmic ratio, and moderate to scant homogeneous bluish cytoplasm. Nuclei were round and eccentric, with coarsely dispersed chromatin and occasionally a single prominent nucleolus. Anisocytosis and anisokariosis were moderate, and mitoses were rare. Numerous bi- and multinucleated atypical cells were also observed (Fig. B). Histologically, the parenchyma of both testicles was diffusely effaced and replaced by a densely cellular, multilobular, poorly demarcated, unencapsulated, infiltrative neoplasm. The neoplasm was composed of round cells arranged in sheets and small clusters, which were variably supported by a thin fibrovascular stroma. Atypical cells were round from 25 to 30 µm in diameter with abundant eosinophilic to amphophilic homogeneous cytoplasm and moderate to high nuclear-cytoplasmic ratio. Nuclei were round to oval, ranged from 15 to 25 µm in diameter, central to paracentral with finely stippled and often marginated chromatin and one occasionally visible eosinophilic nucleolus. Anisocytosis and anisokariosis were moderate to high, and there were 9 mitoses in 2.37 mm 2 ; numerous multinucleated neoplastic cells were also present. Intratumoral necrotic areas were multifocally observed (Fig. ). Considering the spread of koi, and their value as pet animals, an improvement in the veterinary diagnostic algorithmis needed. Neoplastic diseases are described in these animals, and gonadal tumors should be considered in cases of coelomic swelling in koi. In the literature, gonadal tumors in koi are described but as exposed in a recent study by Ott Knüsel et al. conducted in 2016, these tumors are mostly represented by ovarian predominantly sex cord stroma tumors, whereas tumors originating from germ cells account for only 2.5 % of coelomic neoplasms being relatively rare although reported in the literature (Ott Knüsel et al. ). The causes of the onset of these tumors are not yet known. However, the request for particular color varieties has increased the selection and inbreeding of the species; thus, a genetic predisposition has been suggested (Ott Knüsel et al. ). Moreover, since few studies exist on these tumors, environmental factors such as toxic compounds, or viral causes cannot be excluded (Sirri et al. ). In fish, organic pollutants are often absorbed through the gills and skin, and accumulate in lipid-rich tissues, such as liver, brain, gonads, and hypodermal lipid storages. (Baines et al. ) In particular, exposure to substances such as: PAH (7,12-Diniethylbenz[a]anthracene), Ethlynitrosourea, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), PCB’s, pesticides (ß-endosulfan and α-endosulfan), hydrocarbons (oil), heavy metals, are known to be related to the occurrence of gonadal tumors in fish, particularly seminomas and dysgerminomas (Baines et al. ; Bunton and Wolfe ; Spitsbergen et al. , ). In the present case, the neoplasia described was composed of cells that resemble normal germinal epithelium and have oval nuclei, straight cell borders and distinct Golgi complex,. These aspects, together with the presence of intercellular bridges, as seen in normal germinal cells, are present in seminomas (Maxie ). These histological and cytological features allow the clear distinction of these tumors from the differential diagnoses of other testicular tumors such as interstitial or Sertoli cell tumors. Given the histological and cytological findings observed in this case, the present neoplasia was diagnosed as a spontaneous seminoma. Seminomas in fish are reported in literature and are described as tumors composed of typical germ cells similar to those from humans and equivalent mammalian tumors. This enables the comparative oncologist to classify fish tumors on the same bases as mammal tumors (Masahito et al. ). According to the WHO International Histological Classification of the Tumors of the Genital System of Domestic Animals (Kennedy et al. ), the present seminoma could be classified as diffuse, given the lobular arrangement of neoplastic cells divided by a stromal component infiltrated by lymphocytes suggested a similarity with the diffuse form. However, the high malignancy of our seminoma and the probable origin of the neoplastic cells from undifferentiated seminal cells suggest that the present seminoma is ascribable to the classical type, according to the WHO classification of testicular tumors in humans (Mostofi and Sesterhenn ). As occurs for mammals, gonadal tumors are diagnosed histologically supported by cytological examination. However, in order to confirm the cytological and histological diagnosis and, above all, to classify seminomas according to classifications in human and veterinary medicine, an immunohistochemical panel, tested in a previous case of seminoma in a koi described by Sirri et al. in 2010 together with the PAS staining, is available (Sirri et al. ). This panel included several markers including in particular cytokeratin, vimentin, c-KIT, placental alkaline phosphatase (PLAP), and neuronspecific enolase (NSE), revealing an immunoreactivity of seminomatous germ cells to vimentin, PLAP, and c-KIT, but not to NSE and cytokeratin (Feitz et al. ; Foster and Ladds ; Grieco et al. ; Sirri et al. ). PLAP, which is produced ectopically by a variety of malignant tumors including human seminoma, was found to be a specific antibody for neoplastic cells of a classical histotype (Lange et al. ; Grieco et al. ). c-KIT, which is normally expressed by germ cells, has been validated as a marker to distinguish seminoma from Sertoli cell tumors, as it is also expressed by undifferentiated neoplastic seminal cells (Grieco et al. ; Yu et al. ; Sirri et al. ). However, the use of mammalian antibodies in fish tissues has certain limitations related to their specificity. In addition the immunohistochemical panel is useful for classifying the neoplasm whereas, the cytologic and histopathologic diagnosis is itself quite accurate given the particularity of the neoplasm and its very different appearance from the main differential diagnoses. Therefore, the cytological examination, which is quick, inexpensive and can be performed at the surgical site, is an excellent first-stage diagnostic tool. Little is known about the prognosis of these neoplasms as there is only one case report in the literature of a black sea bass in which surgery was performed to remove a seminoma diagnosed by histological examination. In that case, surgery was successful, as an improvement in the patient’s vital parameters and the absence of a recurrence of the neoplasm during follow-up diagnostic investigations eight weeks after surgery have been described (Weisse et al. ). The cases described in the literature concerning surgical procedures for the removal of seminomas in koi and their post-operative prognosis are rare. The present report does not provide any further information in this respect as the koi died during the surgical procedure. There are currently studies in the literature in which new anaesthetic protocols are being tested with the aim of reducing the already high anaesthesiological risk in fish. This risk depends on several factors such as the sensitivity of these species to anaesthetics, drug dosage, anaesthesia monitoring and post-operative hospitalisation (Gladden et al. ). Seminomas in koi carp are diagnosed histologically and classified immunohistochemically, but cytology, a rapid and cheap exam executable in all veterinary clinical facilities, could be a relevant preliminary diagnostic tool that may influence the entire diagnostic process. |
Ocular Nanomedicine | 0ddc53c5-5388-44a0-abfe-5e2c7e8519e2 | 9130902 | Ophthalmology[mh] | Introduction The human eye is an exclusive and intricate organ of the visual senses. Anatomically, the human eye includes unique physiological barriers, including the tear film, cornea, and conjunctival barriers constituting the anterior segment barriers, and the inner limiting membrane and blood‐retinal barrier (BRB), which form the posterior segment barriers. Clinically, millions of individuals worldwide suffer from ocular diseases, including cataracts, dry eye, conjunctivitis, keratitis, myopia, glaucoma, ocular tumors, and vitreous/retinal diseases, especially age‐related macular degeneration (AMD). Most of these conditions can result in severe visual impairment and even blindness, thus substantially impacting the quality of life. The last few decades have witnessed a surge in pathogenic investigations to elucidate various pharmacological interventions for diverse ocular diseases. For example, dexamethasone has been widely employed as a highly effective anti‐inflammatory and antibacterial biopharmaceutical that inhibits ocular inflammatory and bacterial infections. However, owing to existing ocular barriers, intrinsic deficiencies of drug delivery by eye drops or intravitreal administration, such as poor permeation, ineffective distribution, and insufficient bioavailability, limit their clinical efficacy. Additionally, recombinant proteins/peptides, especially anti‐vascular endothelial growth factor (VEGF) agents (e.g., ranibizumab and aflibercept) with high potency and activity, minimize drug–drug interactions, and are routinely recommended to inhibit neovascular fundus diseases. However, these agents require intravitreal administration owing to their strong hydrophilicity and high molecular weight, which impedes their penetration across complex tissue barriers and cell membranes. Furthermore, their rapid physical and chemical degradation raises significant challenges in long‐term therapeutic effectiveness, warranting repeated intravitreal intervention that can induce intraocular bleeding, potential infection, and discomfort, leading to poor patient compliance. Importantly, the inability of available therapeutic strategies to image and diagnose ocular diseases early and precisely monitor post‐administration could result in unsatisfactory vision recovery. Therefore, it is urgent to explore safer and more efficient alternatives to combat eye‐related diseases. With the rapid advancement of material science, nanochemistry, and nanobiotechnology, growing efforts have focused on developing safer and more effective ocular nanomedicines based on versatile nanosystems. Several nanomaterials, which are remarkably distinctive from their bulky counterparts, have been assessed to substantially modulate bioavailability, medical diagnostics, and ocular disease treatment. To a large extent, nanomedicine in ophthalmology can be attributed to its intrinsic therapeutic properties or drug/gene/cell delivery capability. Ocular nanomedicine can be elaborately engineered based on concrete biological scenarios (physicochemical cues, location, immunologic environment) to promote biostability and bioavailability in pathological regions. For instance, metal nanoparticles such as silver nanoparticles with intrinsic anti‐inflammatory, antibacterial, and antiangiogenic functions reportedly improve therapeutic efficacy in related ocular diseases. In terms of nanostructured systems, from traditional liposomes and polymers, emerging quantum dots (QDs) have been extensively used as ocular delivery carriers by expanding the encapsulated space and improving the bioavailability of ocular targeted drugs/molecules. Another considerable advantage of ocular nanomedicine is its ability to permeate across complex ocular barriers, especially the corneal‐retinal barrier and BRB, with minimal unwanted systematic/ocular side effects. Additionally, some engineered “smart” biomaterials, such as mesoporous silica nanoparticles (MSNs) with unique electronic, optical, and catalytic physiochemical properties, could trigger controllable drug release by responding to exogenous physical irradiation (e.g., light and ultrasound) or endogenous biological stimulations (e.g., redox and pH). Moreover, numerous nanomaterials utilized for sensing, imaging, and labeling biomarkers or cells/tissues involved in eye‐related diseases were deemed both evolutionary and revolutionary, along with significant success achieved in nanotechnology. For instance, nanomaterials, particularly inorganic nanoparticles, ranging from 1 to 100 nm, are comparable in size to peptide drugs and various cellular compartments (e.g., mitochondria) and exhibit a large surface area and high intracellular biodistribution, which is beneficial for in vivo imaging, biosensing, and non‐invasive tracking. In ophthalmology, routine use of ocular fluorophores under visible light irradiation may lead to ocular autofluorescence, thereby decreasing the image contrast. Multifunctional nanomaterials such as upconversion nanoparticles and QDs allow fluorescence imaging under the emission of visible and near‐infrared (NIR) light without causing ocular autofluorescence. Herein, we attempted to provide a holistic overview of nanomedicine used in ophthalmology by examining state‐of‐the‐art literature mainly reported between 2016 and 2021. Accordingly, we comprehensively reviewed the recent progress in ocular nanomedicines composed of conventional biomaterials (e.g., liposomes, polymers, metal, and metal oxide nanoparticles) and emerging nanomaterials (e.g., QDs and exosome‐based nanomaterials). Furthermore, efforts have been made to demonstrate their fundamental physicochemical properties, especially size, surface charge, hydrophilicity, hydrophobicity, and biodegradability, which are strongly associated with therapeutic effectiveness in ocular biological/pathological milieu. In addition, developmental strategies for the surface engineering of ocular nanomedicine have been discussed in detail, facilitating personalized ocular medicine, including controllable and targeted release, on‐demand gene delivery, pathology‐oriented diagnostics and therapeutics (theranostic), and side‐effect mitigation for specific paradigms. Furthermore, we emphasized their versatile ophthalmologic applications, as illustrated in Figure , especially demonstrating biomimetic ophthalmology, for example, smart electrochemical eye. Finally, inspired by current trends and therapeutic concepts, we highlighted challenges encountered during ocular nanomedicine and corresponding directions, such as the development of non‐invasive intraocular penetrable nanomaterials or nanomaterials with retrieval and recycling capability (e.g., ocular microrobots). Although the role of ocular nanomedicine remains only partially elucidated, it is highly expected that ocular nanomedicine will confer marked contributions to ocular disease management based on advanced concepts and improved theranostic performance.
Structures/Compositions of Ocular Nanomedicine The family of nanomaterials has enriched diverse nanomedical applications, given their diverse structures and compositions. The most relevant formulations in ocular nanomedicine are composed of synthetic organic nanomaterials (including lipid‐based nanoparticles, dendrimers, polymeric micelles, polyester, and natural biopolymer), inorganic nanomaterials (e.g., metal [oxide] nanoparticles, QDs, carbonous and silica nanoparticles [SiNPs]), and biological components of purified biomolecules (e.g., exosomes). The present section summarizes various ocular nanomedicines characterized by distinct compositions, unique structures, and properties based on recent representative studies, discussing their respective merits/demerits when utilized in the ophthalmologic field. 2.1 Lipid‐Based Nanoparticles Lipid‐based nanoparticles can be employed as colloidal systems and formulated using typical liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs). Liposomes, as non‐covalent aggregates, are composed of one or more phospholipid bilayer membranes and are thus capable of encapsulating both hydrophilic and/or lipophilic biopharmaceuticals. Liposomes have been widely developed for treating the anterior eye segment by topical instillation. Accordingly, cationic liposomes form ionic interactions with the cornea, resulting in high corneal drug absorption by prolonging drug residence time, along with low toxicity and antigenicity. However, drug delivery into the posterior eye segment, especially the retina, remains challenging due to complex ocular barriers. To improve cellular localization retinal distribution, light‐activated liposomes have been formulated using time‐ and site‐specific drug delivery into intraocular tissues. Moreover, liposomal nanoparticles could afford focused protection against ocular diseases by targeting specific signaling (e.g., disease driver common to different types of retinal diseases) or by engineering with a specific antibody to target the pathophysiological niche of unhealthy tissues. Accordingly, Paek et al. first designed azide‐functionalized fluorescent dipalmitoylphosphatidylcholine‐cholesterol liposomal nanoparticles. The liposomal surface was subsequently functionalized with a monoclonal antibody against intercellular adhesion molecule 1 (ICAM‐1) to confer active targeting abilities for specifically binding to induced endothelial cells in the inflamed vasculature ( Figure ). Interestingly, biocompatible liposomes for different drug delivery have been verified in several clinical trials to combat several ocular diseases, including glaucoma, retinoblastoma, and metastatic malignant uveal melanoma (Table ). However, it should be noted that potential shortcomings of liposomes, such as the formation of lipid crystal matrix, gelation tendency, in vivo burst release of cargo, and oxidation of liposomal phospholipids, may result in poor shelf‐life stability, insufficient drug loading, poor batch‐to‐batch reproducibility, and high‐cost manufacturing of liposomal products, which should be overcome in the future. Lipid nanoparticles, especially SLNs and NLCs, are important alternatives to liposomes, exhibiting an overwhelming advantage over liposomes in terms of cost‐effective manufacturing, easy scale‐up, and fewer drug‐leakage issues. Compared with the liquid state, SLNs are composed of physiological and biocompatible solid lipids such as triglycerides, steroids, and fatty acids, thereby providing high superiority to avoid organic solvent formulation. However, a short‐term release window (3 h to 2 weeks) and inadequate therapeutic concentrations of substrates in ocular tissues, such as the interface membrane of tear film/cornea, vitreous body, and retina, have been reported after SLN delivery by parenteral and topical administration. Moreover, some additional drawbacks of SLNs, including limited biopharmaceutical loading and rapid elimination/metabolism by the mononuclear phagocytic system, need to be overcome. As second‐generation lipid nanocarriers, NLCs are composed of biocompatible surfactants, drugs, lipids, or solid lipids. In contrast to SLNs, NLCs are preferred to afford pharmaceutical protection and entrapment efficiency by overcoming expulsion during phase modifications, low drug loading, and high water content of aqueous dispersions. To date, both NLCs and SLNs have shown potential for small molecule delivery into various ocular tissues; however, the capacity of SLNs to deliver peptide‐ and protein‐based biomacromolecules into ocular tissues needs to be further explored. 2.2 Polymeric Micelles Polymeric micelles (10–100 nm) comprise block copolymers with amphiphilic components, typically enabling self‐assembly under aqueous conditions to generate organized supramolecular/core–shell structures. Over the past several years, polymeric micelles, especially those formulated using poly(lactide), acrylic acid, and vinylpyrrolidone, have been routinely used as nanocarriers to improve therapeutic outcomes in different ocular diseases. Given their ability to stabilize and solubilize hydrophobic compounds, polymeric micelles reportedly enhance drug permeation by prolonging retention on the ocular surface. Facile surface modification and targeted delivery to improve drug bioavailability confer additional advantages to polymeric nanoparticles used in ophthalmology. For instance, 1, 2‐distearoyl‐sn‐glycero‐3‐phosphoethano–lamine‐ N ‐[maleimide (polyethylene glycol)‐2000] (DSPE‐PEG 2000 ‐MAL) was functionalized using a cyclic peptide ligand (GRGDSPKC) (cRGD) to develop a tailored DSPE‐PEG 2000 ‐cRGD nanomicelle for encapsulating flurbiprofen. Combining cRGD with integrin receptors on the corneal surface, the fabricated nanomicelle could specifically target the cornea, facilitating robust and rapid mucoadhesion, superior transcorneal penetration performance, and ocular surface retention of nanomicelles ( Figure ). Given the multiplicity of functional groups, some proposed micelle nanocarriers exhibit various polymer block arrangements based on diverse requirements and can deliver poorly aqueous soluble drugs by solubilizing the hydrophobic core. Additionally, this overwhelming advantage provides some important implications for the further development of ophthalmic nanoformulations. Interestingly, small‐molecule drugs such as cyclosporine A have been developed into nanomicelles and extensively studied in clinical trials for efficient control of dry eyes, further suggesting the enormous market for nanomicellar‐based products in ocular nanomedicine (Table ). However, considerable efforts should be made to overcome the limitations of self‐assembled polymeric micelles prior to extensive application in ophthalmology, such as lack of potential and adequate utilization in gene delivery, limited entrapment of macromolecule agents, and unsuitable scale‐up methods. 2.3 Dendrimers Unlike linear polymers, dendrimers with branched and layered tree‐like topologies can be divided into several generations depending on the number or size of branches and functional groups at the terminal surface. These versatile dendrimer topologies confer excellent control over dispersity, stability, specific size, molecular weight, distribution, solubility, and biological activity during dendrimer synthesis. Interestingly, dendrimers with multivalent characteristics endow higher payloads of small biological molecules, biocompatible pharmaceuticals, and imaging agents. Currently, poly(amidoamine) (PAMAM)‐derived dendrimers are the most commonly applied and commercially available systems for ocular applications, conferring enhanced drug bioavailability, tolerability, and biological response and diminishing drug clearance from the body following subconjunctival injection. Recently, Zhou et al. rationally constructed a boronic acid‐rich dendrimer (BARD) based on generation 5 PAMAM dendrimers for ocular intracellular superoxide dismutase (SOD) delivery for oxidative stress reduction in an acute retinal ischemia/reperfusion injury rat model. The authors revealed that the BARD‐mediated SOD nanoformulation could efficiently protect retinal function and reduce cell apoptosis by achieving high levels of cellular uptake without immunogenicity and cytotoxicity, thus suggesting that the PAMAM dendrimer‐based nanoformulation possesses robust efficacy for the intracellular delivery of native peptides and proteins without impairing bioactivities ( Figure ). In addition, it is considerably flexible for precisely regulating the delivery of functional dendrimers of diverse biomedical substances by interacting with highly functional amino, carboxyl, and/or hydroxyl groups on the terminal surface of dendrimers. Using co‐modification with a penetrating and cyclic RGD hexapeptide, a dendrimer‐based PAMAM system has been developed for non‐invasive, targeted penetration into the posterior ocular segment. Dendrimer‐based vehicles also exhibit powerful intrinsic anti‐inflammatory and antioxidant activities in inflamed regions, which could effectively deliver therapeutic substrates to the inflamed ocular tissue for improved therapeutic outcomes. Nonetheless, several underlying disadvantages of dendrimers in terms of toxicological issues, complex multistep synthesis, inadequate evaluations of in vivo quality control, and high cost of preparation significantly prohibit the progression and advancement of dendrimers from laboratory to clinic settings. 2.4 Polyester Nanoparticles Polyester nanoparticles are comparatively stable in vivo, tunable, and reproducible and have been positioned to provide useful solutions to a wide range of medical challenges. In ophthalmology, extensively available polyesters such as poly(lactide‐ co ‐glycoside) (PLGA), polylactic acid (PLA), and poly( ε ‐caprolactone), exhibiting highly favorable biocompatibility and biodegradability, have been extensively employed to synthesize nanoparticles, tissue‐engineered implants, and surgical sutures. In particular, PLGA nanoparticles, which are considered the least toxic, biodegradable synthetic polymers by simple elimination, have been widely employed in ophthalmology for high drug loading, efficient delivery, and long‐term release of diverse cargo molecules, ranging from hydrophobic/hydrophilic small molecules to large biopharmaceuticals. For example, a distinctive DNA/PLGA hybrid hydrogel (HDNA) with a porous structure effectively loaded water‐insoluble dexamethasone for sustained delivery for at least 4 weeks to attenuate ocular inflammatory symptoms following retinal injection ( Figure ). In addition to the various properties discussed, polyester nanoparticles can target delivery into intended intracellular sites/tissues and release the loaded cargo in a sustained fashion. A brief, low‐power, far‐ultraviolet light‐responsive degradable polymer PLGA allowed on‐demand delivery of anti‐angiogenesis molecules, thus suppressing choroidal neovascularization (CNV) in rats. This nanosystem stably retained the encapsulated molecules in the vitreous for up to 30 weeks post‐injection, thereby non‐invasively controlling the timing of drug release in response to 365 nm ultraviolet exposure. Collectively, synthetic polyester polymers exhibit a precisely controlled chemical structure and biological inertness, thus possessing great chemical versatility and high batch‐to‐batch uniformity. Nonetheless, polyester‐based nanosystems lack biological cues, given their biologically inert characteristics, which limits further progress. Accordingly, incorporating functionalities into the polyester structure to improve interactions with targeted cells/tissues remains an essential topic in the ocular and polymeric science field. 2.5 Natural Polymers Naturally occurring polymers are characterized by cell‐activated proteolytic degradation, bioactivity, very low/even no toxicity profiles, and enhanced membrane permeability. Natural biopolymer‐based nanomedicines used for ocular diseases are mainly derived from gelatin, chitosan, and its derivatives, for example, hyaluronic acid (HA). Among them, gelatin‐based hydrogels perfectly mimic the natural dermal extracellular matrix and are often used to support ocular tissue regeneration. Different technologies can readily modify mechanical, biodegradable, and biological properties in the developed cross‐linkable gelatin. For instance, an injectable, photo‐curable gelatin system was fabricated using thiolated gelatin and acrylated gelatin with tunable mechanical properties. The developed system can be employed as a potential cell‐supportive scaffold for focal corneal wound repair, and ultraviolet irradiation showed no obvious harmful effects on ocular tissues. The mechanical properties of generated hydrogels could be readily modified (0.3–22 kPa of the post‐cure shear modulus) by varying the photo intensity, the ratio of acrylate to thiol groups, and solid content. Moreover, the biodegradation times could be tuned by altering the solid content. Additionally, a recent study has reported that an injectable positive‐charge‐tuned gelatin‐tyramine hydrogel with cross‐linking ability was highly effective for siRNA delivery and protection. The carriers significantly reduced subconjunctival scarring in a rabbit model after glaucoma filtration surgery without cytotoxicity. Typically, a novel delivery system based on a charge‐tunable gelatin hydrogel is highly scalable and simple to fabricate, exhibiting strong translational potential for epigenetic silencing therapy. Chitosan possesses abundant functional groups that vary in composition, including hydroxyl, carboxyl, and amino groups that interact with mucosal surfaces to afford mucoadhesion by hydrogen bond formation. The mucoadhesive property allows nanoparticles to markedly prolong the retention time in ocular tissues and impede drug clearance. For example, DexaSite has shown success in clinical trials for treating postoperative inflammation and pain following ocular surgery (NCT03192137), which is mainly attributed to the addition of chitosan to achieve greater viscosity for effective dexamethasone delivery. To improve the time‐consuming and laborious nature of the polysaccharide fabrication process, functional drug‐free and shear flow‐driven layer‐by‐layer (SF‐LbL)‐assembled nanofilms consisting of chitosan and heparin were successfully constructed for corneal modification and defective wound healing. This strategy is expected to afford a versatile and robust nanoplatform for nanofilm surface engineering in ocular nanomedicine ( Figure ). Depending on advantages such as low immunogenicity, high transfection efficacy, and lack of mutational possibility in contrast to virus vectors, chitosan has also been identified as a potential non‐viral nanoplatform for gene delivery. HA is a biodegradable biopolymer that naturally exists within the vitreous of the eye, rendering it non‐immunogenic and highly biocompatible when employed in biomedical systems. Importantly, HA also exhibits ligands for receptors, such as CD44, located in retinal cells, which is particularly significant for ocular delivery. However, HA alone cannot form a gel; thus, hydrogels made from HA depend on cross‐linking or gelling agents and chemical modifications as both static and stimuli‐responsive. For instance, HA with several anionic carboxylic groups can be added to cationic chitosan to form chitosan‐based hybrid hydrogels (chitosan‐HA) by ionic interactions, effectively improving the drug loading efficiency and cargo delivery to the conjunctiva and cornea without apparent ocular irritation. HA hydrogels have been investigated as an ocular delivery system and an artificial vitreous substitute by chemical or enzymatic cross‐linking. Baker et al. produced a vitreous substitute comprising a novel hyaluronan‐oxime cross‐linked hydrogel by chemical modification, exhibiting physical features similar to those of the native vitreous, including density, refractive index, and transparency. The hydrogel also showed no impairment in retinal function after implantation for over 90 days when compared with eyes administered balanced saline solution, thus significantly enhancing the status quo as a vitreous substitute. Although various biodegradable natural polymers have been successfully utilized in several preclinical trials, few passively targeted nanocarriers have been applied for ocular clinical application. In addition, natural polymers such as ocular nanocarriers remain challenging in terms of chemical synthesis, precise purity, and identification of chemical structure/composition, thus inducing batch‐to‐batch variability. 2.6 Inorganic Nanomaterials Although most organic nanosystems display high biocompatibility in ophthalmologic applications, their relatively low chemical and thermal stability may hinder further development. Conversely, a vast array of rapidly progressing inorganic nanoparticles, ranging from metal nanoparticles, metal oxides, semi‐conductive QDs, graphene oxide, and SiNPs, exhibit unique intrinsic properties, especially high physiological stability, tunable morphology and structure, and easy functionalization. These inorganic nanoparticles have attracted extensive attention from the scientific community for expanding ophthalmological applications, especially wearable digital vision systems. Metal nanoparticles, especially gold nanoparticles (AuNPs) of different shapes, are widely accepted as indispensable high‐contrast agents for photothermal therapy, bioimaging, and biosensing to manage ocular disorders. Given the high efficacy of photothermal conversion using palladium‐coated gold nanorods (GNRs@Pd), GNRs@Pd and gelatin were selected as raw materials to develop a photothermal conversion gelatin hydrogel‐based mini‐eye patch. Following adherence to the lacrimal gland, the GNRs@Pd hydrogel eye patch could sense diverse visible light irradiations and spontaneously respond by heating up to secrete more tears, which is beneficial for preventing dry eye, as well as for avoiding preservatives contained in artificial tears. Additionally, based on the remarkable plasmon properties of AuNPs, Wagle et al. designed an AuNP support platform with HA coating to generate vapor nanobubbles on applying mechanical forces exerted by pulsed laser illumination. The results revealed that the nanobubbles could successfully destroy collagen aggregates with ≈1000 times less light energy and prevent damage to normal tissues, exhibiting easier, faster, and safer behavior than typical YAG laser therapy ( Figure ). Like AuNPs, a series of reports have suggested that silver nanoparticles (AgNPs) can be extensively applied in ocular diseases. For instance, AgNPs could serve as ocular bandages to kill infectious pathogens in bacterial keratitis and promote cell proliferation for ocular wound healing. As an effective antioxidant, AgNPs were also efficacious in preventing experimental selenite‐induced opacification of lenses. It has also been reported that AgNPs exert potent antiangiogenic activity and anticancer effects by suppressing cell survival during ocular neovascularization and tumor treatment. These findings suggest that AgNPs could serve as a promising candidate for managing various ocular diseases. Furthermore, nanoceria and fabricated nanomaterials reportedly possess autoregenerative and catalytic features and have been widely used as non‐enzymatic antioxidants to alleviate ocular oxidative stress and inflammation, especially for treating ocular surface diseases and long‐term reduction of intraocular pressure (IOP). These ideal outcomes revealed that metal‐ and metal oxide‐based nanoparticles could act as effective therapeutic agents for managing ocular diseases; however, their potential toxic effects on the normal retina or whole eye need to be resolved. Emerging QDs allowing easy conjugation, cost‐effective production, and stable excitable fluorescence have gained momentum in ocular bioimaging and cell/tissue labeling, electrical stimulation, or targeted delivery. Owing to the rapid development of nanotechnology, 2D transition metal dichalcogenide (XS 2 , X = Mo/W) QDs have shown significant potential for combating drug‐resistant bacteria by ion irradiation. During this process, sulfur atoms in the top layer of XS 2 were sputtered, leaving S‐vacancies and tuning the atomic ratio of S:X (XS 2–y ). S‐vacancies generated more surface electronic states, improving the quantity of charge transport on the surface of QDs; the physical contact of the microbe membrane with WS 2–y QDs (p‐type semiconductor) resulted in p‐n junctions, limiting the one‐way charge transport. Thus, WS 2–y QDs exhibited strong reactive oxygen species (ROS)‐independent oxidative stress, with a rapid response and independent of light activation ( Figure ). Although 2D transition metal nanosheets are regarded as one of the most promising candidates in ophthalmology owing to their intriguing optical properties, large surface area, and easily functionalized surface, their biosafety and long‐term effectiveness in vivo need to be further explored. Recently, a flexible optoelectronic sensor array using a combination of indium‐based colloidal QDs or perovskite QDs (as active materials) was designed for an efficient neuromorphic vision system. The practical artificial device possesses an extraordinary sensitivity to light and a specific detectivity, demonstrating neuromorphic reinforcement learning by training the sensor array with a weak light pulse. Flexibility, high integration, and ultra‐sensitivity are essential for artificial vision systems attempting to mimic biological processing. As a distinctive nanocarbon, 2D structural graphene and its derivatives with a thickness of one atomic layer graphene has also been used as multifunctional platforms for ocular applications, for example, implantable optoelectronic device for retinal stimulation and optical sensing. On account of outstanding electrical properties, graphene‐coated contact lens platform enhanced dehydration protection by monitoring the changed water evaporation rate, and further protected eyes from the damage of electromagnetic waves through dissipating electromagnetic waves in thermal radiation. It is noteworthy that a potential clinical implementation of graphene‐based biomaterials has been greatly restricted attributing to their potential toxicity, relatively poor water solubility, unsatisfied distribution, absorption, and targeted delivery. Nonporous SiNPs have been developed as a promising ophthalmic carrier thanks to their charming properties embracing a stable chemical structure, large surface area to volume ratio, and ease of surface engineering. Recent observations showed that SiNPs with small sizes (5–50 nm) could permeate into corneas. Upon topical administration, SiNPs were utilized as an effective anti‐neovascularization prophylactic agent to inhibit corneal neovascularization suffering from chemical burn. As one of representatives among mesoporous family used in ophthalmology, MSN‐based nanosystems presented a sustained effect on suppressing corneal and retinal neovascularization in vivo by prolonging drug residency in aqueous/vitreous humor and maintaining a long‐lasting drug concentration. Moreover, MSNs could conjugate with various targeted motifs to improve biocompatibility and targeted effect by enhancing cellular internalization. However, their biodegradability in a more reliable and safer performance should be reconsidered for further ocular applications since the retaining of SiNPs in human body may lead to an undesirable cellular toxicity and long‐term health effects. The unique physiochemical characteristics of inorganic nanosystems, especially electronic, optical, acoustic, catalytic, and paramagnetic properties will facilitate the development of advanced theranostic nanoplatforms in ocular photothermal therapy, photodynamic therapy, computed tomography imaging, magnetic resonance imaging, etc. Determining these optimal functionalizations of inorganic nanosystems in the process of ocular theranostics is of high significance, and will occupy an irreplaceable position in the field of ocular nanomedicine. However, their metabolic activities in ocular and systemic elimination, and the cytotoxic mechanisms/effects behind individual inorganic nanosystems in vivo impede their clinical translations and commercialization. 2.7 Exosome‐Based Nanomaterials Several typical nanocarriers, such as the aforementioned dendrimers and inorganic nanoparticles, have achieved considerable success by developing feasible and abundant strategies for versatile ocular applications; however, these exogenous nanosystems generated by artificial fabrication can exhibit apparent heterogeneity in various living organisms, possibly inducing potent immunogenicity and toxicity. In contrast, as numerous endogenous cell‐derived nanovesicles, exosomes naturally endow a low risk of immunological rejection, negligible toxicity, and superior target‐homing specificity. Several types of exosomes from mesenchymal stem cells (MSCs), including bone marrow, umbilical, and adipose MSCs, have been identified. Importantly, their substantial positive effects on anatomical and functional restoration of ocular tissues, such as the cornea, retina, and optic nerve, have been highlighted and are closely associated with different mechanisms, including the regulation of angiogenesis and inflammatory pathways, immunomodulation, and tissue regeneration. Likewise, Mathew et al. reported the neuroprotective effects of MSC‐nanovesicles, suppressing neuroinflammation and apoptosis following intravitreal administration in retinal ischemia in rats, further enhancing retinal functional recovery. Uptake of MSC‐nanovesicles by retinal ganglion cells, retinal neurons, and microglia was observed, detectable in the vitreous for 4 weeks post‐administration; this highlighted the potential of exosome‐based biomaterials to afford neuroprotection and regeneration in retinal disorders ( Figure ). As phospholipid nanocarriers, specific small molecules or large proteins can be immersed in geometrical cytosol or inlaid on exosomal topographical lipid bilayers. Therefore, exosomes exhibit considerable potential for drug/gene delivery and immunotherapy. Upon intravitreal injection, the exosome‐mediated adeno‐associated virus was broadly targeted into the retina, suggesting that exosomes could serve as a robust nanocarrier for gene delivery and expand their application in the field of ophthalmology. Two clinical trials assessing exosomes, that is, exosome‐derived miRNA and MSC‐exosomes, were initiated to further explore their potential application in managing diabetic cataract and macular holes, respectively (Table ). Exosomes can be taken up by cells via four identified mechanisms, including 1) ligand‐receptor combined with cleavage and exosomal cargo release, 2) direct fusion to the cell plasma membrane and subsequent release of exosomal content into the cytoplasm, 3) receptor‐regulated endocytosis, and 4) phagocytosis, which are beneficial to better understand intercellular communication. However, rapid clearance from the body remains a primary drawback. Although the fate of nanovesicles administered as eye drops or via intravitreal or subconjunctival routes has not been comprehensively examined, it can be speculated that a similar rapid clearance of exosomes may also occur under some conditions. On a large scale, a sustained exosome‐based delivery platform greatly relies on producing vesicles of consistent quality and high purity, another challenge that needs to be resolved. Nevertheless, based on the above overwhelming merits, exosome‐based nanosystems afford unique advantages for the rational design of next‐generation nanomedicine, facilitating studies in the interdisciplinary field of ophthalmology and nanomedicine over the next several decades.
Lipid‐Based Nanoparticles Lipid‐based nanoparticles can be employed as colloidal systems and formulated using typical liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs). Liposomes, as non‐covalent aggregates, are composed of one or more phospholipid bilayer membranes and are thus capable of encapsulating both hydrophilic and/or lipophilic biopharmaceuticals. Liposomes have been widely developed for treating the anterior eye segment by topical instillation. Accordingly, cationic liposomes form ionic interactions with the cornea, resulting in high corneal drug absorption by prolonging drug residence time, along with low toxicity and antigenicity. However, drug delivery into the posterior eye segment, especially the retina, remains challenging due to complex ocular barriers. To improve cellular localization retinal distribution, light‐activated liposomes have been formulated using time‐ and site‐specific drug delivery into intraocular tissues. Moreover, liposomal nanoparticles could afford focused protection against ocular diseases by targeting specific signaling (e.g., disease driver common to different types of retinal diseases) or by engineering with a specific antibody to target the pathophysiological niche of unhealthy tissues. Accordingly, Paek et al. first designed azide‐functionalized fluorescent dipalmitoylphosphatidylcholine‐cholesterol liposomal nanoparticles. The liposomal surface was subsequently functionalized with a monoclonal antibody against intercellular adhesion molecule 1 (ICAM‐1) to confer active targeting abilities for specifically binding to induced endothelial cells in the inflamed vasculature ( Figure ). Interestingly, biocompatible liposomes for different drug delivery have been verified in several clinical trials to combat several ocular diseases, including glaucoma, retinoblastoma, and metastatic malignant uveal melanoma (Table ). However, it should be noted that potential shortcomings of liposomes, such as the formation of lipid crystal matrix, gelation tendency, in vivo burst release of cargo, and oxidation of liposomal phospholipids, may result in poor shelf‐life stability, insufficient drug loading, poor batch‐to‐batch reproducibility, and high‐cost manufacturing of liposomal products, which should be overcome in the future. Lipid nanoparticles, especially SLNs and NLCs, are important alternatives to liposomes, exhibiting an overwhelming advantage over liposomes in terms of cost‐effective manufacturing, easy scale‐up, and fewer drug‐leakage issues. Compared with the liquid state, SLNs are composed of physiological and biocompatible solid lipids such as triglycerides, steroids, and fatty acids, thereby providing high superiority to avoid organic solvent formulation. However, a short‐term release window (3 h to 2 weeks) and inadequate therapeutic concentrations of substrates in ocular tissues, such as the interface membrane of tear film/cornea, vitreous body, and retina, have been reported after SLN delivery by parenteral and topical administration. Moreover, some additional drawbacks of SLNs, including limited biopharmaceutical loading and rapid elimination/metabolism by the mononuclear phagocytic system, need to be overcome. As second‐generation lipid nanocarriers, NLCs are composed of biocompatible surfactants, drugs, lipids, or solid lipids. In contrast to SLNs, NLCs are preferred to afford pharmaceutical protection and entrapment efficiency by overcoming expulsion during phase modifications, low drug loading, and high water content of aqueous dispersions. To date, both NLCs and SLNs have shown potential for small molecule delivery into various ocular tissues; however, the capacity of SLNs to deliver peptide‐ and protein‐based biomacromolecules into ocular tissues needs to be further explored.
Polymeric Micelles Polymeric micelles (10–100 nm) comprise block copolymers with amphiphilic components, typically enabling self‐assembly under aqueous conditions to generate organized supramolecular/core–shell structures. Over the past several years, polymeric micelles, especially those formulated using poly(lactide), acrylic acid, and vinylpyrrolidone, have been routinely used as nanocarriers to improve therapeutic outcomes in different ocular diseases. Given their ability to stabilize and solubilize hydrophobic compounds, polymeric micelles reportedly enhance drug permeation by prolonging retention on the ocular surface. Facile surface modification and targeted delivery to improve drug bioavailability confer additional advantages to polymeric nanoparticles used in ophthalmology. For instance, 1, 2‐distearoyl‐sn‐glycero‐3‐phosphoethano–lamine‐ N ‐[maleimide (polyethylene glycol)‐2000] (DSPE‐PEG 2000 ‐MAL) was functionalized using a cyclic peptide ligand (GRGDSPKC) (cRGD) to develop a tailored DSPE‐PEG 2000 ‐cRGD nanomicelle for encapsulating flurbiprofen. Combining cRGD with integrin receptors on the corneal surface, the fabricated nanomicelle could specifically target the cornea, facilitating robust and rapid mucoadhesion, superior transcorneal penetration performance, and ocular surface retention of nanomicelles ( Figure ). Given the multiplicity of functional groups, some proposed micelle nanocarriers exhibit various polymer block arrangements based on diverse requirements and can deliver poorly aqueous soluble drugs by solubilizing the hydrophobic core. Additionally, this overwhelming advantage provides some important implications for the further development of ophthalmic nanoformulations. Interestingly, small‐molecule drugs such as cyclosporine A have been developed into nanomicelles and extensively studied in clinical trials for efficient control of dry eyes, further suggesting the enormous market for nanomicellar‐based products in ocular nanomedicine (Table ). However, considerable efforts should be made to overcome the limitations of self‐assembled polymeric micelles prior to extensive application in ophthalmology, such as lack of potential and adequate utilization in gene delivery, limited entrapment of macromolecule agents, and unsuitable scale‐up methods.
Dendrimers Unlike linear polymers, dendrimers with branched and layered tree‐like topologies can be divided into several generations depending on the number or size of branches and functional groups at the terminal surface. These versatile dendrimer topologies confer excellent control over dispersity, stability, specific size, molecular weight, distribution, solubility, and biological activity during dendrimer synthesis. Interestingly, dendrimers with multivalent characteristics endow higher payloads of small biological molecules, biocompatible pharmaceuticals, and imaging agents. Currently, poly(amidoamine) (PAMAM)‐derived dendrimers are the most commonly applied and commercially available systems for ocular applications, conferring enhanced drug bioavailability, tolerability, and biological response and diminishing drug clearance from the body following subconjunctival injection. Recently, Zhou et al. rationally constructed a boronic acid‐rich dendrimer (BARD) based on generation 5 PAMAM dendrimers for ocular intracellular superoxide dismutase (SOD) delivery for oxidative stress reduction in an acute retinal ischemia/reperfusion injury rat model. The authors revealed that the BARD‐mediated SOD nanoformulation could efficiently protect retinal function and reduce cell apoptosis by achieving high levels of cellular uptake without immunogenicity and cytotoxicity, thus suggesting that the PAMAM dendrimer‐based nanoformulation possesses robust efficacy for the intracellular delivery of native peptides and proteins without impairing bioactivities ( Figure ). In addition, it is considerably flexible for precisely regulating the delivery of functional dendrimers of diverse biomedical substances by interacting with highly functional amino, carboxyl, and/or hydroxyl groups on the terminal surface of dendrimers. Using co‐modification with a penetrating and cyclic RGD hexapeptide, a dendrimer‐based PAMAM system has been developed for non‐invasive, targeted penetration into the posterior ocular segment. Dendrimer‐based vehicles also exhibit powerful intrinsic anti‐inflammatory and antioxidant activities in inflamed regions, which could effectively deliver therapeutic substrates to the inflamed ocular tissue for improved therapeutic outcomes. Nonetheless, several underlying disadvantages of dendrimers in terms of toxicological issues, complex multistep synthesis, inadequate evaluations of in vivo quality control, and high cost of preparation significantly prohibit the progression and advancement of dendrimers from laboratory to clinic settings.
Polyester Nanoparticles Polyester nanoparticles are comparatively stable in vivo, tunable, and reproducible and have been positioned to provide useful solutions to a wide range of medical challenges. In ophthalmology, extensively available polyesters such as poly(lactide‐ co ‐glycoside) (PLGA), polylactic acid (PLA), and poly( ε ‐caprolactone), exhibiting highly favorable biocompatibility and biodegradability, have been extensively employed to synthesize nanoparticles, tissue‐engineered implants, and surgical sutures. In particular, PLGA nanoparticles, which are considered the least toxic, biodegradable synthetic polymers by simple elimination, have been widely employed in ophthalmology for high drug loading, efficient delivery, and long‐term release of diverse cargo molecules, ranging from hydrophobic/hydrophilic small molecules to large biopharmaceuticals. For example, a distinctive DNA/PLGA hybrid hydrogel (HDNA) with a porous structure effectively loaded water‐insoluble dexamethasone for sustained delivery for at least 4 weeks to attenuate ocular inflammatory symptoms following retinal injection ( Figure ). In addition to the various properties discussed, polyester nanoparticles can target delivery into intended intracellular sites/tissues and release the loaded cargo in a sustained fashion. A brief, low‐power, far‐ultraviolet light‐responsive degradable polymer PLGA allowed on‐demand delivery of anti‐angiogenesis molecules, thus suppressing choroidal neovascularization (CNV) in rats. This nanosystem stably retained the encapsulated molecules in the vitreous for up to 30 weeks post‐injection, thereby non‐invasively controlling the timing of drug release in response to 365 nm ultraviolet exposure. Collectively, synthetic polyester polymers exhibit a precisely controlled chemical structure and biological inertness, thus possessing great chemical versatility and high batch‐to‐batch uniformity. Nonetheless, polyester‐based nanosystems lack biological cues, given their biologically inert characteristics, which limits further progress. Accordingly, incorporating functionalities into the polyester structure to improve interactions with targeted cells/tissues remains an essential topic in the ocular and polymeric science field.
Natural Polymers Naturally occurring polymers are characterized by cell‐activated proteolytic degradation, bioactivity, very low/even no toxicity profiles, and enhanced membrane permeability. Natural biopolymer‐based nanomedicines used for ocular diseases are mainly derived from gelatin, chitosan, and its derivatives, for example, hyaluronic acid (HA). Among them, gelatin‐based hydrogels perfectly mimic the natural dermal extracellular matrix and are often used to support ocular tissue regeneration. Different technologies can readily modify mechanical, biodegradable, and biological properties in the developed cross‐linkable gelatin. For instance, an injectable, photo‐curable gelatin system was fabricated using thiolated gelatin and acrylated gelatin with tunable mechanical properties. The developed system can be employed as a potential cell‐supportive scaffold for focal corneal wound repair, and ultraviolet irradiation showed no obvious harmful effects on ocular tissues. The mechanical properties of generated hydrogels could be readily modified (0.3–22 kPa of the post‐cure shear modulus) by varying the photo intensity, the ratio of acrylate to thiol groups, and solid content. Moreover, the biodegradation times could be tuned by altering the solid content. Additionally, a recent study has reported that an injectable positive‐charge‐tuned gelatin‐tyramine hydrogel with cross‐linking ability was highly effective for siRNA delivery and protection. The carriers significantly reduced subconjunctival scarring in a rabbit model after glaucoma filtration surgery without cytotoxicity. Typically, a novel delivery system based on a charge‐tunable gelatin hydrogel is highly scalable and simple to fabricate, exhibiting strong translational potential for epigenetic silencing therapy. Chitosan possesses abundant functional groups that vary in composition, including hydroxyl, carboxyl, and amino groups that interact with mucosal surfaces to afford mucoadhesion by hydrogen bond formation. The mucoadhesive property allows nanoparticles to markedly prolong the retention time in ocular tissues and impede drug clearance. For example, DexaSite has shown success in clinical trials for treating postoperative inflammation and pain following ocular surgery (NCT03192137), which is mainly attributed to the addition of chitosan to achieve greater viscosity for effective dexamethasone delivery. To improve the time‐consuming and laborious nature of the polysaccharide fabrication process, functional drug‐free and shear flow‐driven layer‐by‐layer (SF‐LbL)‐assembled nanofilms consisting of chitosan and heparin were successfully constructed for corneal modification and defective wound healing. This strategy is expected to afford a versatile and robust nanoplatform for nanofilm surface engineering in ocular nanomedicine ( Figure ). Depending on advantages such as low immunogenicity, high transfection efficacy, and lack of mutational possibility in contrast to virus vectors, chitosan has also been identified as a potential non‐viral nanoplatform for gene delivery. HA is a biodegradable biopolymer that naturally exists within the vitreous of the eye, rendering it non‐immunogenic and highly biocompatible when employed in biomedical systems. Importantly, HA also exhibits ligands for receptors, such as CD44, located in retinal cells, which is particularly significant for ocular delivery. However, HA alone cannot form a gel; thus, hydrogels made from HA depend on cross‐linking or gelling agents and chemical modifications as both static and stimuli‐responsive. For instance, HA with several anionic carboxylic groups can be added to cationic chitosan to form chitosan‐based hybrid hydrogels (chitosan‐HA) by ionic interactions, effectively improving the drug loading efficiency and cargo delivery to the conjunctiva and cornea without apparent ocular irritation. HA hydrogels have been investigated as an ocular delivery system and an artificial vitreous substitute by chemical or enzymatic cross‐linking. Baker et al. produced a vitreous substitute comprising a novel hyaluronan‐oxime cross‐linked hydrogel by chemical modification, exhibiting physical features similar to those of the native vitreous, including density, refractive index, and transparency. The hydrogel also showed no impairment in retinal function after implantation for over 90 days when compared with eyes administered balanced saline solution, thus significantly enhancing the status quo as a vitreous substitute. Although various biodegradable natural polymers have been successfully utilized in several preclinical trials, few passively targeted nanocarriers have been applied for ocular clinical application. In addition, natural polymers such as ocular nanocarriers remain challenging in terms of chemical synthesis, precise purity, and identification of chemical structure/composition, thus inducing batch‐to‐batch variability.
Inorganic Nanomaterials Although most organic nanosystems display high biocompatibility in ophthalmologic applications, their relatively low chemical and thermal stability may hinder further development. Conversely, a vast array of rapidly progressing inorganic nanoparticles, ranging from metal nanoparticles, metal oxides, semi‐conductive QDs, graphene oxide, and SiNPs, exhibit unique intrinsic properties, especially high physiological stability, tunable morphology and structure, and easy functionalization. These inorganic nanoparticles have attracted extensive attention from the scientific community for expanding ophthalmological applications, especially wearable digital vision systems. Metal nanoparticles, especially gold nanoparticles (AuNPs) of different shapes, are widely accepted as indispensable high‐contrast agents for photothermal therapy, bioimaging, and biosensing to manage ocular disorders. Given the high efficacy of photothermal conversion using palladium‐coated gold nanorods (GNRs@Pd), GNRs@Pd and gelatin were selected as raw materials to develop a photothermal conversion gelatin hydrogel‐based mini‐eye patch. Following adherence to the lacrimal gland, the GNRs@Pd hydrogel eye patch could sense diverse visible light irradiations and spontaneously respond by heating up to secrete more tears, which is beneficial for preventing dry eye, as well as for avoiding preservatives contained in artificial tears. Additionally, based on the remarkable plasmon properties of AuNPs, Wagle et al. designed an AuNP support platform with HA coating to generate vapor nanobubbles on applying mechanical forces exerted by pulsed laser illumination. The results revealed that the nanobubbles could successfully destroy collagen aggregates with ≈1000 times less light energy and prevent damage to normal tissues, exhibiting easier, faster, and safer behavior than typical YAG laser therapy ( Figure ). Like AuNPs, a series of reports have suggested that silver nanoparticles (AgNPs) can be extensively applied in ocular diseases. For instance, AgNPs could serve as ocular bandages to kill infectious pathogens in bacterial keratitis and promote cell proliferation for ocular wound healing. As an effective antioxidant, AgNPs were also efficacious in preventing experimental selenite‐induced opacification of lenses. It has also been reported that AgNPs exert potent antiangiogenic activity and anticancer effects by suppressing cell survival during ocular neovascularization and tumor treatment. These findings suggest that AgNPs could serve as a promising candidate for managing various ocular diseases. Furthermore, nanoceria and fabricated nanomaterials reportedly possess autoregenerative and catalytic features and have been widely used as non‐enzymatic antioxidants to alleviate ocular oxidative stress and inflammation, especially for treating ocular surface diseases and long‐term reduction of intraocular pressure (IOP). These ideal outcomes revealed that metal‐ and metal oxide‐based nanoparticles could act as effective therapeutic agents for managing ocular diseases; however, their potential toxic effects on the normal retina or whole eye need to be resolved. Emerging QDs allowing easy conjugation, cost‐effective production, and stable excitable fluorescence have gained momentum in ocular bioimaging and cell/tissue labeling, electrical stimulation, or targeted delivery. Owing to the rapid development of nanotechnology, 2D transition metal dichalcogenide (XS 2 , X = Mo/W) QDs have shown significant potential for combating drug‐resistant bacteria by ion irradiation. During this process, sulfur atoms in the top layer of XS 2 were sputtered, leaving S‐vacancies and tuning the atomic ratio of S:X (XS 2–y ). S‐vacancies generated more surface electronic states, improving the quantity of charge transport on the surface of QDs; the physical contact of the microbe membrane with WS 2–y QDs (p‐type semiconductor) resulted in p‐n junctions, limiting the one‐way charge transport. Thus, WS 2–y QDs exhibited strong reactive oxygen species (ROS)‐independent oxidative stress, with a rapid response and independent of light activation ( Figure ). Although 2D transition metal nanosheets are regarded as one of the most promising candidates in ophthalmology owing to their intriguing optical properties, large surface area, and easily functionalized surface, their biosafety and long‐term effectiveness in vivo need to be further explored. Recently, a flexible optoelectronic sensor array using a combination of indium‐based colloidal QDs or perovskite QDs (as active materials) was designed for an efficient neuromorphic vision system. The practical artificial device possesses an extraordinary sensitivity to light and a specific detectivity, demonstrating neuromorphic reinforcement learning by training the sensor array with a weak light pulse. Flexibility, high integration, and ultra‐sensitivity are essential for artificial vision systems attempting to mimic biological processing. As a distinctive nanocarbon, 2D structural graphene and its derivatives with a thickness of one atomic layer graphene has also been used as multifunctional platforms for ocular applications, for example, implantable optoelectronic device for retinal stimulation and optical sensing. On account of outstanding electrical properties, graphene‐coated contact lens platform enhanced dehydration protection by monitoring the changed water evaporation rate, and further protected eyes from the damage of electromagnetic waves through dissipating electromagnetic waves in thermal radiation. It is noteworthy that a potential clinical implementation of graphene‐based biomaterials has been greatly restricted attributing to their potential toxicity, relatively poor water solubility, unsatisfied distribution, absorption, and targeted delivery. Nonporous SiNPs have been developed as a promising ophthalmic carrier thanks to their charming properties embracing a stable chemical structure, large surface area to volume ratio, and ease of surface engineering. Recent observations showed that SiNPs with small sizes (5–50 nm) could permeate into corneas. Upon topical administration, SiNPs were utilized as an effective anti‐neovascularization prophylactic agent to inhibit corneal neovascularization suffering from chemical burn. As one of representatives among mesoporous family used in ophthalmology, MSN‐based nanosystems presented a sustained effect on suppressing corneal and retinal neovascularization in vivo by prolonging drug residency in aqueous/vitreous humor and maintaining a long‐lasting drug concentration. Moreover, MSNs could conjugate with various targeted motifs to improve biocompatibility and targeted effect by enhancing cellular internalization. However, their biodegradability in a more reliable and safer performance should be reconsidered for further ocular applications since the retaining of SiNPs in human body may lead to an undesirable cellular toxicity and long‐term health effects. The unique physiochemical characteristics of inorganic nanosystems, especially electronic, optical, acoustic, catalytic, and paramagnetic properties will facilitate the development of advanced theranostic nanoplatforms in ocular photothermal therapy, photodynamic therapy, computed tomography imaging, magnetic resonance imaging, etc. Determining these optimal functionalizations of inorganic nanosystems in the process of ocular theranostics is of high significance, and will occupy an irreplaceable position in the field of ocular nanomedicine. However, their metabolic activities in ocular and systemic elimination, and the cytotoxic mechanisms/effects behind individual inorganic nanosystems in vivo impede their clinical translations and commercialization.
Exosome‐Based Nanomaterials Several typical nanocarriers, such as the aforementioned dendrimers and inorganic nanoparticles, have achieved considerable success by developing feasible and abundant strategies for versatile ocular applications; however, these exogenous nanosystems generated by artificial fabrication can exhibit apparent heterogeneity in various living organisms, possibly inducing potent immunogenicity and toxicity. In contrast, as numerous endogenous cell‐derived nanovesicles, exosomes naturally endow a low risk of immunological rejection, negligible toxicity, and superior target‐homing specificity. Several types of exosomes from mesenchymal stem cells (MSCs), including bone marrow, umbilical, and adipose MSCs, have been identified. Importantly, their substantial positive effects on anatomical and functional restoration of ocular tissues, such as the cornea, retina, and optic nerve, have been highlighted and are closely associated with different mechanisms, including the regulation of angiogenesis and inflammatory pathways, immunomodulation, and tissue regeneration. Likewise, Mathew et al. reported the neuroprotective effects of MSC‐nanovesicles, suppressing neuroinflammation and apoptosis following intravitreal administration in retinal ischemia in rats, further enhancing retinal functional recovery. Uptake of MSC‐nanovesicles by retinal ganglion cells, retinal neurons, and microglia was observed, detectable in the vitreous for 4 weeks post‐administration; this highlighted the potential of exosome‐based biomaterials to afford neuroprotection and regeneration in retinal disorders ( Figure ). As phospholipid nanocarriers, specific small molecules or large proteins can be immersed in geometrical cytosol or inlaid on exosomal topographical lipid bilayers. Therefore, exosomes exhibit considerable potential for drug/gene delivery and immunotherapy. Upon intravitreal injection, the exosome‐mediated adeno‐associated virus was broadly targeted into the retina, suggesting that exosomes could serve as a robust nanocarrier for gene delivery and expand their application in the field of ophthalmology. Two clinical trials assessing exosomes, that is, exosome‐derived miRNA and MSC‐exosomes, were initiated to further explore their potential application in managing diabetic cataract and macular holes, respectively (Table ). Exosomes can be taken up by cells via four identified mechanisms, including 1) ligand‐receptor combined with cleavage and exosomal cargo release, 2) direct fusion to the cell plasma membrane and subsequent release of exosomal content into the cytoplasm, 3) receptor‐regulated endocytosis, and 4) phagocytosis, which are beneficial to better understand intercellular communication. However, rapid clearance from the body remains a primary drawback. Although the fate of nanovesicles administered as eye drops or via intravitreal or subconjunctival routes has not been comprehensively examined, it can be speculated that a similar rapid clearance of exosomes may also occur under some conditions. On a large scale, a sustained exosome‐based delivery platform greatly relies on producing vesicles of consistent quality and high purity, another challenge that needs to be resolved. Nevertheless, based on the above overwhelming merits, exosome‐based nanosystems afford unique advantages for the rational design of next‐generation nanomedicine, facilitating studies in the interdisciplinary field of ophthalmology and nanomedicine over the next several decades.
Fundamental Physicochemical Properties of Ocular Nanomedicine To date, the ophthalmologic field has evidenced a range of nanoplatforms with diverse compositions and nanostructures, promoted by the unparalleled feasibility and superiority of nanotechnologies. In terms of theranostics in eye‐related diseases, determining how fundamental physicochemical properties and corresponding biological effects of ocular nanomedicine guide interactions with the surrounding environment remains a consistent theme. Bioavailability, penetration, biodistribution, and elimination of ocular nanomedicine in targeted ocular cells/tissues could be significantly affected by their differential fundamental physicochemical properties, mainly including size, hydrophilicity and/or hydrophobicity, surface charge, and biodegradability, which need to be explored to achieve optimal utilization. 3.1 Size Typically, ocular nanomedicine ranges between 1 and 1000 nm, in at least one dimension. Compared with large‐scale particulate systems, corresponding ocular nanomedicines with similar chemical compositions display distinctive chemical/physical properties and biological effects. One overwhelming advantage of nanoparticles over their larger counterparts is improved cellular uptake. In nature, mammalian cells are generally a few microns in diameter, and their organelles appear to be considerably smaller within the nanometer range. The uptake of nanoparticles is a direct size‐dependent internalization and affords enhanced cellular uptake of smaller particles than larger particles. Therefore, it is postulated that nanoparticles can better locate into specific cells and organelles than micron‐sized systems. Following intraocular administration, nanoscale particles penetrate ocular physiological barriers into specific cells or tissues. Compared with larger‐scaled microspheres, considerably more fluorescence‐marked nanoscale spheres were detected in the vitreous 2 weeks after a single intravitreal delivery, with only nanospheres detected at week four following administration; this suggests the important role of biomaterial size for improved intraocular delivery. Considering nanoparticle biodistribution, nanoparticles with a diameter of 100–200 nm were evenly distributed in the vitreous, whereas nanoparticles at 50 nm could surpass retinal barriers and accumulate in the retina. Additionally, the rate constant of the in vivo circulating half‐life has received considerable attention to achieve long‐term therapeutic outcomes, which could also be impacted the biomaterial size. After systemic or topical administration, smaller nanoparticles are more likely excreted by the lymphatic, choroidal blood, or systemic circulation, while larger particles (preferably >100 nm) could avoid rapid clearance, allowing prolonged circulation within ocular tissues. These phenomena highlight that biomaterials of relatively large size are more valuable for sustained release as an ocular delivery system by prolonging their exposure and resident time in surrounding environments. In general, the size of ocular biomaterials is an essential parameter that determines their internalization, penetration, biodistribution, and elimination. 3.2 Surface Charge From an engineering perspective, the surface charge of nanomedicines is another determinant in terms of phagocytosis, penetration, and biodistribution in ophthalmology. Researchers have focused on exploring the effects of electrostatic properties on “nano‐bio” interactions. Cationic nanoparticles interacting with anionic components of intercellular surfaces or membranes could result in improved particle phagocytosis, indicating that nanoparticles with a positive charge might maximize cellular internalization when compared with negatively charged counterparts. Interestingly, the phenomenon of biological interactions and cellular uptake in response to particle charge also demonstrates the potential for targeted delivery in the absence of specifically targeted ligands. For example, the successful delivery of therapeutic antisense oligonucleotides (ASO) or plasmid DNA by a nonbiodegradable cationic polymer has been developed, which can penetrate the cornea into the retina, enabled by electrostatic interaction with the retina. Furthermore, in vivo biodistribution of nanoparticles can be influenced by surface charges through electrostatic interactions with ocular tissues. In the anterior eye segment, cationic nanoparticles, such as chitosan/peptide‐decorated polymeric and triblock copolymeric micelles, can interact with the conjunctiva and cornea (with a negatively charged mucin layer) by electrical attraction, thereby prolonging drug retention and promoting nanoformulation permeability. Conversely, nanoparticles with a negatively charged surface reportedly avoid adhesion to the normal or healthy ocular surface or cells, promoting tear retention time. In the posterior eye segments, the cellular membranes of retinal cells are characterized by negatively charged phospholipids, while the vitreous consists of a net negative charge. Considering electrostatic interactions, cationic nanoparticles are speculated to attract anionic components of the vitreous and remain localized before reaching targeted sites by delaying convective movement and diffusion, whereas anionic particles are often diffused into the vitreous body. Nevertheless, it is worth noting that the electrostatic explanation may not be adaptive to all types of nanomaterials considering other factors, such as stability or introduced ligands that may interfere or dynamically alter electrostatic behavior; this should be considered when further utilizing charged nanomaterials in the ophthalmologic field. 3.3 Hydrophilicity and/or Hydrophobicity The inherent hydrophilic, lipophilic, or amphiphilic properties of nanomaterials exert a vital effect on permeability, endocytosis, and diffusion when nanomaterials communicate with the biological interface of different ocular structures (cornea, sclera, and choroid). In the biological interface membrane of tear film/cornea, increased hydrophilicity of the nanoparticle surface could improve drug bioavailability at the ocular surface after administration. However, it has been reported that amphiphilic nanocarriers, such as peptide amphiphile nanofibers, are especially beneficial for corneal delivery, given their ability to remain at the site of interest for extended periods and the long‐term presentation of bioactive sequences. In contrast to relatively hydrophilic sclera alone, the trend of decreasing permeation with increasing nanoparticle hydrophobicity is remarkable in sclera‐choroid‐retinal pigmented epithelium (RPE) barriers; this indicates that the choroid‐Bruch's combination affords a more challenging obstacle for lipophilic nanomaterials when compared with hydrophilic ones. To achieve non‐invasive drug delivery into intraocular tissues/cells, amphiphilic core‐shell‐based nanocarriers comprising hydrophobic cores (e.g., polycaprolactone, to encapsulate hydrophobic drugs) and a hydrophilic shell (e.g., PEG, to provide excellent water dispersity) have been proposed. These strategies exhibit significant potential for the treatment of posterior segment ocular diseases by topical administration. Additionally, the hydrophilicity and/or hydrophobicity of nanomaterials is considered a critical aspect affecting cellular internalization by accommodating cellular membrane wrapping processes. The underlying basis of biological interactions with cells depends on the hydrophobic nature of nanoparticles, wherein nanosystems with more hydrophobic components than those in cellular membranes allow enhanced endocytosis and substrate combination. Conversely, nanomaterials with hydrophilic surfaces can undergo optimal uptake by retinal cells. Therefore, for ocular delivery, nanomaterials exhibiting hydrophilic, lipophilic, or amphiphilic properties need to be carefully considered under specific conditions prior to application. 3.4 Biodegradability Currently, numerous degradable nanoparticles have achieved improved ocular drug delivery, possibly attributed to their positive effects on prolonging cargo release and retention, along with alleviated cytotoxicity in ocular cells/tissues. The bioavailability and therapeutic window of cargo released from biodegradable ocular nanomedicine are strongly determined by the degradation rate of carriers in the biological milieu, and the degradation rate of nanomedicine greatly depends on their composition, molecular weight, and route of administration. For instance, nanomaterials with a low molecular weight typically undergo rapid degradation, that is, instability tends to disassemble or aggregate, which could cause burst release of cargo and compromised therapeutic outcomes. However, high‐molecular‐weight biomaterials will undergo slow in vivo degradation, inducing the risk of ocular tissue accumulation. Therefore, a controllable degradation rate for ocular nanomedicine in regions of interest remains critical to obtain higher drug availability without cytotoxicity. Accordingly, Li et al. designed microenvironment‐triggered degradable hydrogels based on ultrasmall rare‐earth nanoparticles with enhanced NIR‐II luminescence, capable of drug release by responding to glutathione and heat energy in the tumor microenvironment. The degradability of the hydrogel composites under physiological conditions is conducive for alleviating long‐term biological toxicity and promoting a photothermal‐chemotherapeutic effect ( Figure ). Conversely, the biodegradability of nanomedicine remains a particularly important consideration in gene‐based therapies for repeated gene transfection. Following cell uptake, nanocarriers need to escape intracellular compartments, such as endosomes and lysosomes, to sequentially unpack their gene payloads, such as pDNA and siRNA, via degradation. Nanocarriers possessing non‐degradable bonds and high molecular weight may markedly accumulate in normal cells, resulting in adverse effects on the metabolic activities and transportation of normal cells, eventually causing high cytotoxicity, especially after repeated administration. Thus, nanocarriers with biodegradable features benefit gene delivery processes by effectively unpacking the loaded therapeutic genes, reducing accumulation, and alleviating toxicity in targeted cells. Currently, the biodegradability of nanomaterials is the most critical feature that impacts the clinical applicability of corresponding final biomedical products, given their substantial influence on biocompatibility and biosafety. Despite exhibiting favorable biodegradability and biocompatibility, the development of biodegradable ocular nanomaterials in clinical trials remains an immense challenge. In summary, ocular nanomedicine has undergone tremendous growth based on a gradual understanding of how these parameters collectively influence the “nano‐bio” interface, especially in terms of endocytosis, penetration, cargo release, biodistribution, and elimination after interacting with ocular surroundings. Among the four key factors, the size of ocular nanomedicine plays an essential role in improving cellular uptake and penetration across ocular physiological barriers into ocular cells/tissues. Surface charge significantly influences in vivo biodistribution of nanoparticles by electrostatic interaction with intended ocular tissues. In addition, hydrophilicity and/or hydrophobicity can critically impact permeability and diffusion across biological interfaces of different ocular structures, whereas biodegradability markedly influences biosafety, cargo release, and retention in intended ocular cells. Therefore, the value of optimizing and tailoring these parameters of ocular nanomedicine to improve therapeutic outcomes needs to be highlighted in future applications.
Size Typically, ocular nanomedicine ranges between 1 and 1000 nm, in at least one dimension. Compared with large‐scale particulate systems, corresponding ocular nanomedicines with similar chemical compositions display distinctive chemical/physical properties and biological effects. One overwhelming advantage of nanoparticles over their larger counterparts is improved cellular uptake. In nature, mammalian cells are generally a few microns in diameter, and their organelles appear to be considerably smaller within the nanometer range. The uptake of nanoparticles is a direct size‐dependent internalization and affords enhanced cellular uptake of smaller particles than larger particles. Therefore, it is postulated that nanoparticles can better locate into specific cells and organelles than micron‐sized systems. Following intraocular administration, nanoscale particles penetrate ocular physiological barriers into specific cells or tissues. Compared with larger‐scaled microspheres, considerably more fluorescence‐marked nanoscale spheres were detected in the vitreous 2 weeks after a single intravitreal delivery, with only nanospheres detected at week four following administration; this suggests the important role of biomaterial size for improved intraocular delivery. Considering nanoparticle biodistribution, nanoparticles with a diameter of 100–200 nm were evenly distributed in the vitreous, whereas nanoparticles at 50 nm could surpass retinal barriers and accumulate in the retina. Additionally, the rate constant of the in vivo circulating half‐life has received considerable attention to achieve long‐term therapeutic outcomes, which could also be impacted the biomaterial size. After systemic or topical administration, smaller nanoparticles are more likely excreted by the lymphatic, choroidal blood, or systemic circulation, while larger particles (preferably >100 nm) could avoid rapid clearance, allowing prolonged circulation within ocular tissues. These phenomena highlight that biomaterials of relatively large size are more valuable for sustained release as an ocular delivery system by prolonging their exposure and resident time in surrounding environments. In general, the size of ocular biomaterials is an essential parameter that determines their internalization, penetration, biodistribution, and elimination.
Surface Charge From an engineering perspective, the surface charge of nanomedicines is another determinant in terms of phagocytosis, penetration, and biodistribution in ophthalmology. Researchers have focused on exploring the effects of electrostatic properties on “nano‐bio” interactions. Cationic nanoparticles interacting with anionic components of intercellular surfaces or membranes could result in improved particle phagocytosis, indicating that nanoparticles with a positive charge might maximize cellular internalization when compared with negatively charged counterparts. Interestingly, the phenomenon of biological interactions and cellular uptake in response to particle charge also demonstrates the potential for targeted delivery in the absence of specifically targeted ligands. For example, the successful delivery of therapeutic antisense oligonucleotides (ASO) or plasmid DNA by a nonbiodegradable cationic polymer has been developed, which can penetrate the cornea into the retina, enabled by electrostatic interaction with the retina. Furthermore, in vivo biodistribution of nanoparticles can be influenced by surface charges through electrostatic interactions with ocular tissues. In the anterior eye segment, cationic nanoparticles, such as chitosan/peptide‐decorated polymeric and triblock copolymeric micelles, can interact with the conjunctiva and cornea (with a negatively charged mucin layer) by electrical attraction, thereby prolonging drug retention and promoting nanoformulation permeability. Conversely, nanoparticles with a negatively charged surface reportedly avoid adhesion to the normal or healthy ocular surface or cells, promoting tear retention time. In the posterior eye segments, the cellular membranes of retinal cells are characterized by negatively charged phospholipids, while the vitreous consists of a net negative charge. Considering electrostatic interactions, cationic nanoparticles are speculated to attract anionic components of the vitreous and remain localized before reaching targeted sites by delaying convective movement and diffusion, whereas anionic particles are often diffused into the vitreous body. Nevertheless, it is worth noting that the electrostatic explanation may not be adaptive to all types of nanomaterials considering other factors, such as stability or introduced ligands that may interfere or dynamically alter electrostatic behavior; this should be considered when further utilizing charged nanomaterials in the ophthalmologic field.
Hydrophilicity and/or Hydrophobicity The inherent hydrophilic, lipophilic, or amphiphilic properties of nanomaterials exert a vital effect on permeability, endocytosis, and diffusion when nanomaterials communicate with the biological interface of different ocular structures (cornea, sclera, and choroid). In the biological interface membrane of tear film/cornea, increased hydrophilicity of the nanoparticle surface could improve drug bioavailability at the ocular surface after administration. However, it has been reported that amphiphilic nanocarriers, such as peptide amphiphile nanofibers, are especially beneficial for corneal delivery, given their ability to remain at the site of interest for extended periods and the long‐term presentation of bioactive sequences. In contrast to relatively hydrophilic sclera alone, the trend of decreasing permeation with increasing nanoparticle hydrophobicity is remarkable in sclera‐choroid‐retinal pigmented epithelium (RPE) barriers; this indicates that the choroid‐Bruch's combination affords a more challenging obstacle for lipophilic nanomaterials when compared with hydrophilic ones. To achieve non‐invasive drug delivery into intraocular tissues/cells, amphiphilic core‐shell‐based nanocarriers comprising hydrophobic cores (e.g., polycaprolactone, to encapsulate hydrophobic drugs) and a hydrophilic shell (e.g., PEG, to provide excellent water dispersity) have been proposed. These strategies exhibit significant potential for the treatment of posterior segment ocular diseases by topical administration. Additionally, the hydrophilicity and/or hydrophobicity of nanomaterials is considered a critical aspect affecting cellular internalization by accommodating cellular membrane wrapping processes. The underlying basis of biological interactions with cells depends on the hydrophobic nature of nanoparticles, wherein nanosystems with more hydrophobic components than those in cellular membranes allow enhanced endocytosis and substrate combination. Conversely, nanomaterials with hydrophilic surfaces can undergo optimal uptake by retinal cells. Therefore, for ocular delivery, nanomaterials exhibiting hydrophilic, lipophilic, or amphiphilic properties need to be carefully considered under specific conditions prior to application.
Biodegradability Currently, numerous degradable nanoparticles have achieved improved ocular drug delivery, possibly attributed to their positive effects on prolonging cargo release and retention, along with alleviated cytotoxicity in ocular cells/tissues. The bioavailability and therapeutic window of cargo released from biodegradable ocular nanomedicine are strongly determined by the degradation rate of carriers in the biological milieu, and the degradation rate of nanomedicine greatly depends on their composition, molecular weight, and route of administration. For instance, nanomaterials with a low molecular weight typically undergo rapid degradation, that is, instability tends to disassemble or aggregate, which could cause burst release of cargo and compromised therapeutic outcomes. However, high‐molecular‐weight biomaterials will undergo slow in vivo degradation, inducing the risk of ocular tissue accumulation. Therefore, a controllable degradation rate for ocular nanomedicine in regions of interest remains critical to obtain higher drug availability without cytotoxicity. Accordingly, Li et al. designed microenvironment‐triggered degradable hydrogels based on ultrasmall rare‐earth nanoparticles with enhanced NIR‐II luminescence, capable of drug release by responding to glutathione and heat energy in the tumor microenvironment. The degradability of the hydrogel composites under physiological conditions is conducive for alleviating long‐term biological toxicity and promoting a photothermal‐chemotherapeutic effect ( Figure ). Conversely, the biodegradability of nanomedicine remains a particularly important consideration in gene‐based therapies for repeated gene transfection. Following cell uptake, nanocarriers need to escape intracellular compartments, such as endosomes and lysosomes, to sequentially unpack their gene payloads, such as pDNA and siRNA, via degradation. Nanocarriers possessing non‐degradable bonds and high molecular weight may markedly accumulate in normal cells, resulting in adverse effects on the metabolic activities and transportation of normal cells, eventually causing high cytotoxicity, especially after repeated administration. Thus, nanocarriers with biodegradable features benefit gene delivery processes by effectively unpacking the loaded therapeutic genes, reducing accumulation, and alleviating toxicity in targeted cells. Currently, the biodegradability of nanomaterials is the most critical feature that impacts the clinical applicability of corresponding final biomedical products, given their substantial influence on biocompatibility and biosafety. Despite exhibiting favorable biodegradability and biocompatibility, the development of biodegradable ocular nanomaterials in clinical trials remains an immense challenge. In summary, ocular nanomedicine has undergone tremendous growth based on a gradual understanding of how these parameters collectively influence the “nano‐bio” interface, especially in terms of endocytosis, penetration, cargo release, biodistribution, and elimination after interacting with ocular surroundings. Among the four key factors, the size of ocular nanomedicine plays an essential role in improving cellular uptake and penetration across ocular physiological barriers into ocular cells/tissues. Surface charge significantly influences in vivo biodistribution of nanoparticles by electrostatic interaction with intended ocular tissues. In addition, hydrophilicity and/or hydrophobicity can critically impact permeability and diffusion across biological interfaces of different ocular structures, whereas biodegradability markedly influences biosafety, cargo release, and retention in intended ocular cells. Therefore, the value of optimizing and tailoring these parameters of ocular nanomedicine to improve therapeutic outcomes needs to be highlighted in future applications.
Surface Engineering of Ocular Nanomedicine Unmodified ocular nanomedicines with unique structure/composition and natural physicochemical configurations can enhance ocular penetration and bioavailability and allow sophisticated kinetic release. However, personalized ocular medicine, including controllable and targeted release, on‐demand gene delivery, pathology‐oriented diagnostics and therapeutics (theranostic), and side‐effect mitigation for specific paradigms, remains a challenge. For instance, actively targeting pathological regions by binding targeted agents to nanomedicine is necessary to achieve the high efficacy of synergistic therapy in ophthalmology. Accordingly, the functionalization of ocular nanomedicine with programmable properties using surface engineering with one or more additional entities can provide unprecedented opportunities to meet these requirements. Presently, three specific surface engineering approaches in ocular nanomedicine include: 1) decorating surfaces with certain molecules/polymers, 2) targeted modification of functional ligands/motifs, and 3) loading various therapeutic substances. Considering the first approach, that is, decorating ocular nanomedicine with a certain molecule, such as amino and carboxylic acids, or polymers (such as PEG and chitosan), has been employed to improve the physiological stability and bioavailability by controllable and targeted properties. Amino‐functionalized MSNs as nanocarriers for drug delivery achieved sustained drug release over 8 h, enhancing ocular bioavailability and allowing mucoadhesion owing to the presence of hydroxyl and amino groups. Coating with an immune evasion PEG polymer prevented recognition and uptake of ocular nanomedicine by metabolic organs in the host, with subsequent removal by macrophages in vivo. This strategy significantly improves the bioavailability of nanomaterials, thus contributing to compromised clearance and prolonging blood circulation and retention time prior to the second dose. In addition to polymeric PEG decoration, nanocarriers are directly modified with a mucoadhesive molecule, such as chitosan, to functionalize the sulfobutylether‐ β ‐cyclodextrin nanoparticles, tremendously enhancing the ocular controlled drug release with a high retention time and permeability across the ocular surface. The second approach for surface engineering in ocular nanomedicine is targeted modification of functional ligands/motifs to accomplish site‐specific therapy. The successful development of antibody technologies has increased the specificity of ocular nanomedicine via bioconjugation with different affinity ligands, ranging from biomaterials (e.g., HA), peptides (e.g., RGD peptide), and proteins (e.g., transferrin) to nucleic acids (aptamers or oligonucleotides). For example, high expression of the CD44 receptor on ocular cells, such as Müller cells and the RPE layer, could combine with HA by receptor‐mediated endocytosis, resulting in site‐ and time‐specific drug delivery to posterior ocular tissues. Typically, functionalized QDs with α v β 3 integrin receptor‐specific cyclo peptide significantly improved the combination and cellular uptake in retinal capillary endothelial cells, substantially upregulating the expression of α v β 3 integrin receptor in the early phase of degeneration. Therefore, ocular nanomedicine mediated by specific receptor‐targeted ligands would establish a basis for preventive interventions for ocular disorders at an early stage. Notably, various characteristics of ligand molecules, including solubility, biodistribution, target tissue accumulation, plasma binding, and elimination after incorporation into ocular nanomedicine, should not be restricted by the chemical compositions of nanosystems and should become an in‐part function of physicochemical properties in ocular nanomedicine. The third surface engineering strategy involves encapsulating therapeutic substances, including small molecules, peptides, proteins, nucleic acids, and clinically available biopharmaceuticals, which are then released at targeted pathological sites to accomplish corresponding diagnostic and therapeutic functions. For instance, MSNs used to deliver sodium nitroprusside (a small molecular nitric oxide‐donating drug) readily overcame drawbacks such as short duration of action and poor corneal penetration by delivering the drug to the targeted Schlemm's canal and trabecular meshwork. Typically, bioactive macromolecules, such as nucleic acids (DNA, siRNA, or miRNA), could be a highly efficient host bioreactor to code functional proteins for treating eye‐related diseases; however, they demonstrate relatively poor penetration into desired subcellular compartments, such as endosomal membranes. Thus, nanocarriers must escape from lysosomes prior to endosomal fusion. Additionally, following topical administration, the delivery of large and unstable nucleic acids into intraocular tissues could be significantly obstructed by inherent ocular absorption barriers. Alternatively, cationic liposomal components or polymers can be employed to encapsulate multiple CRISPR components into large particles (typically >100 nm in diameter). Chen et al. designed a customizable nanocapsule (25 nm diameter) based on a thin glutathione‐cleavable covalently cross‐linked polymer, coating around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and a single guide RNA (sgRNA). This customizable nanocapsule efficiently generated robust, targeted gene editing without apparent cytotoxicity, affording in situ delivery of CRISPR RNP complexes ( Figure ). Therefore, safe and effective non‐viral vehicles using different surface engineering methods are promising for ocular on‐demand gene delivery. Collectively, targeted strategies associated with ocular nanomedicine predominantly involve passive and active approaches. The passive targeted strategy (bearing no specific ligands) is directly related to their inherent physicochemical properties (e.g., size, charge), whereas the active targeted strategy is functionalized with specific affinity ligands (therapeutic or imaging agents) to facilitate cellular internalization in a highly specific manner. Additionally, to fulfill the theranostic properties of ocular nanomedicine by surface engineering with programmable entities, several factors should be carefully considered: 1) careful selection of conjugated chemical moieties to avoid unexpected toxicity resulting from residues during the synthetic process; 2) maintaining functional chemical components in ocular nanomedicine to prevent destruction by modified cargo; and 3) on‐demand release of encapsulated substances from the backbone of ocular nanomedicine while retaining functional and therapeutic properties. These proposed strategies will form a basis to achieve personalized ocular medicine in specific paradigms.
Applications of Ocular Nanomedicine Ocular diseases exert a direct impact on visual function and affect the quality of human life. However, most available strategies for targeting ocular diseases exhibit compromised therapeutic efficacy. Over the past several decades, nanotechnology, by employing sophisticated methodologies, has overtly improved the therapeutic effectiveness of ocular nanomedicine by modulating particle structures/compositions and fundamental physicochemical properties or further functionalization using surface engineering to fulfill multiple theranostic applications. The pathogenic‐oriented principle of ocular nanomedicine is an evolutionary concept that greatly depends on whether different pathogenesis involved in ocular diseases can be targeted to attain an effective therapeutic window, as susceptibility often decreases with time. The most prevalent pathological processes in various ocular diseases have been identified and are mainly classified into inflammatory (e.g., uveitis, diabetic cataracts, and dry AMD), elevated IOP (e.g., glaucoma), bacterial infection (e.g., bacterial keratitis and endophthalmitis), neovascularization (e.g., corneal neovascularization and wet AMD), tumors (e.g., retinoblastoma and uveal melanoma), and refractive error (e.g., myopia). Therefore, numerous ocular nanomedicine‐assisted strategies have been investigated to promote ocular therapeutic outcomes based on the pathogenic‐oriented principle ( Table ). 5.1 Ocular Inflammation/Oxidative‐Stress Therapy Human eyes are considered to be sealed from systematic circulation, and the retina is an “immune‐privileged” zone. Therefore, inflammation in the eye plays a pathological role in a series of ocular diseases, including uveitis, diabetic cataracts, and dry AMD, by disrupting phospholipids in the cell membrane and excessive release of inflammatory substances such as prostaglandins. These harmful substances could induce inflammatory ocular disease, accompanied by typical clinical symptoms such as photophobia, opacification, pain, posterior capsule, vasodilatation, increased vascular permeability, and IOP. For example, uveitis is a typical ocular inflammatory disorder in the uvea, possibly induced by virus/bacterial infection or a particulate matter‐induced over‐reactive immune system response. Dexamethasone, corticosteroid, and triamcinolone acetonide exert anti‐inflammatory activity for treating uveitis; however, long‐term release at the targeted site remains an immense challenge. Alternatively, the controllable release of drugs by biodegradable nanocarriers, such as PLGA‐based nanoparticles, has been successfully achieved with sustained anti‐inflammatory drug release in an experimental autoimmune uveitis animal model, thereby suppressing ocular inflammation without complications. More recently, Ganugula et al. designed the receptor‐mediated delivery of curcumin (CUR) assisted by double‐headed gambogic acid (GA)‐coupled PLGA, that is, PLGA‐GA 2 ‐CUR nanoparticles. Oral administration of these nanoparticles in a canine model with lens‐induced uveitis showed a notable drug level in aqueous humor and afforded protection against intraocular inflammation with reduced miosis, aqueous flare, and chemosis in the acute phase (<4 h) when compared with commercial anti‐inflammatory treatment (oral carprofen) ( Figure ). Excessive ROS is undoubtedly involved in the pathological origin of abnormal inflammatory responses during ocular inflammatory disorders, especially diabetic cataracts and AMD. Diabetic cataract is an ocular complication in patients with diabetes who often suffer from diminished visual function, and AMD is the leading cause of vision loss in elderly individuals with progressive photoreceptor death. However, currently available drugs demonstrate limited ability to effectively delay and prevent diabetic cataracts and dry AMD. Several studies have reported that oxidative damage in the crystalline lens and photoreceptor also plays a crucial role in the pathogenesis of diabetic cataract and dry AMD, respectively. Thus, reducing ROS levels could potentially alleviate inflammation‐associated impairment. Currently, two classes of work mechanisms have been investigated in ROS‐mediated inflammatory ocular diseases: 1) endowing ROS‐scavenging ability for guiding detoxification or 2) imparting ROS‐response anti‐inflammatory drug release. However, conventional antioxidants and anti‐inflammatory biopharmaceuticals remain largely inefficacious in ophthalmology, given their poor permeability, nonspecific biodistribution, low retention at pathological sites, and rapid renal excretion. Depending on either the non‐enzymatic or catalytic features, it has been well documented that autoregenerative redox nanoceria can effectively protect lens epithelial cells and photoreceptors from oxidative damage based on the progressive elimination of ROS, thereby maintaining lens transparency in diabetic cataracts and delaying photoreceptor degeneration in AMD for a prolonged period. Interestingly, INVELTYS (Kala Pharmaceuticals), bromfenac DuraSite, and DexaSite are important ocular nanomedicines that have succeeded in clinical trials and/or obtained FDA approval for treating postoperative inflammation and pain following ocular surgery ( Table ). INVELTYS, as a KPI‐121 1% ophthalmic nanosuspension of loteprednol etabonate, is delivered as mucus‐penetrating particles coated with low molecular weight PEG. Primary outcomes in clinical trials revealed that INVELTYS was safe and effectively resolved postoperative ocular inflammation and subject‐rated ocular pain following cataract surgery when administered twice daily for 2 weeks. The observed results could be attributed to mucus‐penetrating particles that allow the drug to efficiently penetrate the mucin layer of the tear film, facilitating drug release to underlying ocular tissues. 5.2 Ocular Hypertension Therapy Glaucoma, a type of optic neuropathy resulting from the damaged optic nerve, affects more than 60 million people worldwide. Several pathogenic parameters have been implicated in glaucoma progression, including elevated IOP, optic nerve ischemia, and oxidative stress‐related activation. Although an elevated IOP is not the only factor attributed to glaucoma development, reducing the elevated IOP directly affords effective neuroprotection and is a primary modifiable target for glaucoma control from a pathophysiological perspective. Daily topical drug application as eye drops to decrease the elevated IOP and oxidative stress is one therapeutic strategy employed in patients with glaucoma. However, developing a product for long‐term medication using eye drops remains a potential challenge in chronic glaucoma. Nanotechnology has been used as a valuable tool to fabricate different nanosystems, such as lipid DNA nanoparticles, hollow ceria nanoparticles, and layer‐by‐layer coated siRNA nanoparticles. Reportedly, nanoparticles could afford long‐term alleviation of glaucomatous damage without disrupting the vesicle shape, owing to potent inherent antioxidant and anti‐inflammatory functions or controllable and sustained release of anti‐IOP drugs or siRNA gene silencing. More recently, a cell‐softening nanotherapy has been proposed to reduce IOP by targeted binding to the vascular endothelial growth factor receptor 3 (VEGFR3)/FLT4 receptor expressed in Schlemm's canal cells. These nanomicelles (tLatA‐MC) are based on a targeted PEG‐ b ‐poly(propylene sulfide) micelle (PEG‐ b ‐PPS) and loaded with the actin depolymerizer latrunculin A (tLatA) to selectively modulate the stiffness of Schlemm's canal cells (an important pathophysiology during glaucoma). tLatA‐MC reduced IOP in a mouse model by 30–50%, indicating the functional efficacy of tLatA‐MC for glaucoma treatment ( Figure ). Conversely, a non‐invasive approach using nanotechnology to monitor the stiffness of the trabecular meshwork in vivo also exhibits significant potential to improve the clinical care of glaucoma. These promising results notably demonstrate the enormous promise of an ocular nanomedicine‐based strategy for chronic glaucoma therapy. 5.3 Ocular Bacterial Infection Therapy Bacteria often play a significant antagonistic and pathogenic role in self‐contained and immune‐privileged ocular tissues by disrupting normal metabolic processes and even causing cell death, collectively considered ocular bacterial infections. The downstream diseases of ocular bacterial infections in the external (e.g., bacterial keratitis) and inner parts of the eyes (e.g., endophthalmitis) may pose a serious threat to visual health. Therefore, it is crucial to employ efficacious therapeutic interventions to address this dilemma. Over several decades, antibiotics have rapidly advanced as ideal terminators of nearly all bacteria‐mediated diseases. However, this expectation has been quickly tempered with the emergence of the first‐rank bacterial pathogens, that is, multidrug‐resistant (MDR) bacteria, particularly Escherichia coli and Klebsiella pneumoniae , which mainly contribute to the existence of biofilms. Biofilms are reportedly involved in 80% of bacterial infectious diseases and are composed of extracellular polymeric substances, water, and bacteria. Among them, bacteria play a vital role in protecting biofilm matrices by markedly increasing the resistance to bactericides. For instance, the dormancy of the bottom bacteria in MDR bacteria significantly undermined bactericide permeability. Accordingly, versatile antibacterial surface/antibiofilm‐based ocular nanomedicines have been developed to kill live bacteria and remove mature biofilms or decrease bacterial adhesion to suppress biofilm development, resulting in potent antibacterial treatment. Patients with bacterial keratitis typically experience unexpected corneal epithelium damage, owing to difficulties in spontaneous recovery after bacterial infection. Therefore, it is crucial to develop an effective approach to eradicate MDR infections and simultaneously promote injury healing. More recently, Qiao et al. proposed a composite cupriferous hollow nanoshell (AuAgCu2O NS) comprising a Cu 2 O shell and a hollow gold‐silver (AuAg) core as a high‐quality photothermal therapeutic agent for treating non‐healing keratitis. The authors revealed that Ag released from the hollow AuAg core exhibited a synergistic effect against MDR bacteria. In addition, copper ions released from the Cu 2 O shell hastened fibroblast cell migration and endothelial cell angiogenesis, augmenting the wound healing efficacy in keratitis models. Endophthalmitis is primarily caused by pathogenic microorganisms, particularly bacteria, which can lead to eyeball damage and distinct inflammation in ocular tissues, especially the uvea and vitreous cavity, seriously threatening the visual function of patients. Currently, comprehensive elimination of endophthalmitis remains a challenge, and clinical antibiotics are only employed to control progressive development. Thus, a strategic approach using targeted phototherapy has been proposed to manage endophthalmitis by biofilm eradication. For example, synergistic photodynamic therapy and chemotherapy to treat endophthalmitis were developed using a pH‐responsive hybrid system, that is, zeolitic imidazolate framework‐8‐polyacrylic acid (ZIF‐8‐PAA)‐methylbenzene blue (MB)@silver nanoparticles (AgNPs)@vancomycin (Van)/NH‐PEG (ZPMAVP). ZIF‐8‐PAA significantly improved the drug‐encapsulation capability, facilitated controllable and targeted delivery of a photosensitizer antibacterial agent MB, and reduced AgNO into AgNPs in situ by dopamine modification. Upon secondary functionalization with Van/NH‐PEG, this platform ultimately formed a synergistic composite. Generally, nanomaterials can rapidly release MB to enrich the local therapeutic concentration and damage bacterial biofilms with good biocompatibility, suggesting the superiority of synergistic photodynamic therapy and chemotherapy over any single therapeutic strategy for bacterial ophthalmic diseases ( Figure ). 5.4 Ocular Neovascularization Therapy Ocular neovascularization, particularly corneal neovascularization and CNV, is one primary pathological phenotype known to occur in corneal and retinal diseases, respectively. The normal cornea is avascular, and corneal neovascularization is often initiated by ocular surface injuries that can disrupt the harmonic balance between angiogenic inhibitors and stimulators, eventually resulting in visual impairment. In wet AMD, pathogenic vessels protrude through Bruch's membrane and then grow into the subretinal space, leading to initial CNV and distorted central vision. For antiangiogenic therapeutics, targeted inhibition of VEGF (a predominant stimulator during angiogenesis) triggers a signal cascade that suppresses migration and proliferation of endothelial cells and decreases vascular permeability. Clinically, monthly or bimonthly intravitreal injection of large protein anti‐VEGF drugs, such as bevacizumab, is a major therapeutic strategy for CNV. However, anti‐VEGF strategies afford a transient therapeutic outcome, owing to their rapid degradation and poor targeting in dysfunctional existing vessels. These drawbacks necessitate repeated administration to maintain effectiveness and subsequently cause potential infection and tissue injury. In addition, ocular nanomedicine‐assisted procedures have been successfully engineered with inherent antiangiogenic properties derived from a natural product, administered via eye drops or conjunctival sac instillation to achieve minimally invasive intraocular delivery, or via targeted and sustained release of anti‐VEGF peptide/antibody and siRNA‐oriented intracellular therapeutic agents at therapeutically relevant concentrations to minimize dosage intervals. Reportedly, externally triggered targeted delivery allows ocular drug delivery with high temporal and spatial resolution. Among external stimulators, light is particularly attractive as an energy source for retinal targeting, as the eye is naturally used to admit light. Wang et al. designed smart photo‐targeted nanoparticles by self‐assembling a chemically modified PEG‐PLA block copolymer, further modified with a targeting moiety, such as a cell‐penetrating peptide for high cellular uptake. After intravenous administration, the nanoparticles are converted to a tissue‐targeting state depending on the irradiation in the eye. This strategy allows the noninvasive treatment of CNV by allowing target drug accumulation in the diseased ocular areas, simultaneously minimizing drug deposition at off‐target sites in healthy ocular/systemic sites. Additionally, oxidative stress and inflammation play a crucial pathogenic role in angiogenesis during AMD pathogenesis. Mitra et al. designed glycol chitosan‐coated antioxidant ceria nanoparticles, which were autoregenerative and more active in blocking laser‐induced CNV models. On downregulating ROS‐activated pro‐angiogenic VEGF expression, the nanoparticles significantly rescued the cumulative oxidative damage and recruited endothelial precursor cells without affording any toxicity ( Figure ). Overall, the ocular nanomedicine‐assisted strategy provides an alternative intervention to optimally inhibit ocular neovascularization without damaging healthy tissues in a non‐invasive or minimally invasive manner while reducing the repeated administration to establish therapeutic control. 5.5 Ocular Tissue Engineering The structure and function of injured cells, tissues, and organs after experiencing trauma, infection, or inflammation can be damaged if healing remains unsatisfactory. For example, retinal degeneration is recognized as one predominant cause underlying irreversible visual impairment and even blindness worldwide, and the death of RPE and/or photoreceptors has been considered common pathogenesis. Hence, RPE/photoreceptor‐based transplantation into diseased sites is necessary to generate monolayer cells and recover the RPE‐photoreceptor interface. Over the past two decades, ocular tissue engineering, including corneal, conjunctival, and retinal regeneration, has been shown to be feasible by transplanting cell/tissue‐derived implants. However, these are limited in terms of specific responses to biological cues and superior reconstruction of normal functions. However, ocular nanomedicine‐based strategies could introduce and guide ocular tissue regeneration by stimulating the signaling pathway by communicating with surroundings or serving as a sensor to monitor/trace transplanted cell replacement and tissue repair. As an emerging nanomaterial, tetrahedral framework nucleic acids (tFNAs) accelerated the re‐epithelialization by promoting the migration and proliferation of human corneal epithelial cells. Moreover, tFNAs showed high efficacy in recovering corneal transparency and corneal wound healing in an established corneal alkali burn animal model. In retinal tissue engineering, a micropatterned PLGA nanosheet with perfect flexibility has been adopted to achieve a good microenvironment for the stable attachment of a transplanted RPE monolayer. By further embedding with magnetic nanoparticles, the nanosheet promoted malleability, optical visualization, and cell morphogenesis. After subretinal injection into the swine eye, this nanosheet exhibited stable attachment to the macula, and the implanted RPE monolayer could differentiate into a cobblestone‐like structure. Thus, nanomedicine‐aided ocular regenerative medicine is expected to establish an innovative therapeutic strategy by guiding the uninjurious cellular organization. Human eyes have exceptional image sensing functions, such as high sensitivity and resolution with low aberration and a spectacularly wide field of view. During the process, the retina efficiently detects light stimuli and pre‐processes image information in parallel, prior to the brain performing more complex actions. Biomimetic eyes with similar superior features are extremely desirable for different technological applications. In recent years, digital vision systems based on charge‐coupled device cameras or conventional, complementary metal‐oxide‐semiconductor imagers have been explored to fulfill computer vision by interface‐interfaced digital processing units on coarsely parallel or serial structures. Recently, a unique biomimetic electrochemical eye (EC‐EYE) using a hemispherical retina has been constructed based on a high‐density array of perovskite nanowires to mimic photoreceptors on an actual retina. A front‐side common contact with nanowires is the ionic liquid electrolyte, and the back‐side contact with the nanowire photosensors is a liquid metal wire to mimic human nerve fibers behind the retina. The artificial visual system possesses an image sensing function and structural similarity to the human eye, with the capacity to obtain a high imaging resolution when individual nanowires are electrically addressed ( Figure ). However, these digital, artificial vision systems are expensive and markedly large for practical applications, as well as consume considerable power. Thus, a new generation of imaging and photosensing neuromorphic vision sensors, based on novel nanomedicine with high detectivity, ultrahigh responsivity, and signal‐to‐noise ratio, could overcome these disadvantages. 5.6 Ocular Cancer Therapy Retinoblastoma is an aggressive and malignant intraocular tumor in infancy and childhood, whereas uveal melanoma (UM) typically occurs in the choroid, iris, and ciliary body, the most common intraocular cancer in adults. The considerably high mortality rate of retinoblastoma and UM warrant the urgent development of efficacious therapeutics. Currently available medical interventions primarily include enucleation (eye removal), external beam/episcleral plaque radiotherapy, chemoreduction, and chemotherapy. Enucleation is the only suitable approach for advanced retinoblastoma, but surgical lesions result in facial deformities. Radiotherapy can induce various side effects, such as dry eye, which requires permanent eye lubrication. Although chemoreduction overcomes several shortcomings associated with other procedures, it can induce severe complications, including temporary pancytopenia, renal toxicity, and secondary cancer. Currently, chemotherapy has been widely utilized to treat ocular cancer, using individual or synergetic administration of anticancer drugs, such as topotecan and vincristine. However, these chemotherapeutics are highly cytotoxic and can cause adverse effects and secondary complications. Moreover, the distinct physiological and anatomical arrangement of the eye presents an enormous barrier for the targeted transport of drugs to cancer tissues. The recent advancement of a single combinatorial nanotechnology to target ocular cancer cells has afforded an unprecedented possibility of reducing off‐target effects and boosting synergistic anti‐tumor efficacy. Accordingly, various biomaterial‐based nanoplatforms composed of nanoceria, MSNs, AuNPs, and functionalized micelles have been extensively exploited in ocular cancer. These materials exert effects by delivering chemotherapy drugs and/or targeted tumor molecules (e.g., microRNAs, oxygen) in response to the extracellular pH to enhance chemotherapeutic activity or excited by two photons to improve photodynamic therapy. An octopus‐like 8‐valent penetratin (8VP < 100 nm) has been designed with extremely flexible cationic penetratin tentacles and branched spatial structure to effectively deliver siRNA or ASO into retinoblastoma‐bearing mice by promoting their stability. This non‐viral vector nanostructure presented overwhelming advantages when compared with commercial transfection reagents in terms of both efficiency and safety by significantly improving cancer cellular internalization (approaching 100%) and transfection rate (over 75%), thus enriching the non‐invasive gene delivery technologies for retinoblastoma treatment ( Figure ). One drawback of ocular radiotherapy is the difficulty in selectively killing retinoblastoma cells during laser irradiation, which may cause injury to healthy ocular tissues, especially the cornea. Accordingly, biocompatible magnesium oxide nanoparticles with desirable thermal conductivities were obtained by computation. The laser beam was emitted after nanoparticle injection along the pupillary axis, and the amount of generated heat was directly proportional to the absorption coefficient in each eye segment. It can be postulated that low‐thermal‐conductive nanoparticles will accumulate around the tumor mass to effectively avoid the protrusion of the lethal zone from the tumor lesion, thus protecting healthy eye tissues from potential hazards of thermal damage. To date, versatile applications of ocular nanomedicine, including synergetic chemotherapeutic, photodynamic therapy, and selective nanothermal therapy for ocular tumors, especially retinoblastoma and UM, remains in the infant stage; therefore, additional advances in nanotechnology are urgently required to provide less toxic, minimally invasive, and highly effective management. 5.7 Refractive Error Correction Refractive error of the cornea, particularly myopia (i.e., short‐sightedness), is a major cause of recoverable/reversible visual impairment worldwide, and a sharp increase in myopia‐related morbidity is inevitable in modern society. Most patients with refractive error wear spectacles to afford refractive error correction; however, wearing spectacles could cause great inconvenience in daily life. Recently, emerging refractive surgeries, especially laser‐assisted in situ keratomileusis (LASIK) surgery), are considered an attractive choice for permanent vision correction. However, a follow‐up investigation of long‐term safety post‐surgery is insufficient, and invasive surgery may cause thin ectatic corneas and corneal flap bonding post‐LASIK surgery, leading to inefficient refractive error correction. Accordingly, two‐photon collagen cross‐linking (2P‐CXL) of intact corneal tissue has been designed using riboflavin and femtosecond laser irradiation. The results revealed that disturbing surrounding non‐irradiated regions exerted no effect on the visualization of the cross‐linked pattern, and the 2P‐CXL‐induced stiffening was similar to that observed in the conventional one‐photon CXL. The 2P‐CXL technique allows selective stiffening of corneal tissue in situ at high spatial resolution and permits 3D control of cross‐linking by nonlinear excitation, which can be readily applied to assist LASIK surgery and selectively modulate corneal curvature for vision error treatment. As an alternative to irreversible LASIK surgery, the application of the Abbe number, high visible light transmittance, and refractive index in contact lenses affords great potential for refractive error correction. Shaker et al. used titanium dioxide nanoparticles (TiO 2 ) as a suitable candidate to maintain high transparency and increase the refractive index of poly(methyl methacrylate)‐TiO 2 contact lenses. Contact lenses with higher doping content contributed to the best vision correction and reduced the generated spherical and chromatic aberrations in the aberrant eye. Additionally, Wang et al. proposed another non‐invasive approach for permanent vision correction based on the femtosecond laser. This strategy produces low‐density plasma and ROS within collagenous tissues to assist the oxidation of surrounding proteins and cross‐link formation, ultimately initiating the alteration of refractive power in the eyes ( Figure ). This pioneering strategy inspired scientists to propose similar feasible ROS‐producing nanomedicines to improve therapeutic outcomes and relieve potential side effects, ultimately favoring future clinical translation for refractive error correction. 5.8 Ocular Imaging Ocular imaging is essential for clinical diagnosis, therapeutics, and real‐time monitoring of ocular disorders at an early stage. For instance, given the rapid progression of intraocular tumors, early imaging diagnosis is necessary to prevent vision loss and even death. However, most available therapeutics lack the capability of imaging and diagnosis before the onset of ocular disorders and precise monitoring. In ocular disease, nanomedicine‐based strategies subjected to a strict screening could be selectively employed to visualize ocular tissues and accurately and quantitatively examine altered levels of small molecules, proteins, or nucleic acids, ultimately determining the physiological state of ocular tissues. Considering that colloidal gold nanoparticles (GNPs) serve as promising contrast agents in photoacoustic imaging, Nguyen et al. designed chain‐like GNPs (CGNP) clusters‐arginine‐glycine‐aspartic acid (RGD) for enhanced molecular imaging by conjugating RGD peptides with ultrapure CGNP clusters, exhibiting a redshift peak wavelength at 650 nm. The synthesized nanoparticles exhibited excellent photostability and biocompatibility and could disassemble to facilitate elimination from the body. Importantly, intravenous injection of CGNP clusters‐RGD via the marginal ear vein bound to CNV, causing an increase of up to 17‐fold in the photoacoustic microscopy signal and a 176% increase in optical coherence tomography signal, which is beneficial for visualizing newly developed blood vessels in the subretinal space ( Figure ). Additionally, smart wearable platforms, such as contact lenses combined with functionalized nanomaterials, help either sensitively identify the altered constituent (e.g., cortisol, Na ions, and IOP) or detect microorganisms such as Staphylococcus aureus , which play a positive role in early diagnosis and prophylactic therapy of various ocular diseases, including glaucoma, dry eye, and edema. Recently, a breakthrough in fabrication methodologies and synthetic chemistry of ocular nanomedicine has facilitated theranostics by integrating imaging diagnosis and therapy, which can help identify the specific period most susceptible to intervention for a given disease. The introduced dual‐functional SiNPs‐RGD is a class of high‐quality theranostic probes for synergetic imaging and treatment of ocular neovascularization by marking angiogenic blood vessels while effectively suppressing corneal and retinal neovascularization with negligible toxicity. However, efforts to improve theranostic nanomaterials should focus on enhanced imaging modalities and more satisfactory diagnostic and prognostic effects.
Ocular Inflammation/Oxidative‐Stress Therapy Human eyes are considered to be sealed from systematic circulation, and the retina is an “immune‐privileged” zone. Therefore, inflammation in the eye plays a pathological role in a series of ocular diseases, including uveitis, diabetic cataracts, and dry AMD, by disrupting phospholipids in the cell membrane and excessive release of inflammatory substances such as prostaglandins. These harmful substances could induce inflammatory ocular disease, accompanied by typical clinical symptoms such as photophobia, opacification, pain, posterior capsule, vasodilatation, increased vascular permeability, and IOP. For example, uveitis is a typical ocular inflammatory disorder in the uvea, possibly induced by virus/bacterial infection or a particulate matter‐induced over‐reactive immune system response. Dexamethasone, corticosteroid, and triamcinolone acetonide exert anti‐inflammatory activity for treating uveitis; however, long‐term release at the targeted site remains an immense challenge. Alternatively, the controllable release of drugs by biodegradable nanocarriers, such as PLGA‐based nanoparticles, has been successfully achieved with sustained anti‐inflammatory drug release in an experimental autoimmune uveitis animal model, thereby suppressing ocular inflammation without complications. More recently, Ganugula et al. designed the receptor‐mediated delivery of curcumin (CUR) assisted by double‐headed gambogic acid (GA)‐coupled PLGA, that is, PLGA‐GA 2 ‐CUR nanoparticles. Oral administration of these nanoparticles in a canine model with lens‐induced uveitis showed a notable drug level in aqueous humor and afforded protection against intraocular inflammation with reduced miosis, aqueous flare, and chemosis in the acute phase (<4 h) when compared with commercial anti‐inflammatory treatment (oral carprofen) ( Figure ). Excessive ROS is undoubtedly involved in the pathological origin of abnormal inflammatory responses during ocular inflammatory disorders, especially diabetic cataracts and AMD. Diabetic cataract is an ocular complication in patients with diabetes who often suffer from diminished visual function, and AMD is the leading cause of vision loss in elderly individuals with progressive photoreceptor death. However, currently available drugs demonstrate limited ability to effectively delay and prevent diabetic cataracts and dry AMD. Several studies have reported that oxidative damage in the crystalline lens and photoreceptor also plays a crucial role in the pathogenesis of diabetic cataract and dry AMD, respectively. Thus, reducing ROS levels could potentially alleviate inflammation‐associated impairment. Currently, two classes of work mechanisms have been investigated in ROS‐mediated inflammatory ocular diseases: 1) endowing ROS‐scavenging ability for guiding detoxification or 2) imparting ROS‐response anti‐inflammatory drug release. However, conventional antioxidants and anti‐inflammatory biopharmaceuticals remain largely inefficacious in ophthalmology, given their poor permeability, nonspecific biodistribution, low retention at pathological sites, and rapid renal excretion. Depending on either the non‐enzymatic or catalytic features, it has been well documented that autoregenerative redox nanoceria can effectively protect lens epithelial cells and photoreceptors from oxidative damage based on the progressive elimination of ROS, thereby maintaining lens transparency in diabetic cataracts and delaying photoreceptor degeneration in AMD for a prolonged period. Interestingly, INVELTYS (Kala Pharmaceuticals), bromfenac DuraSite, and DexaSite are important ocular nanomedicines that have succeeded in clinical trials and/or obtained FDA approval for treating postoperative inflammation and pain following ocular surgery ( Table ). INVELTYS, as a KPI‐121 1% ophthalmic nanosuspension of loteprednol etabonate, is delivered as mucus‐penetrating particles coated with low molecular weight PEG. Primary outcomes in clinical trials revealed that INVELTYS was safe and effectively resolved postoperative ocular inflammation and subject‐rated ocular pain following cataract surgery when administered twice daily for 2 weeks. The observed results could be attributed to mucus‐penetrating particles that allow the drug to efficiently penetrate the mucin layer of the tear film, facilitating drug release to underlying ocular tissues.
Ocular Hypertension Therapy Glaucoma, a type of optic neuropathy resulting from the damaged optic nerve, affects more than 60 million people worldwide. Several pathogenic parameters have been implicated in glaucoma progression, including elevated IOP, optic nerve ischemia, and oxidative stress‐related activation. Although an elevated IOP is not the only factor attributed to glaucoma development, reducing the elevated IOP directly affords effective neuroprotection and is a primary modifiable target for glaucoma control from a pathophysiological perspective. Daily topical drug application as eye drops to decrease the elevated IOP and oxidative stress is one therapeutic strategy employed in patients with glaucoma. However, developing a product for long‐term medication using eye drops remains a potential challenge in chronic glaucoma. Nanotechnology has been used as a valuable tool to fabricate different nanosystems, such as lipid DNA nanoparticles, hollow ceria nanoparticles, and layer‐by‐layer coated siRNA nanoparticles. Reportedly, nanoparticles could afford long‐term alleviation of glaucomatous damage without disrupting the vesicle shape, owing to potent inherent antioxidant and anti‐inflammatory functions or controllable and sustained release of anti‐IOP drugs or siRNA gene silencing. More recently, a cell‐softening nanotherapy has been proposed to reduce IOP by targeted binding to the vascular endothelial growth factor receptor 3 (VEGFR3)/FLT4 receptor expressed in Schlemm's canal cells. These nanomicelles (tLatA‐MC) are based on a targeted PEG‐ b ‐poly(propylene sulfide) micelle (PEG‐ b ‐PPS) and loaded with the actin depolymerizer latrunculin A (tLatA) to selectively modulate the stiffness of Schlemm's canal cells (an important pathophysiology during glaucoma). tLatA‐MC reduced IOP in a mouse model by 30–50%, indicating the functional efficacy of tLatA‐MC for glaucoma treatment ( Figure ). Conversely, a non‐invasive approach using nanotechnology to monitor the stiffness of the trabecular meshwork in vivo also exhibits significant potential to improve the clinical care of glaucoma. These promising results notably demonstrate the enormous promise of an ocular nanomedicine‐based strategy for chronic glaucoma therapy.
Ocular Bacterial Infection Therapy Bacteria often play a significant antagonistic and pathogenic role in self‐contained and immune‐privileged ocular tissues by disrupting normal metabolic processes and even causing cell death, collectively considered ocular bacterial infections. The downstream diseases of ocular bacterial infections in the external (e.g., bacterial keratitis) and inner parts of the eyes (e.g., endophthalmitis) may pose a serious threat to visual health. Therefore, it is crucial to employ efficacious therapeutic interventions to address this dilemma. Over several decades, antibiotics have rapidly advanced as ideal terminators of nearly all bacteria‐mediated diseases. However, this expectation has been quickly tempered with the emergence of the first‐rank bacterial pathogens, that is, multidrug‐resistant (MDR) bacteria, particularly Escherichia coli and Klebsiella pneumoniae , which mainly contribute to the existence of biofilms. Biofilms are reportedly involved in 80% of bacterial infectious diseases and are composed of extracellular polymeric substances, water, and bacteria. Among them, bacteria play a vital role in protecting biofilm matrices by markedly increasing the resistance to bactericides. For instance, the dormancy of the bottom bacteria in MDR bacteria significantly undermined bactericide permeability. Accordingly, versatile antibacterial surface/antibiofilm‐based ocular nanomedicines have been developed to kill live bacteria and remove mature biofilms or decrease bacterial adhesion to suppress biofilm development, resulting in potent antibacterial treatment. Patients with bacterial keratitis typically experience unexpected corneal epithelium damage, owing to difficulties in spontaneous recovery after bacterial infection. Therefore, it is crucial to develop an effective approach to eradicate MDR infections and simultaneously promote injury healing. More recently, Qiao et al. proposed a composite cupriferous hollow nanoshell (AuAgCu2O NS) comprising a Cu 2 O shell and a hollow gold‐silver (AuAg) core as a high‐quality photothermal therapeutic agent for treating non‐healing keratitis. The authors revealed that Ag released from the hollow AuAg core exhibited a synergistic effect against MDR bacteria. In addition, copper ions released from the Cu 2 O shell hastened fibroblast cell migration and endothelial cell angiogenesis, augmenting the wound healing efficacy in keratitis models. Endophthalmitis is primarily caused by pathogenic microorganisms, particularly bacteria, which can lead to eyeball damage and distinct inflammation in ocular tissues, especially the uvea and vitreous cavity, seriously threatening the visual function of patients. Currently, comprehensive elimination of endophthalmitis remains a challenge, and clinical antibiotics are only employed to control progressive development. Thus, a strategic approach using targeted phototherapy has been proposed to manage endophthalmitis by biofilm eradication. For example, synergistic photodynamic therapy and chemotherapy to treat endophthalmitis were developed using a pH‐responsive hybrid system, that is, zeolitic imidazolate framework‐8‐polyacrylic acid (ZIF‐8‐PAA)‐methylbenzene blue (MB)@silver nanoparticles (AgNPs)@vancomycin (Van)/NH‐PEG (ZPMAVP). ZIF‐8‐PAA significantly improved the drug‐encapsulation capability, facilitated controllable and targeted delivery of a photosensitizer antibacterial agent MB, and reduced AgNO into AgNPs in situ by dopamine modification. Upon secondary functionalization with Van/NH‐PEG, this platform ultimately formed a synergistic composite. Generally, nanomaterials can rapidly release MB to enrich the local therapeutic concentration and damage bacterial biofilms with good biocompatibility, suggesting the superiority of synergistic photodynamic therapy and chemotherapy over any single therapeutic strategy for bacterial ophthalmic diseases ( Figure ).
Ocular Neovascularization Therapy Ocular neovascularization, particularly corneal neovascularization and CNV, is one primary pathological phenotype known to occur in corneal and retinal diseases, respectively. The normal cornea is avascular, and corneal neovascularization is often initiated by ocular surface injuries that can disrupt the harmonic balance between angiogenic inhibitors and stimulators, eventually resulting in visual impairment. In wet AMD, pathogenic vessels protrude through Bruch's membrane and then grow into the subretinal space, leading to initial CNV and distorted central vision. For antiangiogenic therapeutics, targeted inhibition of VEGF (a predominant stimulator during angiogenesis) triggers a signal cascade that suppresses migration and proliferation of endothelial cells and decreases vascular permeability. Clinically, monthly or bimonthly intravitreal injection of large protein anti‐VEGF drugs, such as bevacizumab, is a major therapeutic strategy for CNV. However, anti‐VEGF strategies afford a transient therapeutic outcome, owing to their rapid degradation and poor targeting in dysfunctional existing vessels. These drawbacks necessitate repeated administration to maintain effectiveness and subsequently cause potential infection and tissue injury. In addition, ocular nanomedicine‐assisted procedures have been successfully engineered with inherent antiangiogenic properties derived from a natural product, administered via eye drops or conjunctival sac instillation to achieve minimally invasive intraocular delivery, or via targeted and sustained release of anti‐VEGF peptide/antibody and siRNA‐oriented intracellular therapeutic agents at therapeutically relevant concentrations to minimize dosage intervals. Reportedly, externally triggered targeted delivery allows ocular drug delivery with high temporal and spatial resolution. Among external stimulators, light is particularly attractive as an energy source for retinal targeting, as the eye is naturally used to admit light. Wang et al. designed smart photo‐targeted nanoparticles by self‐assembling a chemically modified PEG‐PLA block copolymer, further modified with a targeting moiety, such as a cell‐penetrating peptide for high cellular uptake. After intravenous administration, the nanoparticles are converted to a tissue‐targeting state depending on the irradiation in the eye. This strategy allows the noninvasive treatment of CNV by allowing target drug accumulation in the diseased ocular areas, simultaneously minimizing drug deposition at off‐target sites in healthy ocular/systemic sites. Additionally, oxidative stress and inflammation play a crucial pathogenic role in angiogenesis during AMD pathogenesis. Mitra et al. designed glycol chitosan‐coated antioxidant ceria nanoparticles, which were autoregenerative and more active in blocking laser‐induced CNV models. On downregulating ROS‐activated pro‐angiogenic VEGF expression, the nanoparticles significantly rescued the cumulative oxidative damage and recruited endothelial precursor cells without affording any toxicity ( Figure ). Overall, the ocular nanomedicine‐assisted strategy provides an alternative intervention to optimally inhibit ocular neovascularization without damaging healthy tissues in a non‐invasive or minimally invasive manner while reducing the repeated administration to establish therapeutic control.
Ocular Tissue Engineering The structure and function of injured cells, tissues, and organs after experiencing trauma, infection, or inflammation can be damaged if healing remains unsatisfactory. For example, retinal degeneration is recognized as one predominant cause underlying irreversible visual impairment and even blindness worldwide, and the death of RPE and/or photoreceptors has been considered common pathogenesis. Hence, RPE/photoreceptor‐based transplantation into diseased sites is necessary to generate monolayer cells and recover the RPE‐photoreceptor interface. Over the past two decades, ocular tissue engineering, including corneal, conjunctival, and retinal regeneration, has been shown to be feasible by transplanting cell/tissue‐derived implants. However, these are limited in terms of specific responses to biological cues and superior reconstruction of normal functions. However, ocular nanomedicine‐based strategies could introduce and guide ocular tissue regeneration by stimulating the signaling pathway by communicating with surroundings or serving as a sensor to monitor/trace transplanted cell replacement and tissue repair. As an emerging nanomaterial, tetrahedral framework nucleic acids (tFNAs) accelerated the re‐epithelialization by promoting the migration and proliferation of human corneal epithelial cells. Moreover, tFNAs showed high efficacy in recovering corneal transparency and corneal wound healing in an established corneal alkali burn animal model. In retinal tissue engineering, a micropatterned PLGA nanosheet with perfect flexibility has been adopted to achieve a good microenvironment for the stable attachment of a transplanted RPE monolayer. By further embedding with magnetic nanoparticles, the nanosheet promoted malleability, optical visualization, and cell morphogenesis. After subretinal injection into the swine eye, this nanosheet exhibited stable attachment to the macula, and the implanted RPE monolayer could differentiate into a cobblestone‐like structure. Thus, nanomedicine‐aided ocular regenerative medicine is expected to establish an innovative therapeutic strategy by guiding the uninjurious cellular organization. Human eyes have exceptional image sensing functions, such as high sensitivity and resolution with low aberration and a spectacularly wide field of view. During the process, the retina efficiently detects light stimuli and pre‐processes image information in parallel, prior to the brain performing more complex actions. Biomimetic eyes with similar superior features are extremely desirable for different technological applications. In recent years, digital vision systems based on charge‐coupled device cameras or conventional, complementary metal‐oxide‐semiconductor imagers have been explored to fulfill computer vision by interface‐interfaced digital processing units on coarsely parallel or serial structures. Recently, a unique biomimetic electrochemical eye (EC‐EYE) using a hemispherical retina has been constructed based on a high‐density array of perovskite nanowires to mimic photoreceptors on an actual retina. A front‐side common contact with nanowires is the ionic liquid electrolyte, and the back‐side contact with the nanowire photosensors is a liquid metal wire to mimic human nerve fibers behind the retina. The artificial visual system possesses an image sensing function and structural similarity to the human eye, with the capacity to obtain a high imaging resolution when individual nanowires are electrically addressed ( Figure ). However, these digital, artificial vision systems are expensive and markedly large for practical applications, as well as consume considerable power. Thus, a new generation of imaging and photosensing neuromorphic vision sensors, based on novel nanomedicine with high detectivity, ultrahigh responsivity, and signal‐to‐noise ratio, could overcome these disadvantages.
Ocular Cancer Therapy Retinoblastoma is an aggressive and malignant intraocular tumor in infancy and childhood, whereas uveal melanoma (UM) typically occurs in the choroid, iris, and ciliary body, the most common intraocular cancer in adults. The considerably high mortality rate of retinoblastoma and UM warrant the urgent development of efficacious therapeutics. Currently available medical interventions primarily include enucleation (eye removal), external beam/episcleral plaque radiotherapy, chemoreduction, and chemotherapy. Enucleation is the only suitable approach for advanced retinoblastoma, but surgical lesions result in facial deformities. Radiotherapy can induce various side effects, such as dry eye, which requires permanent eye lubrication. Although chemoreduction overcomes several shortcomings associated with other procedures, it can induce severe complications, including temporary pancytopenia, renal toxicity, and secondary cancer. Currently, chemotherapy has been widely utilized to treat ocular cancer, using individual or synergetic administration of anticancer drugs, such as topotecan and vincristine. However, these chemotherapeutics are highly cytotoxic and can cause adverse effects and secondary complications. Moreover, the distinct physiological and anatomical arrangement of the eye presents an enormous barrier for the targeted transport of drugs to cancer tissues. The recent advancement of a single combinatorial nanotechnology to target ocular cancer cells has afforded an unprecedented possibility of reducing off‐target effects and boosting synergistic anti‐tumor efficacy. Accordingly, various biomaterial‐based nanoplatforms composed of nanoceria, MSNs, AuNPs, and functionalized micelles have been extensively exploited in ocular cancer. These materials exert effects by delivering chemotherapy drugs and/or targeted tumor molecules (e.g., microRNAs, oxygen) in response to the extracellular pH to enhance chemotherapeutic activity or excited by two photons to improve photodynamic therapy. An octopus‐like 8‐valent penetratin (8VP < 100 nm) has been designed with extremely flexible cationic penetratin tentacles and branched spatial structure to effectively deliver siRNA or ASO into retinoblastoma‐bearing mice by promoting their stability. This non‐viral vector nanostructure presented overwhelming advantages when compared with commercial transfection reagents in terms of both efficiency and safety by significantly improving cancer cellular internalization (approaching 100%) and transfection rate (over 75%), thus enriching the non‐invasive gene delivery technologies for retinoblastoma treatment ( Figure ). One drawback of ocular radiotherapy is the difficulty in selectively killing retinoblastoma cells during laser irradiation, which may cause injury to healthy ocular tissues, especially the cornea. Accordingly, biocompatible magnesium oxide nanoparticles with desirable thermal conductivities were obtained by computation. The laser beam was emitted after nanoparticle injection along the pupillary axis, and the amount of generated heat was directly proportional to the absorption coefficient in each eye segment. It can be postulated that low‐thermal‐conductive nanoparticles will accumulate around the tumor mass to effectively avoid the protrusion of the lethal zone from the tumor lesion, thus protecting healthy eye tissues from potential hazards of thermal damage. To date, versatile applications of ocular nanomedicine, including synergetic chemotherapeutic, photodynamic therapy, and selective nanothermal therapy for ocular tumors, especially retinoblastoma and UM, remains in the infant stage; therefore, additional advances in nanotechnology are urgently required to provide less toxic, minimally invasive, and highly effective management.
Refractive Error Correction Refractive error of the cornea, particularly myopia (i.e., short‐sightedness), is a major cause of recoverable/reversible visual impairment worldwide, and a sharp increase in myopia‐related morbidity is inevitable in modern society. Most patients with refractive error wear spectacles to afford refractive error correction; however, wearing spectacles could cause great inconvenience in daily life. Recently, emerging refractive surgeries, especially laser‐assisted in situ keratomileusis (LASIK) surgery), are considered an attractive choice for permanent vision correction. However, a follow‐up investigation of long‐term safety post‐surgery is insufficient, and invasive surgery may cause thin ectatic corneas and corneal flap bonding post‐LASIK surgery, leading to inefficient refractive error correction. Accordingly, two‐photon collagen cross‐linking (2P‐CXL) of intact corneal tissue has been designed using riboflavin and femtosecond laser irradiation. The results revealed that disturbing surrounding non‐irradiated regions exerted no effect on the visualization of the cross‐linked pattern, and the 2P‐CXL‐induced stiffening was similar to that observed in the conventional one‐photon CXL. The 2P‐CXL technique allows selective stiffening of corneal tissue in situ at high spatial resolution and permits 3D control of cross‐linking by nonlinear excitation, which can be readily applied to assist LASIK surgery and selectively modulate corneal curvature for vision error treatment. As an alternative to irreversible LASIK surgery, the application of the Abbe number, high visible light transmittance, and refractive index in contact lenses affords great potential for refractive error correction. Shaker et al. used titanium dioxide nanoparticles (TiO 2 ) as a suitable candidate to maintain high transparency and increase the refractive index of poly(methyl methacrylate)‐TiO 2 contact lenses. Contact lenses with higher doping content contributed to the best vision correction and reduced the generated spherical and chromatic aberrations in the aberrant eye. Additionally, Wang et al. proposed another non‐invasive approach for permanent vision correction based on the femtosecond laser. This strategy produces low‐density plasma and ROS within collagenous tissues to assist the oxidation of surrounding proteins and cross‐link formation, ultimately initiating the alteration of refractive power in the eyes ( Figure ). This pioneering strategy inspired scientists to propose similar feasible ROS‐producing nanomedicines to improve therapeutic outcomes and relieve potential side effects, ultimately favoring future clinical translation for refractive error correction.
Ocular Imaging Ocular imaging is essential for clinical diagnosis, therapeutics, and real‐time monitoring of ocular disorders at an early stage. For instance, given the rapid progression of intraocular tumors, early imaging diagnosis is necessary to prevent vision loss and even death. However, most available therapeutics lack the capability of imaging and diagnosis before the onset of ocular disorders and precise monitoring. In ocular disease, nanomedicine‐based strategies subjected to a strict screening could be selectively employed to visualize ocular tissues and accurately and quantitatively examine altered levels of small molecules, proteins, or nucleic acids, ultimately determining the physiological state of ocular tissues. Considering that colloidal gold nanoparticles (GNPs) serve as promising contrast agents in photoacoustic imaging, Nguyen et al. designed chain‐like GNPs (CGNP) clusters‐arginine‐glycine‐aspartic acid (RGD) for enhanced molecular imaging by conjugating RGD peptides with ultrapure CGNP clusters, exhibiting a redshift peak wavelength at 650 nm. The synthesized nanoparticles exhibited excellent photostability and biocompatibility and could disassemble to facilitate elimination from the body. Importantly, intravenous injection of CGNP clusters‐RGD via the marginal ear vein bound to CNV, causing an increase of up to 17‐fold in the photoacoustic microscopy signal and a 176% increase in optical coherence tomography signal, which is beneficial for visualizing newly developed blood vessels in the subretinal space ( Figure ). Additionally, smart wearable platforms, such as contact lenses combined with functionalized nanomaterials, help either sensitively identify the altered constituent (e.g., cortisol, Na ions, and IOP) or detect microorganisms such as Staphylococcus aureus , which play a positive role in early diagnosis and prophylactic therapy of various ocular diseases, including glaucoma, dry eye, and edema. Recently, a breakthrough in fabrication methodologies and synthetic chemistry of ocular nanomedicine has facilitated theranostics by integrating imaging diagnosis and therapy, which can help identify the specific period most susceptible to intervention for a given disease. The introduced dual‐functional SiNPs‐RGD is a class of high‐quality theranostic probes for synergetic imaging and treatment of ocular neovascularization by marking angiogenic blood vessels while effectively suppressing corneal and retinal neovascularization with negligible toxicity. However, efforts to improve theranostic nanomaterials should focus on enhanced imaging modalities and more satisfactory diagnostic and prognostic effects.
Conclusion and Outlook The intrinsic limitations of conventional management of ocular diseases have propagated the growth of ocular nanomedicine. Herein, we overview the state‐of‐the‐art ocular nanomedicine, predominantly reported in the last 5 years, and provide an in‐depth analysis of how fundamental physicochemical characteristics of nanomaterials affect their effectiveness after interacting with the extra/intraocular milieu. Further discussion of functionalized ocular nanomedicine by engineering suitable entities has also been conducted. Additionally, diverse applications of ocular nanomedicine to combat a wide range of ocular pathogenesis have been comprehensively presented. Typically, the advantages of nanomedicine used in ophthalmology include: 1) prophylactic and antidotic effects by exerting inherent therapeutic properties, 2) possible permeation across complex ocular barriers, 3) targeted and controllable cargo release by avoiding rapid inactivation and burst effects, 4) on‐demand and co‐delivery of various therapeutic cargos with differentiated physicochemical properties, and 5) synergistic effects by integrating imaging with therapeutics. Although considerable breakthroughs have been achieved, several unresolved scientific issues and corresponding perspectives encountered in the design and clinical translation of ocular nanomedicine require elucidation. 1) Current trends in ocular nanomedicine have gradually shifted from in vitro evaluations to preliminary in vivo assays in different animal models. However, the anatomical structure/geometry and composition of ocular tissues are distinct among distinct species, particularly among animal models and humans. Thus, in vivo animal models of eye‐related diseases have a roughly predictive role in humans. For instance, the distinctive space and dimension discrepancy of the vitreous body and the proximity of the vitreous body to the retina is known to exist between rodents and humans, making it difficult to translate preclinical outcomes to humans. Moreover, the thicker retina and smaller vitreous volume in mice and rats than in humans make it difficult to precisely evaluate the actual intraocular drug transport and distribution of ocular nanomedicine. As a commonly used animal model of eye disease, rabbits are comparable in size to the human eye, with different physiological behaviors, such as higher mucus production, lower tear production, and higher surface sensitivity. It is also worth noting that simply converting the equivalent dose of nanoparticles from other species does not ensure safety in humans, given the above differences between animals and humans. Therefore, considerable attention should be paid to higher mammals for preclinical safety and efficacy studies of ocular nanomedicine, especially in non‐human primates whose eyes are similar to the human eye, as well as to reproduce several clinical symptoms consistent with human patients. 2) Although numerous cellular studies and animal investigations have been performed to determine therapeutic outcomes of designed ocular nanomedicine, it is still challenging to completely clarify the underlying mechanisms of nanomedicine on ocular cells/tissues following biomedical analysis, that is, only therapeutic results, rather than the processes, are established. For instance, regardless of the existing evidence supporting the ultimate therapeutic outcomes of nanomaterials, it can be challenging to quantify their reaction kinetics in vivo. These “black boxes” obviously exist in almost all therapeutic processes associated with nanomedicine used for various biomedical applications. Multifunctional optical probes have been developed to evaluate the distribution and therapeutic concentration of specific biochemical ingredients in the biological milieu. These multifunctional diagnostic/therapeutic agents may be beneficial for the precise characterization of therapeutic processes in vivo, consequently clarifying the relationship between ocular nanomedicine and in vivo behavior. 3) The human eye is regarded as one of the most exquisite organs of the body and serves as a perfect platform for nanoparticle delivery, capable of bypassing systemic circulation. Different routes of administration influence nanoparticle biodistribution. Current methods for nanoparticle delivery into the anterior eye segment mainly focus on eye drops; however, frequent blinking and tears limit nanoparticle retention on the ocular surface, leading to poor bioavailability. Subconjunctival administration serves as a less invasive option to overcome blood‐ocular barriers and improve drug bioavailability in specific ocular sites. Subconjunctival implantation has been well‐examined for periocular implants, but this approach may require a second surgery. As chronic retinal disorders require long‐term intervention, subretinal injection provides a relatively high nanoparticle bioavailability; however, it is highly invasive and may be accompanied by complications such as intraocular bleeding. Although the intravitreal procedure avoids anterior eye barriers, the complex inner limiting membrane and vitreoretinal interface may prevent nanoparticle access to the retina. Retinal permeation of intravitreal nanocarriers is limited and probably misleading, given inter‐species differences in retinal structures between animals and humans. In addition, Abrishami et al. recently designed a gelling hypotonic formulation for extended ocular delivery by forming a highly clear and uniform thin layer. Compared with commercial eye drops and conventional thermosensitive gelling formulations, the hypotonic gel layer resisted clearance from blinking and conformed to the ocular surface, thereby enhancing intraocular drug absorption and prolonging ocular surface contact. Based on these findings, further development of penetrable nanomaterials that can permeate into intraocular tissues upon direct eye drop could afford a more effective, non‐invasive, and long‐lasting alternative to conventional therapies. In contrast, given the minimum invasiveness and driving capability of a magnetic field, ocular delivery with the aid of a microrobot confers an inherent advantage for targeted ocular treatment. More importantly, Kim et al. developed a bilayer hydrogel microrobot capable of retrieving magnetic nanoparticles (MNPs) using a magnetic field after drug targeted delivery. This procedure effectively avoids the side effects of MNP retention in conventional microrobots. The sheet‐type microrobot with retrieval and recycling ability demonstrates superior potential as a therapeutic agent against intraocular diseases and affords a new ocular delivery paradigm to develop recycled nanomaterials for ocular disease treatment. 4) As most ocular nanomedicines are unique and under development, a continuous investigation of therapeutic safety and effectiveness is required prior to utilization in clinical practice. Biosafety issues, including genotoxicity, neurotoxicity, and reproductive toxicity of ocular nanomedicine, especially inorganic nanoparticles, remain a prerequisite and top priority. In vivo biocompatibility concerns of elaborately constructed nanomaterials, such as unsterile contaminants in contact with various ocular structures, have been comprehensively reviewed by the Zhu group, demonstrating that the biological compatibility of ocular nanomedicine is closely associated with the synthesized structure/morphology, particle sizes, and dosing parameters ( Figure ). 5) The last and most critical concern associated with ocular nanomedicine is the future clinical translation and successful commercialization. Although numerous fundamental investigations and preclinical evaluations have been reported, to the best of our knowledge, only several types of nanosystems have been approved for clinical trials to combat ocular diseases, including emerging exosome‐based therapeutics for macular edema (Table ). The current large‐scale synthesis of nanomedicines used in biomedical fields is a common issue considering their complex, diverse nanostructures and surface modification, which may be addressed to a certain extent by simplifying the corresponding construction without compromising bioactivity. These challenges and corresponding directions in the progression of both conventional and emerging ocular nanomedicine prompt us to reconsider aspects that should be considered to accelerate clinical translation ( Figure ). Despite continued advances in basic and clinical ophthalmology research and nanomedicine to meet individual patient requirements, it is believed that there remains “plenty of room at the bottom” for developing a satisfactory strategy with broad impact in the future.
The authors declare no conflict of interest.
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Computational Approaches to Enzyme Inhibition by Marine Natural Products in the Search for New Drugs | 23f6d6f8-d540-492e-a30b-a877146f267c | 9961086 | Pharmacology[mh] | Both pharmacology and basic cell biology have traditionally benefited from the continuous identification and biochemical characterization of active principles obtained from natural sources. The scanty primitive chemical libraries of natural products (NPs), consisting mostly of the alkaloids and heterosides isolated from terrestrial plants that provided the foundations of modern pharmacology , were progressively enriched with a multitude of small- to medium-sized molecules present in numerous living creatures, both big and small, including those inhabiting seas and oceans, which together make up a huge water mass that covers >70% of Earth’s total surface and hosts ~80% of all living species . Nonetheless, and despite a notable renaissance in recent years , the list of marine natural products (MNPs) that have been approved or are currently found in the global marine pharmaceutical clinical pipeline ( https://www.midwestern.edu/departments/marinepharmacology/clinical-pipeline , accessed on 20 December 2022) is still very limited, and only a few of these drugs actually target an enzyme. The vastness of the largely unexplored chemical space existing in marine environments poses daunting challenges in terms of (i) sample recollection, (ii) compound isolation, (iii) chemical characterization, (iv) evaluation in as many biochemical and/or biological assays as possible, preferably using validated targets and high-throughput state-of-the-art technologies , and (v) the identification and validation of pharmacologically relevant targets. Given the precedents of successful marine leads as a source of useful medicinal agents and biochemical probes, it can be argued that it makes sense to continue exploiting over four billion years of evolution in nature’s combinatorial chemistry, often subjected to unique ecological pressures and nutrient availability, that led to selective survival advantages in the producing organisms . The best studied phylogenetically diverse living beings from marine habitats include green, brown, and red algae; sponges; coelenterates (i.e., jellyfishes, corals, and sea anemones); bryozoans (i.e., invertebrates known as moss animals); the Ascidiacea class (commonly known as the ascidians, tunicates, and sea squirts); mollusks; echinoderms; phytoplankton; and innumerable bacteria and fungi. Secondary metabolites are specialized organic compounds that are not considered essential for normal growth or reproduction (under laboratory culture conditions) but instead play roles in evolution, communication (as chemical cues), and competition, or else appear to be used as chemical weaponry against prey or natural enemies in their natural environments. MNPs often feature unique scaffolds and carbocyclic skeletons, and many have been discovered following their bioassay-guided isolation, although the paucity of material usually prevents the full profiling of bioactivity , which is often limited to some rudimentary tests (e.g., phenotype-oriented antimicrobial or cytotoxic assays , and/or inhibitory activity against one enzyme or a limited set of enzymes). In this respect, it has been pointed out that micromolar activities detected in extracts should be critically analyzed because of potential artefactual assay readouts due to unspecific aggregation , hence the recommendation to use β-lactamase and malate dehydrogenase as counter-screening enzymes , among other precautionary measures. Recent progress in understanding the genetic basis of MNP biosynthesis and the ever-increasing availability of genomic information have created unique opportunities to develop sequence-based approaches for the discovery of novel bioactive molecular entities . Polyketide synthases (PKSs) and multimodular nonribosomal peptide synthetases (NRPS) stand out among the enzymes that are ultimately responsible for the highly efficient synthesis of three large subclasses of important NPs (PKs, NRPs, and PK/RP or NRP/PK hybrids) through the concerted assembly of relatively simple carboxylic acid and amino acid building blocks, respectively . Type I PKSs consist of multiple modules, with each module minimally containing three core domains: acyltransferase (AT) domain, ketosynthase (KS) domain, and thiolation (T) domain [aka acyl carrier protein (ACP) domain] . These (mega)enzymes are encoded in biosynthetic gene clusters (BGCs), which have been identified for hundreds of bacterial and fungal metabolites and are highly evolved for horizontal exchange . Besides, attention continues to be drawn to two facts that have significantly expanded the area of MNP research, namely (i) that some isolated MNPs are bioaccumulated in the target organism from dietary sources, e.g., algae ; and (ii) that a significant number of MNPs are actually produced by microbes and/or microbial interactions with the “host from whence it was isolated” . The growing emphasis on the study of compounds from microbial sources (both terrestrial and marine) has been fueled by interest in (i) the central role that microorganisms play in mediating both interspecies interactions and host-microbe relationships ; and (ii) their natural ability to produce ribosomally synthesized and post-translationally modified peptides (RiPPs), which often contain noncanonical amino acids and structural motifs that give rise to a currently under-represented class of biologically active molecules . Modern science (and the world at large) is overly dependent on computer and internet technologies. Computers have a long history in data management, as well as in information storage, processing, retrieval, and dissemination, and for these purposes their use has expanded enormously in recent years and has contributed to shaping the current research landscapes in bioscience and biomedicine as we know them today. The World Wide Web has become a central source of (i) information on all possible subjects that is stored and (ideally) curated in extensively hyperlinked databases; (ii) educational and research tools; and (iii) services that are intended to make life easier not only for the general public, but also for scientists, including those devoted to chemical biology, medicinal chemistry, and drug discovery. Devices ranging from pocket computers, also known as mobile or cellular phones which have superseded earlier personal digital assistants (PDA), to tablets, laptops, desktops, mainframes, and supercomputers dominate many aspects of our lives and complement human skills in numerous applications designed to utilize an ever-growing torrent of biological and chemical data in effective manners. While this is the driving force behind the increasing use of high-performance computing, machine learning and artificial intelligence for processing tons of data in a way that compensates for the inherent constraints of human cognition , better-informed decision making in drug discovery and development still largely relies (or so I like to believe) on the power of human judgement and life-long expertise. The concise Guide to Pharmacology ( https://www.guidetopharmacology.org/ ; latest release 13 October 2022, accessed on 20 December 2022) presented by the International Union of Basic and Clinical Pharmacology (IUPHAR) and the British Pharmacological Society (BPS) includes enzymes (Nature’s catalysts essential to the chemistry of life) as one of the six major classes of pharmacological targets, the others being G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, and transporters (including the very large SLC superfamily of solute carriers) . Over one thousand distinct human enzymes are described in the Universal Protein Knowledgebase (UniProtKB) , therefore representing almost half of all current human targets. Fortunately, the three-dimensional (3D) structures of many of these enzymes or closely related counterparts from other species—both in their apo forms and in complexes with ligands—have been solved and deposited in the Worldwide Protein Data Bank (wwPDB) , a continuously enlarging global repository established in 1971. These 3D structures facilitate the elucidation of functional mechanisms, aid in understanding the binding mode of inhibitors, and enable virtual screening (VS) and structure-based drug design (SBDD) technologies. For other enzymes of interest, we still depend on several homology modeling approaches , neural network-based models, such as those generated by AlphaFold , and artificial intelligence, which was recently employed to build the ESM Metagenomic Atlas ( https://esmatlas.com/ , accessed on 20 December 2022), with more than 617 million structures from all kingdoms of life . Following the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB, https://www.qmul.ac.uk/sbcs/iubmb/enzyme/ ; accessed on 20 December 2022), the wwPDB assigns Enzyme Commission (EC) numbers to protein chains in macromolecular structures according to the type of chemical reaction that they catalyze. The main classes are oxidoreductases (EC 1), transferases (EC 2), hydrolases (EC 3), lyases (EC 4), isomerases (EC 5), ligases (EC 6), and translocases (EC 7), with subclasses (with up to 4 digits) being defined on the basis of the specific donors and receptors of chemical groups that participate in the reactions and additional considerations. The main collection of functional enzyme and metabolism data is possibly BRENDA ( https://www.brenda-enzymes.org/ , accessed on 20 December 2022), which was established in 1987 and selected as an ELIXIR Core Data Resource in 2018 . In addition, the merging of MACiE (Mechanism, Annotation and Classification in Enzymes), a database of enzyme mechanisms, and CSA (Catalytic Site Atlas), a database of catalytic sites of enzymes, has resulted in the M-CSA Mechanism and Catalytic Site Atlas ( http://www.ebi.ac.uk/thornton-srv/m-csa/browse/?sort=ec , accessed on 20 December 2022) , which consolidates a body of knowledge on enzyme structures, gene sequences, reaction mechanisms, metabolic pathways, and kinetic data that any researcher working on enzyme inhibitors should be familiar with. Building on this introductory background information, the following sections will separately cover each of the abovementioned aspects for which specialized computer technologies have been developed in the field of enzyme inhibition by MNPs . Chemical libraries encompassing millions of compounds include the Chemical Abstracts Service (CAS) REGISTRY database ( http://www.cas.org/expertise/cascontent/registry/index.html , accessed on 20 December 2022), which is updated on a daily basis and contains >250,000 NPs out of >150 million chemical substances, PubChem (including PCSubstance, PCCompound, and PCBioAssay) , ChEMBL (a manually curated database of >2,300,000 bioactive molecules with drug-like properties, last update July 2022) , and ChemSpider (with various levels of partial to complete stereochemistry) . The free-to-access resource DrugBank is a web-enabled database ( https://go.drugbank.com/ , accessed on 20 December 2022) that incorporates comprehensive molecular information about drugs, their mechanisms, their interactions, and their targets. First described in 2006 as a knowledgebase for drugs, drug actions, and drug targets , DrugBank has evolved over time in response to improvements in web standards and changing needs for drug research and development. The latest update, DrugBank 5.0 , was expanded to cover not only drug binding data, numerous investigational drugs, drug-drug and drug-food interactions, and SNP-associated drug effects, but also information on the influence of hundreds of drugs on metabolite levels (pharmacometabolomics), gene expression levels (pharmacotranscriptomics), and protein expression levels (pharmacoproteomics). Enzyme inhibitors (DBCAT000003) are described as “compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction”. Reviews on MNPs have been published on a regular basis in the scientific literature . The renewed upsurge of interest in NPs, and MNPs in particular, over the last two decades has led to a rapid multiplication of databases in both the private sector and the public domain that compile general-purpose or thematic information on these naturally occurring compounds, often incorporating supplementary material published in scientific papers. A dedicated, searchable, and continuously updated database (MarinLit, https://marinlit.rsc.org/ , accessed on 20 December 2022) that was established in the 1970s by Prof. John Blunt and Prof. Murray Munro (University of Canterbury, New Zealand) has been maintained by the Royal Society of Chemistry (UK) since 2014. MarinLit covers ~40,000 compounds from marine macro- and microorganisms and about the same number of references to journal articles. Among the specialized MNP databases, the Dictionary of Marine Natural Products (DMNP) appeared as the first of its kind in 2008 and encompassed a subset of data from the Dictionary of Natural Products (DNP, one of several Chapman & Hall chemical dictionaries) based on the biological source of the compounds. DMNP was marketed as a book together with a CD-ROM for a desktop version, and the searchable web-based version CHEMnetBASE ( https://dmnp.chemnetbase.com/ , accessed on 20 December 2022) is still available (v. 31.1; updated in 2022), but only to subscribing institutions. Virtual chemical libraries of NPs can be categorized into (i) encyclopedic and general NP databases; (ii) special subsets within fully enumerated, ultra-large scale chemical libraries specifically built to facilitate VS campaigns, e.g., ZINC ; (iii) compound collections enriched with NPs used in traditional medicines; and (iv) specialized databases focused on specific habitats, geographical regions, organisms, biological activities, or even specific NP classes. Unfortunately, many NP databases belonging to the latter two categories are rather ephemeral or rapidly become either outdated or unavailable to the scientific community , and the same criticism applies to many bioinformatics web services related to NPs . This is most likely due to (i) a lack of funds (and/or human resources) for their sustained management and continuous upgrading, and (ii) the current overwhelming “data deluge”. For these reasons, there is an urgent need for nonredundant, community-wide efforts that optimize the use of contemporary bioinformatic and chemoinformatic capabilities, as exemplified by the recently established open platform LOTUS ( https://lotus.naturalproducts.net , accessed on 20 December 2022), a knowledgebase that is expected to have strong transformative potential for research on NPs and beyond . In this praiseworthy initiative, data sharing within the Wikidata framework broadens interoperability and facilitates access to >750,000 referenced structure-organism pairs. Another large and freely available NP database is Super Natural II ( https://bioinf-applied.charite.de/supernatural_new/index.php ; last updated: October 2022, accessed on 20 December 2022), which provides two-dimensional (2D) structures and physicochemical properties for ~326,000 molecules, as well as information about the pathways associated to their synthesis, degradation, and mechanisms of action with respect to structurally similar drugs . An additional recent compilation of 400,000 non-redundant NPs was made available in 2021 as the open-access COlleCtion of Open NatUral producTs (COCONUT, https://coconut.naturalproducts.net/ , accessed on 20 December 2022). One important goal of these NP databases is to facilitate a quick assessment of novelty for any newly identified compound in a natural extract. To distinguish between known and unknown compounds, it is important to have rapid and trustworthy “dereplication” methods, which rely heavily on the interpretation of molecular mass and molecular formula, as well as UV and NMR spectral data . Nevertheless, the dereplication process can be problematic sometimes because (i) the present validity and accuracy of the collected information is only as good as that of the original data source; and (ii) stereochemical information on NPs is often inaccurate or incomplete. In the field of MNPs alone, it was recently reported that more than 200 structures were misassigned in the last ten years only . A comparative analysis of the original and the revised structures revealed that major pitfalls still plague the structural elucidation of small molecules and, consequently, that quite a few 3D molecular structures present in databases may be inaccurate. This finding emphasizes the roles of total synthesis, X-ray crystallography, as well as chemical and biosynthetic logic, to complement spectroscopic data. Nevertheless, it is noteworthy that a much lower incidence of “impossible” structures was found in MNPs compared to NPs of plant origin. The utilization of computer-assisted structure elucidation (CASE) programs can minimize the risk of misassignment and help identify truly novel compounds (the “unknown unknowns”) by generating all structures that are consistent with key data from 2D correlation spectroscopy (COSY), heteronuclear multiple bond correlation (HMBC), and 1,1-adequate sensitivity double-quantum spectroscopy (ADEQUATE) NMR experiments, and by ranking the resulting structures in order of probability. The algorithms may additionally benefit from both stereospecific NMR data and use of optimized geometries and predicted chemical shifts provided by density funtional theory (DFT) quantum mechanical calculations . The absolute configuration of an MNP can be unequivocally confirmed by crystallographic analysis and, in the case of noncrystalline compounds containing a pseudo-meso core structure that results in a specific rotation ([a] D ) of almost zero (e.g., elatenyne), it may be necessary to absorb the compound into a porous coordination network (a “crystalline sponge”) . The exploration of the identities and biological activities of metabolites present in complex mixtures has benefited enormously in recent years from scalable native and functional metabolomics approaches . Novel techniques, such as affinity selection mass spectrometry (MS), complemented with pulsed ultrafiltration, size exclusion chromatography, and magnetic microbead affinity selection screening, now allow the separation of non-covalent ligand-receptor complexes from other nonbinding compounds . Recognizing the need for community-wide platforms to effectively share and analyze raw, processed, or identified tandem MS (MS/MS or MS 2 ) data of NPs, in an analogous fashion to what has been achieved in genomics and proteomics research with the GenBank ® at the National Center for Biotechnology Information (NCBI) and the UniProtKB , the open-access knowledgebase known as Global Natural Products Social Molecular Networking (GNPS, http://gnps.ucsd.edu , accessed on 20 December 2022) was presented in 2017 . The spectral libraries enable unambiguous dereplication (by matching spectral features of the unknown compound(s) to curated spectral databases of reference compounds, i.e., identification of “known unknowns”) , variable dereplication (approximate matches to spectra of related molecules), and the identification of spectra in molecular networks. Importantly, GNPS allows for the community-driven, iterative re-annotation of reference MS/MS spectra in a wiki-like fashion, and therefore it will contribute to library improvements and eventual convergence of all curated MS/MS spectra. The visualization of molecular networks in GNPS represents each spectrum as a node, and spectrum-to-spectrum alignments as edges (connections) between nodes. Secondary metabolites can be considered genetically encoded small molecules that play a variety of roles in cell biology and therefore have the potential to become chemical probes or drug leads. Their identification and characterization can benefit from a growing number of databases and genomics-based computational tools that have been compiled and hyperlinked at the Secondary Metabolite Bioinformatics Portal (SMBP ( http://www.secondarymetabolites.org/ , accessed on 20 December 2022) website . Inherent limitations related to their low production and difficult detection, and also high rediscovery rates, can be addressed, at least in part, by searching for BGCs in genomic data and unveiling their (sometimes cryptic) metabolic potential . However, the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis, hence the need for new bioinformatic tools. An example is the Natural Product Domain Seeker (NaPDoS) web service ( https://npdomainseeker.sdsc.edu/napdos2/ , accessed on 20 December 2022), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from genes encoding PKS and NRPS, respectively. The sequence tags correspond to PKS-derived KS domains and NRPS-derived condensation (C) domains and are compared to an internal database of experimentally characterized biosynthetic genes, so that genes associated with uncharacterized biochemistry can be identified . The latest update (NaPDoS2) greatly expands the taxonomic and functional diversity represented in the webtool database and allows larger datasets to be analyzed. Importantly, NaPDoS2 can be used to detect genes involved in the biosynthesis of specific structural classes or new biosynthetic mechanisms, and also to predict biosynthetic potential . The key role of marine microbial symbionts of invertebrates in MNP biosynthesis has been increasingly recognized and “genome mining” (i.e., the exploitation of genomic information for the discovery of biosynthetic pathways) provides unique opportunities for (i) the identification of yet undisclosed specialized metabolites and their chemical variants ; (ii) the genetic engineering of BGCs to obtain novel “unnatural” NPs ; and (iii) the heterologous expression of secondary metabolic pathways that remain silent or are poorly expressed in the absence of a specific trigger or elicitor . In fact, the results of a variety of genome sequencing projects have unveiled the metabolic diversity of microorganisms (which may be overlooked under standard fermentation and detection conditions) and their tremendous biosynthetic potential. Furthermore, studies on the evolutionary history of BGCs in relation to that of the bacteria harboring them (“comparative genomics”) beautifully illustrate the mechanisms by which chemical diversity is created in nature and how some NPs represent ecotype-defining traits while others appear selectively neutral . Novel algorithms have been devised to systematically identify BGCs in microbial genomic sequences . A network analysis of the predicted BGCs in Proteobacteria (aka Pseudomonadota, a major phylum of Gram-negative bacteria) has revealed large gene cluster families, and the experimental characterization of the most prominent one revealed two subfamilies consisting of hundreds of BGCs encoding the biochemical machinery for the synthesis of a series of remarkably conserved lipids with an aryl head group conjugated to a polyene tail (i.e., aryl polyenes) that are likely to play important roles in Gram-negative cell biology . The systematic study of BGCs in Actinobacteria (actinomycetes mainly associated to sponges in marine habitats) is complicated by numerous repetitive motifs. By combining several metrics, a method for the global classification of these gene clusters into families (GCFs) has been developed, and the biosynthetic capacity of the resulting GCF network has been validated in hundreds of strains by correlating confident MS detection of known NPs with the presence or absence of their established BGCs . The Minimum Information about a Biosynthetic Gene cluster (MIBiG, https://mibig.secondarymetabolites.org/ , accessed on 20 December 2022) specification is a data standard that facilitates the consistent and systematic deposition and retrieval of metadata on BGCs and their molecular products . MIBiG is a Genomic Standards Consortium project that builds on the Minimum Information about any Sequence (MIxS) framework to (i) identify which genes are responsible for the biosynthesis of which chemical moieties, thus systematically connecting genes and chemistry; (ii) understand the natural genetic diversity of BGCs within their environmental and ecological context; and (iii) develop an evidence-based parts registry for engineering biosynthetic pathways and gene clusters through synthetic biology. The MIBiG standard contains dedicated class-specific checklists for gene clusters encoding pathways to produce alkaloids, saccharides, terpenes, polyketides, NRPs, and RiPPs . Natural antimicrobial peptides (AMPs) have been found not only in marine fish but also in marine invertebrates as major components of their innate host defense systems. The Antimicrobial Peptide Database (APD, https://aps.unmc.edu/ , accessed on 20 December 2022), online since 2003 and last updated in June 2022 , defines four unified classes of AMPs on the basis of the polypeptide chain’s connection patterns: (I) linear polypeptide chains (e.g., cathelicidins) ; (II) sidechain-linked peptides, such as disulfide-containing defensins and lantibiotics (i.e., lanthionine-containing antibiotics, e.g., microbisporicin, produced by the soil actinomycete Microbispora corallina and mathermycin from the marine actinomycete Marinactinospora thermotolerans ); (III) polypeptide chains with side chain to backbone connection (e.g., bacterial lassos and fusaricidins); and (IV) circular peptides with a seamless backbone, i.e., N- and C-termini linked by a peptide bond (e.g., plant cyclotides and animal θ-defensins) . The manually curated Database of Antimicrobial Activity and Structure of Peptides (DBAASP, http://dbaasp.org , accessed on 20 December 2022) provides detailed information (including chemical structure and activity against specific targets) on experimentally tested peptides (both natural and synthetic) that have shown antimicrobial activity as monomers, multimers, or multi-peptides . The Collection of Antimicrobial Peptides (CAMP), CAMPSign, and ClassAMP are open-access resources that have been developed to advance our current understanding of AMPs, from N- and C-terminal modifications and the presence of unusual amino acids to 3D structures thorough family-specific signatures that facilitate AMP identification and classification as antibacterial, antifungal, or antiviral . Synthetic AMPs are substantially enriched in residues with physicochemical properties known to be critical for antimicrobial activity, such as high α-helical propensity, positive charge, and hydrophobicity. The Natural Products Atlas was created as an open-access centralized knowledgebase encompassing ~25,000 microbially produced NPs using a combination of manual curation and automated data mining approaches, and was developed as a community-supported resource under findable, accessible, interoperable, and reusable (FAIR) principles. It contains referenced data for molecular structure, source organism, isolation, total synthesis, and instances of structural reassignment for compounds of bacterial, fungal, and cyanobacterial origin. Its associated web interface ( https://www.npatlas.org , v. 2.3.0, accessed on 20 December 2022) allows users to search by structure, substructure, and physical properties, as well as to explore the chemical space of these NPs from a variety of perspectives. The NP Atlas is integrated with other NP databases, including the MIBiG repository and the GNPS platform cited above. The NP Atlas was recently updated and currently embodies (i) >32,000 compounds; (ii) a full RESTful (REST is an acronym for REpresentational State Transfer and an architectural style for distributed hypermedia systems) application programming interface (API); (iii) full taxonomic descriptions for all microbial taxa; (iv) integrated data from external resources, including CyanoMetDB ( https://www.eawag.ch/en/department/uchem/projects/cyanometdb/ , accessed on 20 December 2022), a comprehensive public database of secondary metabolites from cyanobacteria (aka “blue-green algae”) ; and (v) chemical ontology terms from both ClassyFire (see below) and NPClassifier (a deep-learning tool for the automated structural classification of NPs from their counted Morgan fingerprints) . Finally, more than seven terabases of metagenomic data from samples collected in epipelagic and mesopelagic water locations across the globe by the Tara ( https://fondationtaraocean.org/en/foundation/ , accessed on 20 December 2022) Oceans project have been used to generate an ocean microbial reference gene catalog ( http://ocean-microbiome.embl.de/companion.html , accessed on 20 December 2022) with >40 million nonredundant sequences from viruses, prokaryotes, and picoeukaryotes. Remarkably, almost three quarters of ocean microbial core functionality is shared with the human gut microbiome, and epipelagic community composition was found to be mostly driven by water temperature rather than geography or any other environmental factor . A more recent analysis of 214 metagenome-assembled genomes (MAGs) recovered from the polar seawater microbiomes revealed strains that are prevalent in the polar regions while nearly undetectable in temperate seawater . The long-established Gene Ontology (GO) resource describes our knowledge of the “universe” of biology with respect to (i) molecular functions, (ii) cellular locations, and (iii) biological processes of gene products, in terms of a dynamic, controlled vocabulary that can be applied to prokaryotes and eukaryotes, as well as to single and multicellular organisms. Along the same vein, a standardized and purely structure-based chemical ontology (ChemOnt) was recently developed to automatically assign over 77 million compounds to a taxonomy consisting of >4800 different categories by means of a computer program named ClassyFire ( http://classyfire.wishartlab.com/ , accessed on 20 December 2022) that is freely accessible as a web server . This new taxonomy for chemical substances consists of up to 11 different levels (kingdom, superclass, class, subclass, etc.), with each of the categories defined by unambiguous, computable structural rules. As a follow-on, the Chemical Functional Ontology (ChemFOnt), another FAIR-compliant, web-enabled resource ( https://www.chemfont.ca , accessed on 20 December 2022), describes the functions and actions of >341,000 biologically important chemical substances, including primary and secondary metabolites, as well as drugs and NPs. The functional hierarchy within ChemFOnt consists of four functional “aspects” (physiological effect; disposition; process; and role), which are subdivided into twelve functional categories (health effects and organoleptic effects; sources, biological locations, and routes of exposure; environmental, natural, and industrial processes; adverse biological roles, normal biological roles, environmental roles, and industrial applications) and a total of >170,000 functional terms. At the time of publishing, ChemFOnt contained almost four million protein-chemical relationships and more than ten million chemical-functional relationships that can be adopted by other databases and software tools and be of utility not only to general chemists but also to researchers involved in genomics, metagenomics, proteomics, and metabolomics . NPs are the result of nature’s exploration of biologically relevant chemical space through eons of evolutionary time, hence their high diversity regarding atom connectivity and functional groups. Because they cover a broad range of sizes, 3D structures, and physicochemical properties that can be related to drug-likeness (including favorable ADME characteristics), NPs are considered not only as potential drugs, but also as an invaluable source of chemical inspiration for the development of new bioactive small molecules useful in chemical biology and medicinal chemistry research. The structural diversity of drugs was early assessed by making use of shape description methods and grouping the atoms of each drug molecule into ring, linker, framework (or scaffold) , and side chain . A methodology that calculated the NP-likeness score—a Bayesian measure of similarity with respect to the structural space covered by NPs—proved capable of efficiently separating NPs from synthetic (i.e., man-made) molecules in a cross-validation experiment . Nevertheless, rule-based procedures applied to the automated assignment of NPs to different classes, such as alkaloids, steroids, and flavonoids, have unveiled database-dependent differences in the coverage of chemical space . Beyond that, several cheminformatics techniques have been used to analyze NPs and decompose them into fragments in the belief that their unique substructural features and chemical properties are likely to be optimized for protein recognition and enzyme inhibition. A recent cheminformatic analysis of the structural and physicochemical properties of NP-based drugs in comparison to top-selling brand-name synthetic drugs revealed that macrocycles occupied distinctive and relatively underpopulated regions of chemical space, while chemical probes largely overlapped with synthetic drugs . Ideally, molecular diversity in drug discovery efforts should be focused on what is usually considered drug-like chemical space (aka “drug space”), which may (or may not) fully comply with Lipinski’s “rule of five” . A pioneering initiative to map this space made use of 72 descriptors accounting for size, lipophilicity (calculated log P o/w ), polarizability, charge, flexibility (number of nonterminal rotatable bonds), rigidity (total number of rings and rigid bonds), and hydrogen bonding abilities for a set of ~400 compounds encompassing both representative drugs (“core structures”) and a number of “satellite molecules” intentionally placed outside of the drug space (i.e., possessing extreme values in one or several of the desired properties, while containing drug-like chemical fragments). By means of principal component analysis (PCA) and projections to latent structures (PLS) it was possible, after some iterations that involved the inclusion of additional randomly selected active molecules, to extract map coordinates in the form of t -score values and construct a chemical global positioning system (ChemGPS) . The ChemGPS scores were found to describe well the latent structures extracted with PCA from a large set of compounds and appeared to be suitable for comparing multiple libraries and for keeping track of previously explored regions of chemical space. Later work (largely based on cyclooxygenase 1 and/or cyclooxygenase 2 (COX-1/2) inhibition) proposed an expansion of ChemGPS to better cover space for NPs, giving birth to ChemGPS-NP , which was further tuned for the improved handling of the chemical diversity encountered in NP research with a view to increasing the probability of hit identification . The public ChemGPS-NP Web tool ( http://chemgps.bmc.uu.se/ , accessed on 20 December 2022) was then developed to allow for the exploration of NPs by navigating in a consistent 8-dimensional global map of structural characteristics built by means of PCA . Following a different philosophy to chart the known chemical space explored by nature, the structural classification of natural products (SCONP) was devised to accomplish a hierarchical grouping of the scaffolds present in ~170,000 entries from the DNP by establishing parent–child relationships between them and arranging the scaffolds in a tree-like fashion . Some previous processing was necessary that included structure cleansing (i.e., separation from accompanying molecules) and deglycosylation (in the case of glycosides whose active component is the aglycon part). Unfortunately, stereochemistry could not be considered in this early cheminformatic analysis so that the different possible configurations of the NP scaffolds had to be treated as being equivalent. The conversion of the resulting NP scaffolds to SMILES (simplified molecular-input line-entry system) strings allowed for the comparison with those of standard synthetic molecules represented by over 10 million drug-like commercially available samples from the ZINC database . This analysis revealed interesting differences not only between natural and synthetic (i.e., man-made) molecules, but also between scaffolds originating from distinct classes of organisms, i.e., plants, bacteria, and fungi. Visual comparisons of the respective structural features were effectively displayed by plotting the scaffolds according to their frequency distributions . Moreover, a flexible analytics framework named Scaffold Hunter ( https://scaffoldhunter.sourceforge.net/ , accessed on 20 December 2022) generates and enables the visualization of virtual scaffold trees in bioactive compound collections that easily allow for the identification of new starting points for the design and synthesis of biology-oriented small molecule libraries . Interestingly, a recent cluster analysis of chemical fingerprints and molecular scaffolds of >55,000 compounds reportedly isolated from marine and terrestrial microorganisms showed that three quarters of the MNPs are closely related to compounds isolated from their terrestrial counterparts . The cheminformatic deconstruction of hundreds of thousands of NPs has allowed for the definition of thousands of fragment groups that represent a large portion of the chemical space defined by NPs and may guide the synthesis of “non-natural” NPs or pseudo-NPs, that is, molecules made in the lab that contain at least some of the structural features present in NPs but have not yet been found in living organisms . In this regard, we must bear in mind that the prototype “antimetabolite” 6-thioguanine, which was synthesized in 1955 by Nobel Prize winners Elion and Hitchings , was found in 2013 to be biosynthetically produced by Erwinia amylovora , the bacteria responsible for fire blight pathogenesis in apple and pear trees . In fact, a recent cheminformatic analysis revealed that a significant portion of biologically active synthetic compounds can be regarded as pseudo-NPs and, as such, the result of human-directed “chemical evolution” of NP structure . Once again, humans imitate nature by (i) performing atom/group replacement and/or decorating with novel fragments what are thought to be privileged scaffolds for bioactivity ; or (ii) combining fragment-sized NPs and/or NP fragments to provide “hybrid NPs” . Historically, the total synthesis of NPs followed by derivative synthesis (“active analogue approach” or “analogue-oriented synthesis” ) and semisynthetic procedures aimed at modifying the chemical structure of complex fermentation products have enabled a deeper understanding of structure–activity relationships (SAR). In contrast, the de novo combination of NP fragments in unique arrangements, often by virtue of innovative strategies such as “diversity-oriented synthesis” , “target-oriented and diversity-oriented organic synthesis” , and “synthesis-informed design” , has been shown to generate focused NP-like libraries containing compounds endowed with bioactivities unrelated to those of the guiding NP(s) . Examples of successful workflows of pseudo-NP design and development are “biology-oriented synthesis” and “pharmacophore-directed retrosynthesis” . In applying the latter approach, a key first step is to elaborate a tentative pharmacophore, i.e., “an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a specific biological target and to trigger (or block) its biological response”, as defined by the International Union of Pure and Applied Chemistry (IUPAC) , and then devise a retrosynthetic procedure that ensures that the proposed pharmacophore is present in multiple intermediates of increasing complexity, ultimately leading to the NP. An important goal of these synthetic approaches is to find structurally simplified and optimized derivatives with lower molecular weights that can overcome commonly observed limitations, such as poor oral absorption, short half-life, and low blood–brain barrier permeability. The popular term “druggable genome” refers to the genes (or, more appropriately, gene products) that are known or predicted to interact with drugs, ideally resulting in a therapeutic benefit. Although drugs are intended to be selective (i.e., have high affinity for one single target), it is not uncommon for many molecules to bind to more than one protein, giving rise to polypharmacology and side effects. Due to the fact that many drug-target combinations are theoretically possible, the computational exploration of possible interactions can help identify potential targets. Because the systematic identification of drug targets for NPs, regardless of their origin, using a battery of experimental binding or affinity assays, is both costly and time-consuming, a substantial amount of effort has gone into devising in silico tools that allow for the construction of global networks that connect active compounds to their cellular targets. It is expected that, by using these methods, the resulting system’s pharmacology infrastructure will help to predict new drug targets for pharmacologically uncharacterized NPs and identify secondary targets (off-targets) that can aid in the rationalization of side effects of known molecules . The Drug-Gene Interaction Database (DGIdb 4.0, https://www.dgidb.org/ , accessed on 20 December 2022) provides information on drug-gene interactions and druggable gene products collected from publications, databases, and other web sites . The latest update mostly focused on (i) the integration with crowdsourced efforts (e.g., Wikidata) to facilitate term normalization and with the open-data web platform Drug Target Commons ( https://dataverse.harvard.edu/dataverse/dtc2tdc , accessed on 20 December 2022) to enable the upload of community-contributed interaction data; and (ii) export to a Network Data Exchange (NDEx) infrastructure for storing, sharing and publishing biological network knowledge. The tool named substructure-drug-target network-based inference (SDTNBI) was devised to prioritize potential targets for old drugs (“drug repositioning”), failed drugs, and new chemical entities by bridging the gap between new chemical entities and known drug-target interactions (DTIs) . A later modification (wSDTNBI) uses weighted DTI networks, whose edge weights are correlated with binding affinities, and network-based VS, which does not rely on the receptors’ 3D structures . The publicly available SwissTargetPrediction web server ( http://www.swisstargetprediction.ch , accessed on 20 December 2022) also attempts to predict the most likely target(s) (in mice, rats, or human beings) for a SMILES-defined input molecule by using a computational method that combines different measures of similarities (both in 2D chemical structure and in 3D molecular shape) with known ligands . All of these approaches, together with highly efficient receptor-based ligand docking , can be useful to narrow down the number of potential targets, but strict experimental confirmation and validation are needed . The attention initially drawn to certain synthetic molecules that were responsible for disproportionate percentages of hits in enzyme-based bioassays but, on closer inspection, turned out to be false actives and therefore nonprogressible hits, leading to the PAINS acronym (Pan Assay INterference compoundS) , was later extended to NPs . As a result, some NPs have been designated as “invalid metabolic panaceas” and the concept of “residual complexity” ( http://go.uic.edu/residualcomplexity , accessed on 20 December 2022) has emerged . Nowadays, compounds with a PAINS chemotype can be recognized and excluded from bioassays by the judicious use of electronic substructure filters and machine learning approaches (e.g., Hit Dexter, https://nerdd.univie.ac.at/hitdexter3/ , accessed on 20 December 2022). Because the best link connecting NPs to their targets is arguably the experimentally determined 3D structure of the respective complexes, in the following section, I will provide some examples of MNPs and synthetic analogues that were selected on the basis of chemical novelty and submicromolar inhibition data, preferably supported by structural evidence of complex formation with pharmacologically relevant enzyme targets. The road from the research laboratory to the drug pipeline is long and winding. Quite often, molecules originally assayed for one biological activity end up showing promise for another unintended indication, either fortuitously or by following one of the computational approaches outlined in the previous sections. Bengamides A and B were first described as heterocyclic anthelmintics naturally present in the sponge Jaspis cf. coriacea , and later on, not only in other sponges from many biogeographic sites, but also in the terrestrial Gram-negative bacterium Myxococcus virescens . Decades of further research have shown that methionine aminopeptidases MetAP1 and MetAP2 (essential metalloenzymes that remove the initiator amino-terminal methionine from nascent proteins) are molecular targets for bengamides, which also display notable antiproliferative and antiangiogenic properties . In fact, a synthetic analogue of bengamide B, LAF389, was the subject of a phase I anticancer clinical trial that, unfortunately, demonstrated no objective responses and also the occurrence of unanticipated cardiovascular events. The high-resolution 3D structures of both human MetAP1 and MetAP2 enzymes in complex with bengamide derivatives, including LAF389 (PDB entry 1QZY) , have been solved and show these compounds bound in a manner that mimics the binding of peptide substrates, with three key hydroxyl groups on the inhibitor coordinating the di-Co(II) center in the enzyme active site. Renewed interest in bengamides is currently focused on their antibacterial activities against various drug-resistant Mycobacterium tuberculosis and Staphylococcus aureus strains . Incidentally, the mycotoxin fumagillin, first isolated from Aspergillus fumigatus and originally studied also as an antiangiogenic agent and human MetAP2 inhibitor , has been widely used for more than 60 years in apiculture to control nosema disease in honey bees effectively because the microsporidian Nosema apis lacks MetAP1 and targeting MetAP2 suppresses infection. Another showcase example is provided by gracilin A , a nor-diterpene metabolite originally isolated from the Mediterranean sponge Spongionella gracilis , that was initially reported as a potent phospholipase A 2 (PLA 2 ) inhibitor and later shown to mimic the immunosuppressive effects of cyclosporin A through interaction with cyclophilin A (CypA) . In a recent pharmacophore-directed retrosynthesis application, a theoretically derived pharmacophore of gracilin A was chosen as an early synthetic target. Then, sequential increases in the complexity of this minimal structure enabled SAR profiling and the identification of structurally less complex derivatives of gracilin A that displayed selectivity for mitochondrial CypD over CypA inhibition as well as significant neuroprotective and/or immunosuppressive activities . The sesterterpenoids are metabolites first isolated from marine sponges of the Thorectidae family, which includes the genera Cacospongia , Fasciospongia, Luffariella , and Thorecta , that often contain biologically active butenolide and hydroxybutenolide groups in their structures . The anti-inflammatory activity of manoalide and luffolide ( a) was related to the inactivation of secretory PLA 2 , whereas for cacospongionolide F this biological effect was shown to involve the inhibition of the nuclear factor-κB (NF-κB) pathway as well . In contrast, the related dysidiolide ( b) from the Caribbean sponge Dysidea etherea de Laubenfels (Dysideidae family) was the first known natural inhibitor of the cyclin-dependent kinase (CDK)-activating phosphatases cdc25A and cdc25B, with IC 50 values in the micromolar range . Later on, dysidiolide and distinctly decorated analogues prepared from ent -halimic acid following a classical “active analogue approach” were shown to cause stage-specific arrest of proliferating cancer cells, but again at low micromolar concentrations . Because cdc25A was found to be present in the same protein structure similarity cluster (PSSC) as 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1, an enzyme that catalyzes the conversion of cortisone to cortisol), the SCONP-guided selection of the 1,2,3,4,4 a ,5,6,7-octahydronaphthalene scaffold present in dysidiolide led to a focused compound library ( b) that showed the submicromolar inhibition of 11βHSD1 and selectivity over 11βHSD2 . The bisulfide bromotyrosine- and oxime-containing derivatives psammaplin A and bisaprasin were originally characterized as nanomolar inhibitors of histone deacetylases and DNA methyltransferase enzymes , but it is known today that they are used by marine sponges, such as Pseudoceratina purpurea and Aplysinella rhax, in their chemical communication and quorum sensing systems to prevent biofilm formation and attenuate virulence factor expression by pathogenic microorganisms. A number of MNPs are potent inhibitors of proteases, an important drug target class in human diseases that is integrated in MEROPS ( http://www.ebi.ac.uk/merops/ , accessed on 20 December 2022), a database of proteolytic enzymes, their substrates and inhibitors . Gallinamide A , a metabolite of the marine cyanobacterium Schizothrix sp. that originally displayed modest antimalarial activity, was subsequently reisolated and characterized as a potent and irreversible inhibitor of the human cysteine protease cathepsin L ( k i = 9000 ± 260 M −1 s −1 ), with 8- to 320-fold greater selectivity over the closely related cathepsins V or B . Docking-guided modifications to improve the binding affinity resulted in notably enhanced potency against cathepsin L ( K i = 0.0937 ± 0.01 nM and k inact / K i = 8,730,000). Gallinamide and its analogs also displayed the potent inhibition of the highly homologous cruzain, an essential Trypanosoma cruzi cysteine protease, as well as cytotoxic activity on intracellular T. cruzi amastigotes . Importantly, the biochemical data indicated that inhibitor potency was driven by the rate of formation of the reversible enzyme:inhibitor complex, rather than by the rate of covalent modification. The resolution of the 3D co-crystal structure of the complex formed between cruzain and gallinamide A later confirmed the proposed binding pose and revealed the expected covalent bond formed between the drug’s Michael acceptor enamide and the active site Cys25 thiol (PDB entry 7JUJ) . The in vitro antiplasmodial activity of an extract of the sponge Theonella aff. swinhoei collected in Madagascar was ascribed, in part, to the previously known actin-binding metabolite swinholide A . Further work disclosed the presence of three unusual cyclic peptides, cyclotheonellazoles A–C , containing six nonproteinogenic amino acids out of the eight composing units (of which the most novel were 4-propenoyl-2-tyrosylthiazole and 3-amino-4-methyl-2-oxohexanoic acid). These macrocyclic peptides are thought to be produced by hybrid PKS-NRPS enzymes from symbiotic bacteria and were found not to be active against Plasmodium , but instead displayed the nanomolar and subnanomolar inhibition of chymotrypsin and elastase, respectively . This latter enzyme has been considered an important target to prevent acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in COVID-19 patients, and the inhibition of its activity by cyclotheonellazole A has been recently shown to reduce lung edema and pathological deterioration in an ALI mouse model, comparing favorably with the clinically approved elastase inhibitor sivelestat . The (Ahp)-containing cyclodepsipeptide family of cyanobacterial NPs biosynthesized by NRPS is noteworthy for the ability of many of its members to inhibit several serine proteases, most notably human neutrophil elastase and kallikreins , by virtue of mimicking the natural substrates. The 3-amino-6-hydroxy-2-piperidone (Ahp) unit serves as the general pharmacophore, whereas the adjacent ( Z )-2-amino-2-butenoic acid confers selectivity for elastase . The depsipeptide molassamide was purified and characterized from cyanobacterial assemblages of Dichothrix utahensis as a new analogue of the cytostatic depsipeptide dolastatin 13 (originally isolated from the sea hare Dolabella auricularia ) that inhibited elastase and chymotrypsin at submicromolar concentrations, but not trypsin . The analysis of the X-ray crystal structure of porcine elastase in complex with lyngbyastatin 7 (PDB code 4GVU) and SAR studies resulted in the synthesis of symplostatin 5, whose activity was comparable to that of sivelestat in short-term assays and more sustained in longer-term assays . Complex fractionation guided by MS 2 metabolomics (molecular networking) , together with HPLC, NMR, and chiral chromatography, allowed for the identification of tutuilamides A and B from Schizothrix sp., along with tutuilamide C from a Coleofasciculus sp. These novel structures , which are also potent elastase inhibitors, bind reversibly to this enzyme, as shown in the co-crystal structure of tutuilamide A in complex with porcine elastase (PDB code 6TH7), despite the fact that they feature an unusual vinyl chloride-containing residue. An additional hydrogen bond relative to lyngbyastatin 7 has been proposed as the element responsible for its enhanced inhibitory potency . More recently, yet another family of new Ahp-cyclodepsipeptides, the rivulariapeptolides, with nanomolar potency as serine protease inhibitors, was identified from an environmental cyanobacteria community using a scalable, bioactivity-focused, native metabolomics approach . The human proteasome, a multicatalytic enzyme complex that is responsible for the regulated non-lysosomal degradation of cellular proteins, gained notorious pharmacological relevance when the synthetic boron-containing bortezomib (originally developed by ProScript to treat muscle weakness and muscle loss associated with AIDS, as well as muscular dystrophy) was approved in 2003 by the FDA as Velcade ® (co-developed by Millennium/Takeda and Janssen-Cilag) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma. Carfilzomib , an α′,β′-epoxyketone-containing analog of the NP epoximicin—first identified in an Indian soil actinomycete strain —was also approved in 2012 as Kyprolis ® (Onyx Pharmaceuticals) for clinical use in MM patients, in combination with lenalidomide and dexamethasone. Marizomib (aka salinosporamide A) is a structurally and pharmacologically unique MNP that contains a β-lactone-γ-lactam and is produced by the marine actinomycete Salinispora tropica . Marizomib not only inhibits the chymotrypsin-like activity of the proteasome (via a novel mechanism involving the acylation of the O γ in the N-terminal catalytic Thr residue followed by the displacement of chloride) but also those of the caspase-like and trypsin-like subunits . In addition to its “pan-proteasome” pharmacodynamic activity, marizomib crosses the blood-brain barrier, and for these reasons it has been extensively studied, first preclinically , and then in phase I–III clinical trials, both alone and in combination. Many other MNP scaffolds continue serving as inspiration for the design and synthesis of potent 20S human proteasome inhibitors, including carmaphycins A and B from a marine cyanobacterium Symploca species and fellutamide B, originally isolated from Penicillium fellutanium , a fungus found in the gastrointestinal tract of the marine fish Apogon endekataenia . In the development of potent covalent inhibitors of the proteasome, ligand docking and binding energy calculations have highlighted the importance of the optimization of the prior noncovalent binding mode, through conformational restraints, in a pose close to that found in the transition state . Protein kinases are validated drug targets because (i) kinase deregulation plays an essential role in many disease states, and (ii) many inhibitors have already shown therapeutic benefit (almost one hundred are currently approved for clinical use). This bioactivity is of broad scope and has been reported for various MNPs obtained from different sources, including bacteria and cyanobacteria, fungi, algae, soft corals, sponges, and animals . Of note, a significant number of them were originally isolated from terrestrial sources and subsequently found in marine organisms too, and vice versa. For example, the pan-kinase inhibitor staurosporine (a pentacyclic indolo(2,3- a )carbazole first discovered in 1977 from the bacterium Streptomyces staurosporeus ) was one of the early tools used to probe the cellular effects of blocking the ATP-binding pocket in different protein kinases. In 2002, 11-hydroxystaurosporine was reported to be present in an ascidian Eudistoma species collected in Micronesia and to be a more potent inhibitor of protein kinase C than staurosporine itself . Another early potent pan-kinase inhibitor is ( Z )-hymenialdisine, which owes its name to Hymeniacidon aldis , the sponge where it was originally found. These and many other MNPs inspired synthetic work on analogues and novel scaffolds that paved the ground for the discovery of imatinib (Gleevec ® ), a landmark drug that has (i) significantly improved the outcomes of patients with chronic myelogenous leukemia by inhibiting the oncogenic BCR–ABL tyrosine kinase; (ii) shown remarkable clinical efficacy in the treatment of other malignancies; (iii) helped establish the concept of “targeted therapy” in the field of cancer research; (iv) fostered the concept of “precision medicine”, i.e., tailor the chemotherapeutic treatment to the unique genetic changes in an individual’s cancer cells; and (v) fuel the extremely rich and rewarding research on protein kinase inhibitors . A top priority in the development of novel protein kinase inhibitors is to understand selectivity so that the tendency of one given drug to bind to other unintended kinases (off-targets) can be suppressed or attenuated. To this end, kinome-wide inhibitory selectivity profiling is necessary because small assay panels cannot provide a robust measure of selectivity . Binding site similarity searches, as performed in the KinomeFEATURE ( https://simtk.org/projects/kdb , accessed on 20 December 2022) and KID databases, along with machine learning models that map the activity profile of inhibitors across the entire human kinome , as exemplified by Drug Discovery Maps , can be of help not only to gain insight into the structural basis of kinase cross-inhibition, but also to predict the binding affinities of novel kinase inhibitors. Cortistatin A ( a) was isolated as an antiangiogenic steroidal alkaloid from the marine sponge Corticium simplex and should not be confused with the somatostatin-like cortistatin neuropeptides. It consists of a 9(10→19)- abeo -androstane and isoquinoline skeleton and was originally shown to inhibit the proliferation of human umbilical vein endothelial cells at nanomolar concentrations . It was later found that this MNP selectively inhibits the mediator-associated cyclin-dependent kinase CDK8 and disproportionately induces the upregulation of superenhancer-associated genes in acute myeloid leukemia cell lines . The crystal structure of the ternary complex of CDK8 bound to cyclin C and cortistatin A (PDB code 4CRL) revealed exquisite shape complementarity between this alkaloid and the ATP-binding pocket of CDK8 ( b), with the crucial isoquinoline making essential hydrogen bonding interactions with the peptide backbone. The last example in this review is provided by sphaerimicin A , a complex macrocyclic uridine nucleoside derivative isolated from Sphaerisporangium sp. SANK60911 using a genome mining approach focused on the enzyme uridine-5′-aldehyde transaldolase . Even though this is a terrestrial actinomycete, it is closely related to other marine species that contain similar BGCs, hence its inclusion in this section. Sphaeromicin A exhibits nanomolar inhibitory activity on bacterial MraY, an integral membrane enzyme that catalyzes the transfer of phospho- N -acetylmuramyl pentapeptide from UDP- N -acetylmuramyl pentapeptide (Park’s nucleotide) to the phospholipid undecaprenyl phosphate during the lipid cycle of peptidoglycan biosynthesis. In an elegant example of molecular design assisted by theoretical conformational analysis and NMR data, the simplified analogues with defined stereochemistry SPM-1 and SPM-2 were synthesized . The fact that SPM-1 turned out to be 54-fold more potent than SPM-2 against MraY from Aquifex aeolicus (MraY AA ) revealed the importance of the conformationally restrained macrocycle for target binding, an aspect that was clarified even further when the 3D structure of the MraY AA :SPM-1 complex was solved by X-ray crystallography. Therefore structure-based optimization is now feasible in order to develop MraY inhibitors with the potential of becoming novel antibiotics against drug-resistant bacteria. Computational methodologies play indispensable roles in the exploration of the vast chemical space covered by MNPs by helping, among many other tasks, to (i) elucidate their chemical composition and 3D structure; (ii) store, process, curate, and organize huge amounts of information related to source organisms, biosynthesis, and bioactivity; and (iii) connect biological activities with both molecular scaffolds and target binding sites . The limits of biologically relevant chemical space for enzyme inhibitors are defined by the specific binding interactions taking place between small- and medium-sized molecules (e.g., terpenoids, alkaloids, polyketides, non-ribosomal peptides, and RiPPs) and a number of selected orthosteric and allosteric pockets in macromolecular catalysts that have evolved over billions of years . Some of the molecular entities recently found in marine microbiota can easily defy and outperform a chemist’s imagination and ingenuity, and also be endowed with unexpected, and even unprecedented, bioactivities that may inspire more synthetic creativity. A historical example is the clinically used cytarabine (aka cytosine arabinoside or arabinosyl cytosine, Ara-C), a synthetic pyrimidine nucleoside that was developed in the imitation of spongothymidine, a nucleoside originally isolated from the Caribbean sponge Tethya crypta . Many other analogues, however, did not follow the same fate and, in fact, the potential of MNPs as therapeutic agents for human diseases has been realized only in a few cases, which attests to the enormous difficulties of progressing many of these compounds through the drug pipeline with the final goal of demonstrating an acceptable benefit-risk balance in clinical trials and thereafter. We must be confident that the new generations of cross-disciplinary trained scientists working in community-wide networks (e.g., Ocean Medicines, https://cordis.europa.eu/project/id/690944 , accessed on 20 December 2022) will overcome existing hurdles to find valuable new medicines inspired by, or based on, MNPs. It seems clear that the integration of information from various sources, including high-throughput phenotypic screening and BGC engineering, using computational methods has revolutionized NP research and can speed up the process of discovering new biologically active molecules from marine and terrestrial sources. A major bottleneck in these efforts is to identify the macromolecular target that is responsible for the observed (or assigned) mechanism of action, a problem that is usually aggravated when dealing with complex mixtures of MNPs. The recent success in the functional characterization of several NPs and the identification of bioactive metabolites upon integrating results from untargeted metabolomics, high-content image analysis of perturbation-treated cells, and gene expression signatures on a data-driven multi-platform raises the hope for the accelerated discovery of novel pharmacologically active MNPs in the near future. |
Screening of serum biomarkers in patients with PCOS through lipid omics and ensemble machine learning | 6fb72df1-e414-46e4-8152-d058c47dda56 | 11706364 | Biochemistry[mh] | Polycystic ovary syndrome (PCOS) is a multifactorial, complex, and heterogeneous disorder characterized by hyperandrogenism, menstrual irregularities, infertility, and polycystic ovaries on ultrasound . As an endocrine disorder, PCOS affects about 7%-8% of women of reproductive age . Essential factors in its pathogenesis are hyperinsulinemia, insulin resistance, and dyslipidemia . Endocrine and metabolic abnormalities, including infertility, obesity, type 2 diabetes, and increased luteinizing hormone (LH), are common in women with PCOS . In many cases, lifestyle changes and complementary and alternative medicines are the first-line treatments of choice . Despite the high incidence of PCOS, its attendant morbidity is likely to be underdiagnosed. Therefore, the search for molecular markers that can identify and treat PCOS early and understand its pathogenesis is conducive to taking appropriate interventions for early treatment and providing new therapeutic targets. Biomarkers are crucial in diagnosing and monitoring PCOS . For example, elevation of the anti-Mullerian hormone (AMH) level is associated with the diagnosis of PCOS, which can be used as an indicator to assess ovarian reserve function . A decrease in lipocalin levels has been associated with insulin resistance and abnormal lipid metabolism in patients with PCOS . Abnormal lipid metabolism is also common in patients with PCOS . Studies have shown specific changes in the lipid profile of PCOS patients, such as abnormal phosphatidylcholine (PC) metabolism, decreased polyunsaturated fatty acids (PUFAs), and increased long-chain saturated fatty acids (LSFAs) . Therefore, identifying non-invasive PCOS biomarkers has become a priority in PCOS research. Metabolomics looks for relative relationships between specific metabolites, diseases, and their phenotypic changes through qualitative and quantitative analysis of blood, urine, feces, and other body fluids . It is a powerful exploratory tool for discovering new diagnostic molecules or biomarkers, providing evidence for diagnosing and treating diseases, including PCOS . The rapid development of metabolomics has led to renewed interest in metabolism and the role of small molecule metabolites in many biological processes . Untargeted metabolomics is a “hypothesis-generating discovery strategy” that compares sample groups (e.g., cases versus controls), identifies metabolome, and establishes (early indications) perturbations . Therefore, this study further screened the lipid metabolites in the serum of PCOS patients using untargeted lipidomics. Machine learning is an emerging field of medicine, and numerous resources are devoted to the fusion of computer science and statistics to medical problems . It is a branch of computer science and statistics that generates predictive or descriptive models by learning from training data rather than through rigorous programming . Computational modeling and simulations can help interpret and understand critical biomarkers of physiological significance extracted from machine learning . In the study of PCOS, Silva IS et al. used a machine learning model to classify clinical and laboratory variables associated with novel phenotypes of PCOS into phenotypically distinct clusters, helping to guide more personalized and effective treatments for PCOS . Zhang X et al. used Raman spectroscopy combined with a machine learning algorithm to analyze and characterize metabolic changes in follicular fluid and plasma samples from PCOS patients . These studies demonstrate the critical role of machine learning in diagnosing PCOS markers. Based on the above background, this study is the first to combine untargeted lipidomics with machine learning as a powerful strategy for efficient and accurate screening of POCS biomarkers. In this study, we screened the key lipid metabolites in the serum of PCOS patients. We constructed a more efficient diagnostic marker panel model based on the feature selection technology of ensemble learning for clinical application.
Clinical samples 152 PCOS patients (PCOS, EG) and 50 non-PCOS subjects (Control, CG) from Shenzhen Longhua District Central Hospital participated. summarises the clinical characteristics of PCOS patients and non-PCOS subjects. PCOS was diagnosed by the European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE/ASRM) Rotterdam’s Criteria 2003 . This study collected general information about the study subjects, including gender, age, body mass index, and related hormone levels. Diagnostic criteria included scanty menstruation, amenorrhea, or irregular uterine bleeding; fulfilling one of the two criteria (hyperandrogenic clinical manifestations or hyperandrogenemia; ultrasound showing bilateral or unilateral ovarian polycystic changes and/or increased volume), and excluding other diseases that might cause hyperandrogenism and ovulatory abnormalities. Other inclusion criteria included Han nationality, not taking any hormonal drugs (e.g., estrogen, progesterone, birth control pills, etc.) in the last 6 months. Exclusion criteria included hyperadrenocorticism, tumors secreted by the ovaries or adrenal glands, and pharmacological hyperandrogenism. The study was performed after obtaining verbal informed consent from all participants and was approved by the Shenzhen Longhua District Central Hospital Committee (Approval number: 2022-005-01). Samples were collected from March 1, 2022, until December 31, 2022. All participants provided the written informed consent. Untargeted lipidomics analysis Serum samples were collected from PCOS patients and non-PCOS subjects. The blood samples were stored in anticoagulation tubes, centrifuged at 3000 rpm for 10 min, and then the upper serum was transferred to Eppendorf tubes and stored at -80°C until further processing. Lipids were extracted using the MTBE method. Briefly, samples were homogenized with 200 μL water and 240 μL methanol. Then 800 μL of MTBE was added, and the mixture was ultrasound 20 min at 4°C followed by sitting still for 30 min at room temperature. The solution was centrifuged at 14000 g for 15 min at 10°C, and the upper organic solvent layer was obtained and dried under nitrogen. Reverse phase chromatography was selected for LC separation using the CSH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters). The samples were separated by UHPLC Nexera LC-30A and analyzed by mass spectrometry using a Q-Exactive Plus in positive and negative mode, respectively . The data obtained from the positive and negative ion patterns were analyzed using Lipid Search. The lipid class and the number of lipid species identified in each class were identified by positive and negative ion patterns. Changes in lipid content reflect changes in lipid function, and functional discussions and subsequent biofunctional studies were conducted on each of the changed lipid classes. Feature weight calculation Integration was based on the combination of multiple feature selection methods. Substances (such as proteins and metabolites) frequently selected in feature selection methods were rewarded. The reward scores obtained in each feature selection method were combined to calculate each substance’s comprehensive weight value. Selection of candidate biomarkers The distribution of lipid metabolites in different classifications was first demonstrated to select candidate biomarkers efficiently. Orthogonal partial least squares discriminant analysis (OPLS-DA) modelling of lipid metabolites was performed on samples from both groups with 100 simulation replications. Lipid metabolites were compared between the two groups and screened for metabolites with fold change > 2 or fold change < 0.5 and adjusted P value < 0.05. The lipid metabolites in the OPLS-DA model were ranked according to the magnitude of the Variable Importance for the Projection (VIP) value, and the top 20 were selected. The VIP value obtained from the OPLS-DA model can be used to measure the strength of the influence of the expression pattern of each lipid molecule on the categorical discrimination of each group of samples and the ability to explain them and to excavate biologically significant differential lipid molecules. Generally, lipid molecules with VIP > 1 contribute substantially to model interpretation. To avoid overfitting the supervised model during the modelling process, a permutation test was utilized to test the model and ensure its validity. After obtaining the classifications of differential lipid metabolites, we analyzed the associations between the classifications of these differential metabolites and the clinical indexes using the Mantel test. We detected the associations between the classifications of the differential metabolites using Pearson correlation analysis. A receiver operating characteristic (ROC) analysis was utilized to assess the strength of the influence of candidate markers on the model’s AUC value. AUC value was a familiar indicator performed to evaluate the pros and cons of a two-class model. The full name of AUC was the area under the curve, which was the area under the ROC curve. A higher AUC value usually indicates a better classification effect for the model. Validation and evaluation of biomarkers Three commonly used machine learning models were used: logistic regression (LR), random forest (RF), and support vector machine (SVM) to evaluate the effect of candidate biomarkers on the classification model. The candidate markers were validated respectively. LR is particularly suited to binary classification problems. In lipidomic data analysis, constructing a logistic model by LR allows for assessing how individual lipids or lipid combinations affect specific biological phenotypes. It can be used to determine the association between a particular lipid or lipid module and a specific phenotype (e.g., presence or absence of disease). This has important implications for understanding how specific lipids affect disease risk. RF improves prediction accuracy by building multiple decision trees and combining their predictions, and it is ideally suited for dealing with complex datasets with many variables. RF can be used to identify the essential lipids that differentiate between different phenotypes and assess the importance of each lipid in the model, helping us identify the lipids that have the most significant impact on the phenotype. SVM is suitable for complex classification problems because it best distinguishes different classes by separating hyperplanes in multidimensional space. In lipidomic data, SVM can accurately classify samples based on lipid expression levels, such as separating diseased patients from healthy controls. SVM is particularly well suited to datasets with a small number of samples but a large number of features. ROC curve analysis on the horizontal axis indicated a false positive rate (FPR, 1-specificity), which referred to the percentage of accurate negative samples incorrectly identified as positive. The vertical axis representss the actual positive rate (TPR), also known as sensitivity, which refers to the proportion of actual positive samples correctly judged as positive. The single model index mainly depended on the area value (AUC value) under the ROC curve, specificity, and sensitivity. The importance coefficients of candidate biomarkers were calculated using the classification model constructed by the RF algorithm, which was used to compare the contribution of each biomarker to the model. The expression levels of candidate biomarkers in each comparison group were calculated. Pearson correlation coefficient was calculated for the expression levels of the candidate biomarkers. Generally speaking, the lower the correlation between the biomarkers in diagnostic panels, the lower the overlap between the selected biomarkers, and the more optimized the panel combination. Construction and validation of the diagnostic panel model The biomarkers diagnostic panel model was constructed using the LR algorithm. LR is a commonly used classification model that can predict the probability of an event and analyze its influencing factors. Biomarkers LR model formula: p = 1/(1+e -z ). The expression level value of the biomarker was brought into the probability value p calculated by the panel model formula. If the p-value exceeded the cutoff value, it was considered a positive diagnosis. To obtain this cutoff, we used Youden’s index to determine the optimal cutoff value for diagnostic decisions . When the sensitivity and specificity were given the same weight, the cutoff value corresponding to the maximum Youden’s index was the best critical point for biomarker identification ability , because the sum of sensitivity and specificity was the maximum at this point. The best cutoff could have good sensitivity and specificity at the same time. Finally, based on the expression of candidate biomarkers in the sample (the original data set was divided into training set and test set, or new validation set data was provided), ROC analysis was performed using the diagnostic model constructed above. Statistical analysis Graphpad Prism8.0 software was used for statistical analysis. If the normal distribution was met, data were expressed as (x±s), and a t-test was used between groups. If it did not conform to a normal distribution, data were expressed as average (minimum, maximum), and the Mann-Whitney U test was used between groups. P value < 0.05 indicated that the difference was statistically significant.
152 PCOS patients (PCOS, EG) and 50 non-PCOS subjects (Control, CG) from Shenzhen Longhua District Central Hospital participated. summarises the clinical characteristics of PCOS patients and non-PCOS subjects. PCOS was diagnosed by the European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE/ASRM) Rotterdam’s Criteria 2003 . This study collected general information about the study subjects, including gender, age, body mass index, and related hormone levels. Diagnostic criteria included scanty menstruation, amenorrhea, or irregular uterine bleeding; fulfilling one of the two criteria (hyperandrogenic clinical manifestations or hyperandrogenemia; ultrasound showing bilateral or unilateral ovarian polycystic changes and/or increased volume), and excluding other diseases that might cause hyperandrogenism and ovulatory abnormalities. Other inclusion criteria included Han nationality, not taking any hormonal drugs (e.g., estrogen, progesterone, birth control pills, etc.) in the last 6 months. Exclusion criteria included hyperadrenocorticism, tumors secreted by the ovaries or adrenal glands, and pharmacological hyperandrogenism. The study was performed after obtaining verbal informed consent from all participants and was approved by the Shenzhen Longhua District Central Hospital Committee (Approval number: 2022-005-01). Samples were collected from March 1, 2022, until December 31, 2022. All participants provided the written informed consent.
Serum samples were collected from PCOS patients and non-PCOS subjects. The blood samples were stored in anticoagulation tubes, centrifuged at 3000 rpm for 10 min, and then the upper serum was transferred to Eppendorf tubes and stored at -80°C until further processing. Lipids were extracted using the MTBE method. Briefly, samples were homogenized with 200 μL water and 240 μL methanol. Then 800 μL of MTBE was added, and the mixture was ultrasound 20 min at 4°C followed by sitting still for 30 min at room temperature. The solution was centrifuged at 14000 g for 15 min at 10°C, and the upper organic solvent layer was obtained and dried under nitrogen. Reverse phase chromatography was selected for LC separation using the CSH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters). The samples were separated by UHPLC Nexera LC-30A and analyzed by mass spectrometry using a Q-Exactive Plus in positive and negative mode, respectively . The data obtained from the positive and negative ion patterns were analyzed using Lipid Search. The lipid class and the number of lipid species identified in each class were identified by positive and negative ion patterns. Changes in lipid content reflect changes in lipid function, and functional discussions and subsequent biofunctional studies were conducted on each of the changed lipid classes.
Integration was based on the combination of multiple feature selection methods. Substances (such as proteins and metabolites) frequently selected in feature selection methods were rewarded. The reward scores obtained in each feature selection method were combined to calculate each substance’s comprehensive weight value.
The distribution of lipid metabolites in different classifications was first demonstrated to select candidate biomarkers efficiently. Orthogonal partial least squares discriminant analysis (OPLS-DA) modelling of lipid metabolites was performed on samples from both groups with 100 simulation replications. Lipid metabolites were compared between the two groups and screened for metabolites with fold change > 2 or fold change < 0.5 and adjusted P value < 0.05. The lipid metabolites in the OPLS-DA model were ranked according to the magnitude of the Variable Importance for the Projection (VIP) value, and the top 20 were selected. The VIP value obtained from the OPLS-DA model can be used to measure the strength of the influence of the expression pattern of each lipid molecule on the categorical discrimination of each group of samples and the ability to explain them and to excavate biologically significant differential lipid molecules. Generally, lipid molecules with VIP > 1 contribute substantially to model interpretation. To avoid overfitting the supervised model during the modelling process, a permutation test was utilized to test the model and ensure its validity. After obtaining the classifications of differential lipid metabolites, we analyzed the associations between the classifications of these differential metabolites and the clinical indexes using the Mantel test. We detected the associations between the classifications of the differential metabolites using Pearson correlation analysis. A receiver operating characteristic (ROC) analysis was utilized to assess the strength of the influence of candidate markers on the model’s AUC value. AUC value was a familiar indicator performed to evaluate the pros and cons of a two-class model. The full name of AUC was the area under the curve, which was the area under the ROC curve. A higher AUC value usually indicates a better classification effect for the model.
Three commonly used machine learning models were used: logistic regression (LR), random forest (RF), and support vector machine (SVM) to evaluate the effect of candidate biomarkers on the classification model. The candidate markers were validated respectively. LR is particularly suited to binary classification problems. In lipidomic data analysis, constructing a logistic model by LR allows for assessing how individual lipids or lipid combinations affect specific biological phenotypes. It can be used to determine the association between a particular lipid or lipid module and a specific phenotype (e.g., presence or absence of disease). This has important implications for understanding how specific lipids affect disease risk. RF improves prediction accuracy by building multiple decision trees and combining their predictions, and it is ideally suited for dealing with complex datasets with many variables. RF can be used to identify the essential lipids that differentiate between different phenotypes and assess the importance of each lipid in the model, helping us identify the lipids that have the most significant impact on the phenotype. SVM is suitable for complex classification problems because it best distinguishes different classes by separating hyperplanes in multidimensional space. In lipidomic data, SVM can accurately classify samples based on lipid expression levels, such as separating diseased patients from healthy controls. SVM is particularly well suited to datasets with a small number of samples but a large number of features. ROC curve analysis on the horizontal axis indicated a false positive rate (FPR, 1-specificity), which referred to the percentage of accurate negative samples incorrectly identified as positive. The vertical axis representss the actual positive rate (TPR), also known as sensitivity, which refers to the proportion of actual positive samples correctly judged as positive. The single model index mainly depended on the area value (AUC value) under the ROC curve, specificity, and sensitivity. The importance coefficients of candidate biomarkers were calculated using the classification model constructed by the RF algorithm, which was used to compare the contribution of each biomarker to the model. The expression levels of candidate biomarkers in each comparison group were calculated. Pearson correlation coefficient was calculated for the expression levels of the candidate biomarkers. Generally speaking, the lower the correlation between the biomarkers in diagnostic panels, the lower the overlap between the selected biomarkers, and the more optimized the panel combination.
The biomarkers diagnostic panel model was constructed using the LR algorithm. LR is a commonly used classification model that can predict the probability of an event and analyze its influencing factors. Biomarkers LR model formula: p = 1/(1+e -z ). The expression level value of the biomarker was brought into the probability value p calculated by the panel model formula. If the p-value exceeded the cutoff value, it was considered a positive diagnosis. To obtain this cutoff, we used Youden’s index to determine the optimal cutoff value for diagnostic decisions . When the sensitivity and specificity were given the same weight, the cutoff value corresponding to the maximum Youden’s index was the best critical point for biomarker identification ability , because the sum of sensitivity and specificity was the maximum at this point. The best cutoff could have good sensitivity and specificity at the same time. Finally, based on the expression of candidate biomarkers in the sample (the original data set was divided into training set and test set, or new validation set data was provided), ROC analysis was performed using the diagnostic model constructed above.
Graphpad Prism8.0 software was used for statistical analysis. If the normal distribution was met, data were expressed as (x±s), and a t-test was used between groups. If it did not conform to a normal distribution, data were expressed as average (minimum, maximum), and the Mann-Whitney U test was used between groups. P value < 0.05 indicated that the difference was statistically significant.
Characteristics of control and patients with PCOS First, we assessed the subjects’ clinical information. shows the essential clinical information of control (n = 50) and PCOS (n = 152) subjects. Among them, Height, Weight, Body mass index (BMI), Progesterone (P), Prolactin (PRL), and Estradiol (E2) had no significant difference between the control and PCOS groups ( P value>0.05). While Age, LH, follicle-stimulating hormone (FSH), LH/FSH, and Testosterone (T) were significantly different between the control and PCOS groups ( P value <0.05). Classification of differential lipid metabolites We collected serum from Control (CG) and PCOS (EG) subjects for untargeted lipidomics analysis to screen candidate biomarkers for PCOS. First, the distribution of lipid metabolites was shown in different classifications . Then, OPLS-DA modeled the lipid metabolites of the two groups of samples, and 100 simulation repetitions were conducted. As shown in , the model’s predictive ability (Q2 value) was significant, and the explanation ability of sample differentiation between groups was significant and good (R2Y). The lipid metabolites between the two groups were compared, and the metabolites with fold change > 2 or fold change < 0.5 and adjusted P value < 0.05 were screened. The top 8 lipid metabolites of difference included 28POS, 26POS, 161POS, 86POS, 763NEG, 140POS, 765NEG, and 739POS . Next, the lipid metabolites in the OPLS-DA model were sorted according to the VIP value. The top 20 (112POS, 512POS, 946NEG, 576POS, 620POS, 985NEG, 628POS, 1033NEG, 665POS, 667POS, 662POS, 369POS, 1034NEG, 990NEG, 833NEG, 994NEG, 271POS, 1048NEG, 1045NEG, and 257POS) were selected, suggesting that these metabolites were important for distinguishing Control from PCOS groups . In addition, the detected differential lipid metabolites were annotated to show the classification of differential lipid metabolites between the Control and PCOS groups . Associations between differential lipid metabolites and clinical indexes After obtaining the classification of differential lipid metabolites, we used the Mantel test to analyze the association between the classification of these differential metabolites and clinical indexes and Pearson correlation analysis to detect the association between the classification of differential metabolites. The results showed that LPC and LPE were significantly correlated with BMI, PI and SM were correlated considerably with LH, TG was significantly correlated with testosterone (T), and PI was significantly associated with LH/FSH. Moreover, DG and MGDG classification, PE and PS, PI and DG were significantly positively correlated. There was a significant negative correlation between LPE and PS and between LPE and PC . Previously, we obtained the central metabolites that differ between the Control and PCOS groups and the top 20 that contribute significantly to the OPLS-DA model . Therefore, we examined the relationship between these different lipid metabolites and clinical indexes. We found that 620POS and 628POS were significantly associated with clinical indexes (BMI, LH, T, and LH/FSH), and 112POS and 576POS were significantly associated with clinical indexes (T and LH/FSH, ). These results indicate that these metabolites differ between groups and have close effects on corresponding clinical indicators. Screening of candidate biomarkers for PCOS Furthermore, shows the ranking of candidate biomarkers by weight value. The larger the weight value, the more significant the contribution of the substance in distinguishing the Control sample from the PCOS sample. Among them, the top 4 were PI (18:0/20:3)-H, PE (18:1p/22:6)-H, PE (36:2p)-H, and PI (16:0 /22:4)-H. To effectively select candidate biomarkers, we used ROC analysis to evaluate the impact strength of each substance on the AUC value of the model. AUC accumulation curve showed that the substances with the top 2 weights notably facilitated sample classification ability; In contrast, substances ranked after 2nd place no longer contribute significantly to classification ability, so PI (18:0/20:3)-H and PE (18:1p/22:6)-H were selected as candidate biomarkers . Validation of biomarkers for PCOS To evaluate the effect of candidate biomarkers on classification models, three commonly used machine learning models, LR, RF, and SVM, were used to verify the above screening results. showed the three models’ AUC, specificity and sensitivity results in boxplots and ROC curves, respectively. Among them, the closer the area under the ROC curve was to 1, the greater the clinical diagnostic efficacy, and the higher the specificity and sensitivity indicators, the better the efficacy. The results showed that all evaluation indicators had satisfactory results, indicating that the selected subsequent biomarkers had better classification ability and effect. further shows the accuracy, sensitivity, and specificity values of the three ROC models and the curve diagram of the relationship between the cutoff values. The ROC curve comprised multiple sensitivity and false favourable rates (1-specificity). In , each group of accuracy, sensitivity and specificity in the three models has a corresponding cutoff value, and the ROC curve can be used to determine the best cutoff value and its corresponding accuracy, sensitivity and specificity. Characterization of biomarkers for PCOS Then, we used the classification model constructed by the above RF algorithm to calculate the importance coefficient of candidate biomarkers and compare the contribution of each biomarker to the model. The higher the importance coefficient of candidate biomarkers, the closer the relationship between the biomarker and the classification group, and the more significant the contribution to distinguishing different groups . The results of expression level analysis showed that the selected candidate biomarkers PI (18:0/20:3)-H and PE (18:1p/22:6)-H had highly significant differential expression in the comparison group . shows the correlation between biomarkers in the diagnostic panel. The results showed that candidate biomarkers had low correlation, indicating that the overlap between the selected biomarkers was low, and the combination of panels was more optimized. Construction and ability evaluation of PCOS diagnosis panel model The biomarkers diagnostic panel model was constructed using the LR algorithm. shows the LR coefficient of biomarkers. The expression levels of the biomarkers were put into the probability p calculated by the panel model formula (biomarkers LR model formula: p = 1/(1+e -z ), z = -19.62541206+2.249933588*NEG1033+0.634268314*NEG877) if the p-value exceeds the cutoff value, the diagnosis was positive. We used Youden’s index to determine the optimal cut-off value for diagnostic decisions to obtain this cut-off value. shows the variation in the relationship between accuracy and cutoff value, as well as sensitivity and specificity and cutoff value. The cutoff value was 0.83. ROC analysis was performed using the diagnostic model constructed above based on the expression levels of the biomarkers in the samples, and the results are shown in . The AUC value of the training set was 0.822, and that of the test set was 0.815. This indicated that the LR model of candidate biomarkers PI (18:0/20:3)-H and PE (18:1p/22:6)-H had a good effect on the sample classification of the test set. When the cutoff of the binary classification model was 0.83, the accuracy of the model in the test set was 0.74, the specificity was 0.88, and the sensitivity was 0.7.
First, we assessed the subjects’ clinical information. shows the essential clinical information of control (n = 50) and PCOS (n = 152) subjects. Among them, Height, Weight, Body mass index (BMI), Progesterone (P), Prolactin (PRL), and Estradiol (E2) had no significant difference between the control and PCOS groups ( P value>0.05). While Age, LH, follicle-stimulating hormone (FSH), LH/FSH, and Testosterone (T) were significantly different between the control and PCOS groups ( P value <0.05).
We collected serum from Control (CG) and PCOS (EG) subjects for untargeted lipidomics analysis to screen candidate biomarkers for PCOS. First, the distribution of lipid metabolites was shown in different classifications . Then, OPLS-DA modeled the lipid metabolites of the two groups of samples, and 100 simulation repetitions were conducted. As shown in , the model’s predictive ability (Q2 value) was significant, and the explanation ability of sample differentiation between groups was significant and good (R2Y). The lipid metabolites between the two groups were compared, and the metabolites with fold change > 2 or fold change < 0.5 and adjusted P value < 0.05 were screened. The top 8 lipid metabolites of difference included 28POS, 26POS, 161POS, 86POS, 763NEG, 140POS, 765NEG, and 739POS . Next, the lipid metabolites in the OPLS-DA model were sorted according to the VIP value. The top 20 (112POS, 512POS, 946NEG, 576POS, 620POS, 985NEG, 628POS, 1033NEG, 665POS, 667POS, 662POS, 369POS, 1034NEG, 990NEG, 833NEG, 994NEG, 271POS, 1048NEG, 1045NEG, and 257POS) were selected, suggesting that these metabolites were important for distinguishing Control from PCOS groups . In addition, the detected differential lipid metabolites were annotated to show the classification of differential lipid metabolites between the Control and PCOS groups .
After obtaining the classification of differential lipid metabolites, we used the Mantel test to analyze the association between the classification of these differential metabolites and clinical indexes and Pearson correlation analysis to detect the association between the classification of differential metabolites. The results showed that LPC and LPE were significantly correlated with BMI, PI and SM were correlated considerably with LH, TG was significantly correlated with testosterone (T), and PI was significantly associated with LH/FSH. Moreover, DG and MGDG classification, PE and PS, PI and DG were significantly positively correlated. There was a significant negative correlation between LPE and PS and between LPE and PC . Previously, we obtained the central metabolites that differ between the Control and PCOS groups and the top 20 that contribute significantly to the OPLS-DA model . Therefore, we examined the relationship between these different lipid metabolites and clinical indexes. We found that 620POS and 628POS were significantly associated with clinical indexes (BMI, LH, T, and LH/FSH), and 112POS and 576POS were significantly associated with clinical indexes (T and LH/FSH, ). These results indicate that these metabolites differ between groups and have close effects on corresponding clinical indicators.
Furthermore, shows the ranking of candidate biomarkers by weight value. The larger the weight value, the more significant the contribution of the substance in distinguishing the Control sample from the PCOS sample. Among them, the top 4 were PI (18:0/20:3)-H, PE (18:1p/22:6)-H, PE (36:2p)-H, and PI (16:0 /22:4)-H. To effectively select candidate biomarkers, we used ROC analysis to evaluate the impact strength of each substance on the AUC value of the model. AUC accumulation curve showed that the substances with the top 2 weights notably facilitated sample classification ability; In contrast, substances ranked after 2nd place no longer contribute significantly to classification ability, so PI (18:0/20:3)-H and PE (18:1p/22:6)-H were selected as candidate biomarkers .
To evaluate the effect of candidate biomarkers on classification models, three commonly used machine learning models, LR, RF, and SVM, were used to verify the above screening results. showed the three models’ AUC, specificity and sensitivity results in boxplots and ROC curves, respectively. Among them, the closer the area under the ROC curve was to 1, the greater the clinical diagnostic efficacy, and the higher the specificity and sensitivity indicators, the better the efficacy. The results showed that all evaluation indicators had satisfactory results, indicating that the selected subsequent biomarkers had better classification ability and effect. further shows the accuracy, sensitivity, and specificity values of the three ROC models and the curve diagram of the relationship between the cutoff values. The ROC curve comprised multiple sensitivity and false favourable rates (1-specificity). In , each group of accuracy, sensitivity and specificity in the three models has a corresponding cutoff value, and the ROC curve can be used to determine the best cutoff value and its corresponding accuracy, sensitivity and specificity.
Then, we used the classification model constructed by the above RF algorithm to calculate the importance coefficient of candidate biomarkers and compare the contribution of each biomarker to the model. The higher the importance coefficient of candidate biomarkers, the closer the relationship between the biomarker and the classification group, and the more significant the contribution to distinguishing different groups . The results of expression level analysis showed that the selected candidate biomarkers PI (18:0/20:3)-H and PE (18:1p/22:6)-H had highly significant differential expression in the comparison group . shows the correlation between biomarkers in the diagnostic panel. The results showed that candidate biomarkers had low correlation, indicating that the overlap between the selected biomarkers was low, and the combination of panels was more optimized.
The biomarkers diagnostic panel model was constructed using the LR algorithm. shows the LR coefficient of biomarkers. The expression levels of the biomarkers were put into the probability p calculated by the panel model formula (biomarkers LR model formula: p = 1/(1+e -z ), z = -19.62541206+2.249933588*NEG1033+0.634268314*NEG877) if the p-value exceeds the cutoff value, the diagnosis was positive. We used Youden’s index to determine the optimal cut-off value for diagnostic decisions to obtain this cut-off value. shows the variation in the relationship between accuracy and cutoff value, as well as sensitivity and specificity and cutoff value. The cutoff value was 0.83. ROC analysis was performed using the diagnostic model constructed above based on the expression levels of the biomarkers in the samples, and the results are shown in . The AUC value of the training set was 0.822, and that of the test set was 0.815. This indicated that the LR model of candidate biomarkers PI (18:0/20:3)-H and PE (18:1p/22:6)-H had a good effect on the sample classification of the test set. When the cutoff of the binary classification model was 0.83, the accuracy of the model in the test set was 0.74, the specificity was 0.88, and the sensitivity was 0.7.
PCOS is a multifactorial disease associated with reproductive and endocrine organs that may lead to infertility and metabolic abnormalities during reproductive age . The application of metabolomics provides a broad perspective for PCOS research. It is a valuable and rapidly expanding tool enabling the discovery of new metabolites. This approach also improves the diagnostic process, making treatment more effective . Machine learning has also received increasing attention in medical applications; in addition to being widely used in image recognition, language processing and data mining, it is also used in various techniques for analyzing and interpreting large amounts of data, which can then be used to create predictive models . This study screened the serum biomarkers PI (18:0/20:3)-H and PE (18:1p/22:6)-H in PCOS patients by untargeted lipidomics-integrated machine learning. Based on the feature selection technology of ensemble learning, a more efficient diagnostic marker panel model is constructed for clinical application. Machine learning methods provide new techniques for integrating and analyzing various omics data, enabling the discovery of new biomarkers. These biomarkers can potentially contribute to accurate disease prediction, patient stratification, and delivery of precision medicine . Ensemble learning method to select biomarkers by integrating multiple machine learning algorithms of the same or different types to choose those potential target analyses that frequently appear in classification models with high accuracy, thus maximizing the stability of biomarkers . Overall, ensemble learning has superior performance compared to conventional single-dimensional learning. This study uses a feature selection technique based on ensemble learning, which integrates statistical tests and a variety of feature selection algorithms currently used in the mainstream. This technology can select a robust set of diagnostic substances (such as proteins, metabolites, etc.) and build a more efficient diagnostic marker panel model. Analyzing the classification of differential lipid metabolites and the association between differential lipid metabolites and clinical indexes, we demonstrated that different lipid metabolites differ between groups and have close effects on corresponding clinical indexes. Compared to traditional diagnostic methods, integrated learning methods also have multiple advantages in biomarker discovery in biomedical research, especially in processing complex, high-dimensional biomedical data . For example, biomedical data contains multiple types, such as genome, proteome, metabolome, etc. Integrated learning methods can effectively integrate these multimodal data and reveal complex relationships between different data types. Integrated learning methods usually have stronger generalization capabilities than single models and can reduce prediction errors and improve diagnostic accuracy by combining multiple predictions. Integrated learning methods can improve the speed and efficiency of data processing by processing data in parallel, which is particularly important for biomedical research that requires processing large amounts of data. Integrated learning models are capable of discovering potential biomarkers from large amounts of complex biomedical data that may be difficult to detect in traditional methods. In our study, we screened the key serum biomarkers PI (18:0/20:3)-H and PE (18:1p/22:6)-H through an ensemble learning approach. While the diagnostic panel model had an AUC value of 0.815 in the test set, it had an accuracy of 0.74, a specificity of 0.88, and a sensitivity of 0.7. This indicates that a more efficient diagnostic marker panel model has been constructed. Biofilms are mainly composed of structural glycerophospholipids, such as phosphatidylinositol (PI) and phosphatidylethanolamine (PE) . PI is the product of different phosphatidylinositol kinases and phosphorylation states at positions D3, D4 and D5 of the inositol ring . It is necessary for many cell signaling pathways of membrane-associated proteins . PE is the major phospholipid of all eukaryotic cells. It can be synthesized by the CDP-ethanolamine branch of the Kennedy pathway, by decarboxylation of phosphatidylserine, or by base exchange with phosphatidylserine . Insulin resistance in PCOS is characterized by abnormal post-receptor signaling in the PI3-kinase pathway . PE plays a vital role in contractile ring disassembly at the cleavage furrow during cytokinesis in mammalian cells . Moreover, PE tends to form a non-lamellar membrane structure and modulates the membrane curvature . Studies have shown that apoptosis of ovarian granulosa cells could be reduced by regulating lipid metabolism to improve ovulation dysfunction in PCOS patients . Qian Y et al. collected follicular fluid and granular cell samples of PCOS women and ordinary women who underwent in vitro fertilization and embryo transfer and analyzed lipid metabolomics, showing abnormal metabolism of glycerol, phospholipid and sphingosphayelin in follicular fluid of PCOS women . There are significant lipid differences between normal-weight women with PCOS and normal-weight women without PCOS at the same time, and these different lipid species belong to lipid subclasses, including PE and PI . The PCOS vs luteal phase model showed decreased levels of PE in PCOS women compared with controls . Furthermore, Moran LJ, et al reported that lipidomic signatures were mainly associated with central adiposity and free androgen index in women with and without PCOS. Free androgen index was positively associated with PE and PI . In this paper, our study identifies the critical roles of serum biomarkers PI (18:0/20:3)-H and PE (18:1p/22:6)-H in diagnosing PCOS. Additionally, different machine learning feature selection approaches could be applied effectively to identify biomarkers. However, each approach had advantages and disadvantages, and biomarkers produced using various feature selection approaches were usually inconsistent due to the multifactorial nature of the features . Therefore, we utilize an ensemble framework, an ensemble learning approach for multi-step data mining that integrates multiple statistical and machine learning methods. This ensemble algorithm identifies and characterizes robust and precise biomarkers with broad applicability and could be applied to build biomarker models on different omics datasets. Simultaneously, biomarkers screened by ensemble machine learning have good classification performance, strong stability and a minor number. Overall, accurate identification of important biomarkers associated with diseases can provide valuable insights into their underlying etiology and offer great potential for early diagnosis of diseases, personalized medicine, drug response prediction, clinical trials, drug development, and accurate prediction of disease risk. These application prospects indicate that the application of integrated learning in biomedical research has a wide range of potential to improve the efficiency and effectiveness of disease management significantly. With technological advances and improved data analysis methods, future biomarker studies will be more accurate and efficient. However, in this study, the error metrics (accuracy, sensitivity, and specificity) show a relatively lower model performance compared with other works, such as those by Yu Y et al. . In another work, even only with symptoms and anthropomorphic variables, the Boosting models can achieve a better performance . The performance and variables selected in this study are too few. Variable selection is not a priority either; the two most discriminant features were chosen without considering multiple tests. In the future, to improve the current study, using more variables in the classifier model may potentially improve the performance. As in , Age LH, FSH, LH/FSH, and T have small P values. Including these and other substance variables in correlation study and PCA might improve the prediction model. Our study has an additional limitation. The clinical samples collected in this study were randomly collected. Statistically, we found that PCOS patients were younger than non-PCOS subjects. Age might affect the results of this study, but PCOS patients were younger. The age difference might be related to the small sample size, and we will also increase the sample size for further research in future studies. This study’s dataset needs to be more balanced, with a 3:1 ratio. The unbalanced dataset is known to be demanding for this kind of ML approach. ROC may not be the most appropriate way to evaluate performance, and precision-recall curves are needed as a complement in the future. Furthermore, in a metabolomics study, we should include one or more replication cohorts to validate in the future.
In this experiment, two biomarkers, PI (18:0/20:3)-H and PE (18:1p/22:6)-H, were finally screened through the analysis of lipidomic data. When the AUC value of the test set of the diagnostic panel model constructed by biomarker was 0.815, the accuracy, specificity, and sensitivity of the model in the test set were 0.74, 0.88, and 0.7, respectively.
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Mind the gap between low- and middle-income countries (LMICs) and high-income countries (HICs): fostering pediatric anesthesia globally | df3c19de-a5f8-4b97-9eed-f9467d51c3e4 | 11342752 | Pediatrics[mh] | Vinícius Caldeira Quintão and Maria José Carvalho Carmona serve as Associate Editors of the Brazilian Journal of Anesthesiology. The other authors declare no conflicts of interest. |
Use of multiple metrics to assess antibiotic use in Italian children’s hospitals | 8a886ef1-68b5-4d97-9f97-1c06bdc255e7 | 7878731 | Pediatrics[mh] | Improving the use of antibiotics is an important patient safety and public health issue, as well as a global priority .The misuse of antibiotics, in fact, has contributed to the growing problem of antibiotic resistance, which has become one of the most serious and growing public health threats , . Children represent an excellent population for the selection of resistant bacterial pathogens after recent antimicrobial use , . Therefore, the investigation of antibiotic consumption in the pediatric population is crucial, especially in the hospital setting where the risk of the emergence and transmission of bacterial resistance is dramatically increased . A growing body of evidence demonstrates that hospital based antibiotic stewardship programs (ASPs) can both optimize the treatment of infections and reduce adverse events associated with antibiotic use – . Quantification of antibiotic utilization is an integral part of ASPs. Hospital antibiotic consumption is commonly measured by calculating the daily defined doses (DDDs), as defined by the World Health Organization Collaborating Center for Drug Statistics Methodology, divided by a denominator indicating clinical activity (e.g. DDDs per 100 patient-days) , . DDD is calculated as the assumed average maintenance dose per day for a drug used for its main indication in adults. Even if DDDs allow the comparison of drug use between different settings and between different drugs, they do not reflect the dosage actually used for individual patients or specific patient populations , and their use is not recommended in children . To overcome these issues, in-hospital antibiotic use in children is frequently estimated by conducting point-prevalence surveys (PPS). Although the PPS design has been extensively used to evaluate antibiotic use, it cannot capture treatment duration. Other antibiotic consumption indicators are days of therapy (DOTs), length of therapy (LOTs) and prescribed daily doses (PDDs). DOTs are computed summing up the duration (in days) of each antibacterial drug received for treatments. LOTs are the number of days that a patient receives an antibacterial drug irrespective of the number of different drugs. DOTs and LOTs give an accurate estimation of polydrug therapy and duration of therapy, respectively; since they do not consider whether a nonstandard dose was given , they could be considered as accurate metrics of drug use for children. The PDD provides the average daily amount of a drug that is actually prescribed . In this multicentric study, we adopted different metrics to evaluate inpatient antibiotic use in hospitalized children. The study objectives were to describe point prevalence of antibiotic use by indication and patient characteristics, to estimate DOTs, LOTs and PDDs as additional metrics to quantify antibiotic use, and to compare PDDs to DDDs by antibiotic and age group.
Point prevalence of antibiotic use In our study, we included 810 children. Patients were mostly males (n = 422, 52%), aged ≥ 10 years years (n = 233, 29%) and with a length of hospital stay ≤ 7 days (n = 458, 57%) (Table ). About 38% of the patients (306/810) had at least one chronic disease; the most frequent chronic diseases were congenital heart diseases (70/306; 23%), genetic syndromes (38/306; 12%), chronic respiratory diseases (22/306; 7%), neuromuscular (19/306; 6%), and chronic renal diseases (18/306; 6%). Two hundred and thirty-one patients (29%) underwent at least one surgical procedure during hospitalization, for a total of 331 surgical procedures. The majority of patients (450/810; 56%), were admitted to medical units, 32% (261/810) to surgical units and 12% (99/810) to intensive care units (Table ). Of the 810 patients, 380 (46.9%; CI 95%: 43.4–50.4) received one or more antibiotics. The proportion of patients receiving antibiotics significantly varied by hospital, age, sex, comorbidities and surgical procedures. In particular, the prevalence of use was significantly higher in patients aged ≥ 2 years compared to those in the first year of life, in males with respect to females, in patients without comorbidities compared to those who had at least one chronic disease, and in patients who underwent at least one surgical procedure (Table ). Prophylaxis was the main indication for antibiotic use (217/380; 57.1%). Children receiving antibiotics for surgical or medical prophylaxis accounted for 14.3% (116/810) and 12.7% (103/810) of the hospitalized patients, respectively. Surgical prophylaxis lasted more than 24 h in 76.7% of the patients (89/116). Patients treated for CAIs were 13.2% (107/810) of children, followed by patients treated for HAIs (61/810; 7.5%). Overall, 587 drugs were issued to the 380 patients receiving antibiotics (1.5 antibiotic per patient), mainly administered intravenously (465/587; 79.2%). Prescriptions for treatments were 42.8% (251/587); of those, the majority of antibiotics was administered for CAIs (147/251; 58.6%), and the remaining for HAIs (104/251; 41.4%). Overall, microbiological cultures were performed before starting the antibiotic therapy in 54.5% of the treatments (137/251). In 23.1% of the prescriptions (58/251), the results of the antibiogram were available and the antibiotic therapy could be considered as targeted, whereas 68.1% (171/251) of the treatments were empirical. The main infection sites were respiratory tract infections (both low tract and pneumonia), bloodstream infections, and urinary tract infections, which were reported as the reason for therapy in respectively 29.9%, 23.5% and 2.4% of the antibiotic treatments for infections. The top three antibiotic classes were combinations of penicillins including β-lactamase inhibitors (J01CR; 134/587, 22.8%), third-generation cephalosporins (J01DD; 73/587, 12.4%) and aminoglycosides (J01GB; 69/587, 11.8%, Fig. ). The pattern of antibiotic use varied by indication: about 98.0% (42/43) of first-generation cephalosporin prescriptions (J01DB) were issued for surgical prophylaxis, followed by second-generation cephalosporins (J01DC; 9/12, 75.0%) and imidazole derivatives (J01XD; 11/19, 57.9%) (Fig. ). Combinations of sulphonamides and trimethoprim (J01EE), fluoroquinolones (J01MA) and combinations of penicillins including β-lactamase inhibitors (J01CR) were mainly prescribed for medical prophylaxis (84.4%, 43.8% and 34.3% of the treatments, respectively) (Fig. ). CAIs represented the main indication of use for macrolides (J01FA; 19/25, 76.0%), third generation cephalosporins (J01DD; 29/73, 39.7%) and combinations of penicillins including β-lactamase inhibitors (J01CR; 47/134, 35.1%) whereas HAIs were mainly reported for penicillins with extended spectrum (J01CA; 13/26, 50.0%), glycopeptides (J01XA; 19/39, 48.7%) and aminoglycosides (J01GB; 28/69, 40.6%). Estimation of DOTs and LOTs DOTs and LOTs were estimated considering 414 inpatient antibiotic treatments, over a total of 9458 inpatient days, retrospectively evaluated considering the 30 days prior to the collection of the point prevalence data on antibiotic use. Overall, we estimated 30.5 DOTs per 100 patient days and a 19.1 LOT per 100 patient days resulting in a DOT/LOT ratio of 1.6 (Table ). DOT and LOT/100 inpatient days varied significantly by hospital, age, sex, type of ward, and comorbidities. In detail, antibiotic use was higher in males with respect to females, in children aged < 10 years with respect to those aged ≥ 10 years and in the ICUs with respect to medical and surgical wards (Table ). DOT/100 patient days by type of drug confirmed that penicillins, aminoglycosides, carbapenems and cephalosporins were the antibiotics most frequently prescribed for treatment, with estimated DOT/100 patient days of 7, 6 and 4 (Fig. ). Targeted therapies had lower DOT/100 patient days than empirical therapies (8.0 DOT/100 vs. 21.0 DOT/100). The overall DOT/LOT ratio of 1.6 indicated that the combination antibacterial treatment was common (Table ). Estimation of PDDs and comparison with DDDs Table shows the median PDDs by age groups for the eight most frequently prescribed treatments. The PDDs increased with age: the ratio between PDDs estimated in children aged ≥ 10 years and PDDs in children aged 0–11 months did greatly vary by antibiotic, and ranged from a minimum of 2 for sulfamethoxazole and trimethoprim, to a maximum of 25 for meropenem. The PDD approached the DDD only in children aged ≥ 10 years: in this age group the PDDs were equal to the DDDs for gentamicin and ceftriaxone, while the median PDD was lower than the DDD for piperacillin/amoxicillin and beta-lactamase inhibitors, sulfamethoxazole and trimethoprim, amikacin and cefazolin. The PDD was higher than the DDD only for meropenem.
In our study, we included 810 children. Patients were mostly males (n = 422, 52%), aged ≥ 10 years years (n = 233, 29%) and with a length of hospital stay ≤ 7 days (n = 458, 57%) (Table ). About 38% of the patients (306/810) had at least one chronic disease; the most frequent chronic diseases were congenital heart diseases (70/306; 23%), genetic syndromes (38/306; 12%), chronic respiratory diseases (22/306; 7%), neuromuscular (19/306; 6%), and chronic renal diseases (18/306; 6%). Two hundred and thirty-one patients (29%) underwent at least one surgical procedure during hospitalization, for a total of 331 surgical procedures. The majority of patients (450/810; 56%), were admitted to medical units, 32% (261/810) to surgical units and 12% (99/810) to intensive care units (Table ). Of the 810 patients, 380 (46.9%; CI 95%: 43.4–50.4) received one or more antibiotics. The proportion of patients receiving antibiotics significantly varied by hospital, age, sex, comorbidities and surgical procedures. In particular, the prevalence of use was significantly higher in patients aged ≥ 2 years compared to those in the first year of life, in males with respect to females, in patients without comorbidities compared to those who had at least one chronic disease, and in patients who underwent at least one surgical procedure (Table ). Prophylaxis was the main indication for antibiotic use (217/380; 57.1%). Children receiving antibiotics for surgical or medical prophylaxis accounted for 14.3% (116/810) and 12.7% (103/810) of the hospitalized patients, respectively. Surgical prophylaxis lasted more than 24 h in 76.7% of the patients (89/116). Patients treated for CAIs were 13.2% (107/810) of children, followed by patients treated for HAIs (61/810; 7.5%). Overall, 587 drugs were issued to the 380 patients receiving antibiotics (1.5 antibiotic per patient), mainly administered intravenously (465/587; 79.2%). Prescriptions for treatments were 42.8% (251/587); of those, the majority of antibiotics was administered for CAIs (147/251; 58.6%), and the remaining for HAIs (104/251; 41.4%). Overall, microbiological cultures were performed before starting the antibiotic therapy in 54.5% of the treatments (137/251). In 23.1% of the prescriptions (58/251), the results of the antibiogram were available and the antibiotic therapy could be considered as targeted, whereas 68.1% (171/251) of the treatments were empirical. The main infection sites were respiratory tract infections (both low tract and pneumonia), bloodstream infections, and urinary tract infections, which were reported as the reason for therapy in respectively 29.9%, 23.5% and 2.4% of the antibiotic treatments for infections. The top three antibiotic classes were combinations of penicillins including β-lactamase inhibitors (J01CR; 134/587, 22.8%), third-generation cephalosporins (J01DD; 73/587, 12.4%) and aminoglycosides (J01GB; 69/587, 11.8%, Fig. ). The pattern of antibiotic use varied by indication: about 98.0% (42/43) of first-generation cephalosporin prescriptions (J01DB) were issued for surgical prophylaxis, followed by second-generation cephalosporins (J01DC; 9/12, 75.0%) and imidazole derivatives (J01XD; 11/19, 57.9%) (Fig. ). Combinations of sulphonamides and trimethoprim (J01EE), fluoroquinolones (J01MA) and combinations of penicillins including β-lactamase inhibitors (J01CR) were mainly prescribed for medical prophylaxis (84.4%, 43.8% and 34.3% of the treatments, respectively) (Fig. ). CAIs represented the main indication of use for macrolides (J01FA; 19/25, 76.0%), third generation cephalosporins (J01DD; 29/73, 39.7%) and combinations of penicillins including β-lactamase inhibitors (J01CR; 47/134, 35.1%) whereas HAIs were mainly reported for penicillins with extended spectrum (J01CA; 13/26, 50.0%), glycopeptides (J01XA; 19/39, 48.7%) and aminoglycosides (J01GB; 28/69, 40.6%).
DOTs and LOTs were estimated considering 414 inpatient antibiotic treatments, over a total of 9458 inpatient days, retrospectively evaluated considering the 30 days prior to the collection of the point prevalence data on antibiotic use. Overall, we estimated 30.5 DOTs per 100 patient days and a 19.1 LOT per 100 patient days resulting in a DOT/LOT ratio of 1.6 (Table ). DOT and LOT/100 inpatient days varied significantly by hospital, age, sex, type of ward, and comorbidities. In detail, antibiotic use was higher in males with respect to females, in children aged < 10 years with respect to those aged ≥ 10 years and in the ICUs with respect to medical and surgical wards (Table ). DOT/100 patient days by type of drug confirmed that penicillins, aminoglycosides, carbapenems and cephalosporins were the antibiotics most frequently prescribed for treatment, with estimated DOT/100 patient days of 7, 6 and 4 (Fig. ). Targeted therapies had lower DOT/100 patient days than empirical therapies (8.0 DOT/100 vs. 21.0 DOT/100). The overall DOT/LOT ratio of 1.6 indicated that the combination antibacterial treatment was common (Table ).
Table shows the median PDDs by age groups for the eight most frequently prescribed treatments. The PDDs increased with age: the ratio between PDDs estimated in children aged ≥ 10 years and PDDs in children aged 0–11 months did greatly vary by antibiotic, and ranged from a minimum of 2 for sulfamethoxazole and trimethoprim, to a maximum of 25 for meropenem. The PDD approached the DDD only in children aged ≥ 10 years: in this age group the PDDs were equal to the DDDs for gentamicin and ceftriaxone, while the median PDD was lower than the DDD for piperacillin/amoxicillin and beta-lactamase inhibitors, sulfamethoxazole and trimethoprim, amikacin and cefazolin. The PDD was higher than the DDD only for meropenem.
Out of more than 800 children included in our study, 47% were treated with at least one antibiotic. This estimate is higher than reported in European hospitalized children, where the point prevalence of antibiotic use was 39.6% . We showed how hospital and patient characteristics, such as age and comorbidities, are related to the use of antibiotics, and other authors have shown that the prevalence of antibiotic use is higher in third-level hospitals where patients generally have more complex conditions . As patient population characteristics are major determinants of antibiotic consumption, risk adjustment should be adopted to compare antibiotic consumption data among hospitals and over-time. In our results, previous surgery was significantly associated with antibiotic use and surgical prophylaxis was the most frequent indication. As previously documented – , we showed a suboptimal appropriateness of antibiotic surgical prophylaxis; in fact, there was still a proportion of patients treated with non-recommended broad-spectrum active substances, such as III-IV generation cephalosporins. Another worrying aspect is the duration of surgical prophylaxis; in fact, although a prolonged exposure does not reduce the risk of surgical site infections, it represents a relevant ecological risk . In our study, approximately 77% of the patients undergoing surgical prophylaxis received surgical prophylaxis for more than 24 h. This is in line with previous prevalence surveys, which estimated a proportion of children subject to prolonged surgical prophylaxis (> 1 day) fluctuating between 67 and 78% in Europe , . These data show that even in the most documented evidence-based area of practice, which recommends a surgical antimicrobial prophylaxis duration of less than 24 h, clinicians tend to overprescribe, and this is a global phenomenon not exclusively limited to Europe . Combinations of sulfonamides and trimethoprim and combinations of penicillins including beta-lactamase inhibitors have been the most widely used active substances for medical prophylaxis, being widely used to prevent opportunistic infections in neutropenic patients . Medical prophylaxis, as well as infection treatments, include a wide range of diseases and it is therefore difficult to identify unambiguous recommendations in patients with very different clinical conditions. Although point prevalence surveys are useful to assess the pattern of in-hospital antibiotic prescriptions, antibiotic use should be preferably expressed with multiple metrics . We collected DOT, LOT and PDDs as more specific antibiotic indicators for infection treatments with respect to DDDs, which are not recommended for children because doses used are typically much smaller than in adult populations, so this measure underestimates patient exposure to drugs . DOT and LOT have been used to monitor antibiotic use and to assess the impact of antibiotic stewardship programs on improving appropriateness of prescriptions in children , . We showed that calculation of DOT and LOT by retrospectively reviewing medical records was feasible. DOT/100 patient days was comparable with antibiotic prescriptions for infections in a secondary pediatric hospital in the Netherlands, where the average DOT/100 in neonatal and pediatric wards was between 23 and 45 . Intensive care units were the setting with the highest antibiotic use, with LOT and DOT rates per 100 days of 28.5 and 47.2, respectively. Moreover, a DOT/LOT ratio of 1.6 confirms the frequent resort to multi-therapies found in the prevalence survey where an equal average number of active substances per patient was detected. Both LOTs and DOTs included the underlying assumption that antibiotic dosing was appropriate. PDDs represented the real prescribed doses of drugs in children. As reported also in a previous study , our study showed that PDD increased with age and approached to DDD only in children aged ≥ 10 years. Although there was no difference between DDDs and PDDs of antibiotic drugs belonging to the class of aminoglycosides and cephalosporins in this age group, a difference was observed for other antibiotic drugs. PDDs lower than DDDs could be explained considering that children’s weight is lower than the standard weight used to compute DDDs, otherwise PDDs higher than DDDs could be reconducted to the patients’ clinical conditions to be treated. It could be the case of meropenem that is frequently used to treat serious healthcare associated bloodstream infections requiring more aggressive dosages. This multicentre study was conducted in four stand-alone tertiary care children’s hospitals located in Regions in the North, Centre, and South of Italy. Trained investigators collected data regarding antibiotic prescriptions from clinical records minimising the effect of information bias on our study results. Additionally, in contrast to studies that are based on information collected directly from patients medical clinical records, we had no need to restrict the sample size. The study limitations are inherent to the epidemiological methods of our cross-sectional survey, where the main purpose was to describe prescribing patterns in hospitals. The overall rate provided is the average. We did not control for patient case mix, disease incidence, prevalence of different types of infections, variations in resistance levels, institutional factors, all of which can influence antibiotic use patterns . In conclusion, our results confirm that DOT, LOT and PDD are better alternatives to DDD in children. The availability of electronic clinical records can greatly enhance data accessibility to calculate DOT, LOT and PDD data and use those data within antibiotic stewardship programs, as alternative to DDDs in pediatric population.
Study design and setting This study was conducted between November and December 2016 and involved four tertiary care children’s hospitals in Italy: Ospedale Infantile Regina Margherita (Turin, in the Region of Piedmont), Ospedale dei Bambini di Brescia (Brescia, in the Region of Lombardy), Bambino Gesù Children’s Hospital (Rome, in the Region of Lazio) and Ospedale Pediatrico Giovanni XXIII (Bari, in the Region of Apulia). These tertiary care children’s hospitals have a total number of beds ranging from a minimum of 157 to a maximum of 607, with an annual number of inpatients ranging from 5700 to 27,000. Pediatric surgeries and pediatric intensive care units are present in all the participating centres; Onco-Haematological Units and Neonatal Intensive Care Units are present in three centres. Data collection The data collection was carried out during a calendar week and involved all children hospitalized on the study days. Data were collected by trained personnel from patients’ paper medical charts. Information collected for each patient included: age, sex, length of hospital stay, and ward type. To assess point prevalence, we collected information about antibiotics administered during the days of data collection. To estimate DOT and LOT, we retrospectively recorded information about antibiotics administered during hospitalization, up to 30 inpatient days prior to the week of data collection. Information on antibiotics included Anatomical Therapeutic Chemical Classification code, ATC J01) , drug brand name, route of administration, dosage, number of doses per day, and indication for antibiotic administration, defined as community-acquired infections (CAIs), hospital-acquired infections (HAIs), medical or surgical prophylaxis. For antibiotics used for treating infections, collected information included also the site of infection and whether microbiological investigations were performed prior to starting antibiotic therapy. The protocol of this study was approved by the Ethical Committee of the Bambino Gesù Children’s Hospital (N° 1238_OPBG_2016) and all methods were performed following relevant guidelines and regulations. Since data were collected by reviewing medical charts and were analyzed anonymously, informed consent was not deemed necessary. The need for informed consent was waived by the Ethical Committee of the Bambino Gesù Children’s Hospital. All the collected data were uploaded on REDCap (Research Electronic Data Capture) database which is a secure web application for building and managing online surveys and databases, available at no charge to not-for-profit institutions . All the data were analyzed anonymously. Statistical analysis Patients were described according to demographic and clinical factors. Collected data were presented as count and proportions (categorical data) or median and interquartile range (IQR, continuous data). The wards where the patients were hospitalized were categorized as medical (e.g. general neonatal and pediatric wards, oncology and hematology), surgical (e.g. neonatal surgery, pediatric surgery, cardiac surgery, neurosurgery, ENT, orthopedics) and intensive care units (ICUs) (e.g. pediatric intensive care unit, neonatal intensive care unit, and cardiac intensive care unit). The metrics we calculated included point prevalence of antibiotic use, DOTs, LOTs and PDDs. The prevalence of antibiotic use was calculated as the ratio between the number of patients under treatment with at least one antibiotic on the survey day, and the total number of hospitalized patients. Prevalence of use was computed by age group (0–11 months, 12–23 months, 2–4 years; 5–9 years; ≥ 10 years), sex, hospital ward, lenght of hospital stay, comorbidities, surgical procedures during hospitalization, participating centre and indication. Comparisons among groups were conducted using the Chi-squared test. Antibiotic drugs were classified according to the Anatomical Therapeutic Chemical Classification code (ATC: J01); a percentage distribution of antibiotic drugs substances by indication of use was computed considering the antibiotic prescription administered on the survey date and those received in the previous 30 days. DOTs and LOTs were computed on treatments , considering the 30-days study period. For each patient, DOTs were computed summing up the duration (in days) of each antibacterial drug received for treating infections. LOTs were the number of days that a patient received an antibacterial drug irrespective of the number of different drugs . DOTs/100 patient days and LOTs/100 patient days were computed stratifying for sex, age classes, ward type, type of active substances and targeted versus empirical therapy; groups comparisons were performed using Student’s t-test and one way ANOVA test. PDDs were defined as the observed dose received by each patient in grams and was calculated for the most frequently used antibiotics. Due to the extremely large differences in dosing in pediatrics, ranging from pre-term neonates to teenagers, median PDDs, and the respective IQRs, were estimated and stratified into five age groups (1, 0–11 months; 2, 12–23 months; 3, 2–4 years; 4, 5–9 years; 5, ≥ 10 years), as previously described . All statistical analyses were conducted using STATA 13 (Stata Corporation, College Station, Texas, USA).
This study was conducted between November and December 2016 and involved four tertiary care children’s hospitals in Italy: Ospedale Infantile Regina Margherita (Turin, in the Region of Piedmont), Ospedale dei Bambini di Brescia (Brescia, in the Region of Lombardy), Bambino Gesù Children’s Hospital (Rome, in the Region of Lazio) and Ospedale Pediatrico Giovanni XXIII (Bari, in the Region of Apulia). These tertiary care children’s hospitals have a total number of beds ranging from a minimum of 157 to a maximum of 607, with an annual number of inpatients ranging from 5700 to 27,000. Pediatric surgeries and pediatric intensive care units are present in all the participating centres; Onco-Haematological Units and Neonatal Intensive Care Units are present in three centres.
The data collection was carried out during a calendar week and involved all children hospitalized on the study days. Data were collected by trained personnel from patients’ paper medical charts. Information collected for each patient included: age, sex, length of hospital stay, and ward type. To assess point prevalence, we collected information about antibiotics administered during the days of data collection. To estimate DOT and LOT, we retrospectively recorded information about antibiotics administered during hospitalization, up to 30 inpatient days prior to the week of data collection. Information on antibiotics included Anatomical Therapeutic Chemical Classification code, ATC J01) , drug brand name, route of administration, dosage, number of doses per day, and indication for antibiotic administration, defined as community-acquired infections (CAIs), hospital-acquired infections (HAIs), medical or surgical prophylaxis. For antibiotics used for treating infections, collected information included also the site of infection and whether microbiological investigations were performed prior to starting antibiotic therapy. The protocol of this study was approved by the Ethical Committee of the Bambino Gesù Children’s Hospital (N° 1238_OPBG_2016) and all methods were performed following relevant guidelines and regulations. Since data were collected by reviewing medical charts and were analyzed anonymously, informed consent was not deemed necessary. The need for informed consent was waived by the Ethical Committee of the Bambino Gesù Children’s Hospital. All the collected data were uploaded on REDCap (Research Electronic Data Capture) database which is a secure web application for building and managing online surveys and databases, available at no charge to not-for-profit institutions . All the data were analyzed anonymously.
Patients were described according to demographic and clinical factors. Collected data were presented as count and proportions (categorical data) or median and interquartile range (IQR, continuous data). The wards where the patients were hospitalized were categorized as medical (e.g. general neonatal and pediatric wards, oncology and hematology), surgical (e.g. neonatal surgery, pediatric surgery, cardiac surgery, neurosurgery, ENT, orthopedics) and intensive care units (ICUs) (e.g. pediatric intensive care unit, neonatal intensive care unit, and cardiac intensive care unit). The metrics we calculated included point prevalence of antibiotic use, DOTs, LOTs and PDDs. The prevalence of antibiotic use was calculated as the ratio between the number of patients under treatment with at least one antibiotic on the survey day, and the total number of hospitalized patients. Prevalence of use was computed by age group (0–11 months, 12–23 months, 2–4 years; 5–9 years; ≥ 10 years), sex, hospital ward, lenght of hospital stay, comorbidities, surgical procedures during hospitalization, participating centre and indication. Comparisons among groups were conducted using the Chi-squared test. Antibiotic drugs were classified according to the Anatomical Therapeutic Chemical Classification code (ATC: J01); a percentage distribution of antibiotic drugs substances by indication of use was computed considering the antibiotic prescription administered on the survey date and those received in the previous 30 days. DOTs and LOTs were computed on treatments , considering the 30-days study period. For each patient, DOTs were computed summing up the duration (in days) of each antibacterial drug received for treating infections. LOTs were the number of days that a patient received an antibacterial drug irrespective of the number of different drugs . DOTs/100 patient days and LOTs/100 patient days were computed stratifying for sex, age classes, ward type, type of active substances and targeted versus empirical therapy; groups comparisons were performed using Student’s t-test and one way ANOVA test. PDDs were defined as the observed dose received by each patient in grams and was calculated for the most frequently used antibiotics. Due to the extremely large differences in dosing in pediatrics, ranging from pre-term neonates to teenagers, median PDDs, and the respective IQRs, were estimated and stratified into five age groups (1, 0–11 months; 2, 12–23 months; 3, 2–4 years; 4, 5–9 years; 5, ≥ 10 years), as previously described . All statistical analyses were conducted using STATA 13 (Stata Corporation, College Station, Texas, USA).
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Chaotic fun! Promoting active recall of anatomical structures and relationships using the Catch‐Phrase game | 8969d786-d63e-4207-a9f4-4c613668e2be | 11797534 | Anatomy[mh] | Gross anatomy is often viewed as one of the more challenging subjects for health professionals to learn. The challenge in learning anatomy is partially attributed to the high volume of material and time constraints placed on the course, where students are required to learn a plethora of interrelated terms, structures, concepts, and relationships. To add to the difficulty, consolidation of these details must occur within an increasingly shorter time frame. , Students must then efficiently draw upon and apply their anatomical knowledge within the limited time constraints of their written and practical examinations. As a result, the preclinical academic performance of health professional students (e.g., physical therapy, physician assistant, occupational therapy) is highly dependent on their ability to recall their anatomical knowledge quickly and accurately. Once health professional students enter the clinic, their performance may still be dependent on their ability to recall their anatomical knowledge both quickly and accurately. Even with constant access to reference materials, health professionals must quickly recall anatomical knowledge during emergencies and procedures. For example, poor or inaccurate recall of medical knowledge is considered one of the main causes of preventable adverse effects of care , , As such, quick and accurate recall of anatomical information is crucial and should be supported. Active recall is a method that supports both preclinical and clinical learning through the promotion of long‐term retrieval and retention of medical knowledge. Sometimes referred to as retrieval practice, it involves purposefully drawing upon previously consolidated information to answer a specific question. The process of active recall encourages re‐encoding and re‐consolidation of information, resulting in easier and longer‐lasting access to that information in the future. Active recall activities can provide students with numerous educational benefits; for example, it has been shown to increase the long‐term retention and retrieval of knowledge. , , , Students who practiced active recall as part of their studying also performed better on examinations, including medical licensing examinations, when compared with peers who relied on more passive learning techniques. , Periodic engagement in active recall can also enhance the transfer of factual information to higher order thinking. , Ultimately, active recall encourages deeper, more effective learning, and improves learning outcomes for those who practice it. Games‐based learning, which leverages game content and play to enhance knowledge and skills acquisition, is one method of promoting active recall skills in tandem with other educational benefits. , This educational method often includes gamification, which is the incorporation of game elements (e.g., rewards, scoring, and competition) to the process of teaching and learning, and serious games (i.e., full‐fledged games designed to educate or train). , These gamified approaches to active learning can take many forms, including student response systems, escape rooms, board games, and video games, and they each provide educational benefits. Specifically in medical education, games‐based learning has been found to improve motivation, collaborative learning, examination performance, recall of knowledge, and student metacognition. , , , Since most of these activities are low‐stakes, they also encourage students to learn from their mistakes without fear of academic failure or patient injury. , , Lastly, gamification and serious games can bring levity and variety to the learning process and help mitigate the stress of medical professional education. The word guessing party game, Catch‐Phrase, by Hasbro, Inc. has the potential to leverage the principles of both active recall and games‐based learning to promote student learning. Therefore, the purpose of this pilot study was to develop and evaluate an anatomy‐focused adaption of the game, Catch‐Phrase. The primary research question to be addressed in this study is: how do health professional students (i.e., physical therapy, physician assistant, occupational therapy) in a large, dissection‐based gross anatomy course perceive an anatomy‐focused adaption of Catch‐Phrase, including its perceived educational value and benefits? To answer this research question, this study employed quantitative, questionnaire‐based methods. Educational context: Anatomy for healthcare professionals Course overview The Anatomy Catch‐Phrase game was implemented in “Anatomy for Healthcare Professionals,” a graduate‐level interprofessional gross anatomy course in 2022. This course was designed to introduce Doctor of Physical Therapy (DPT, n = 41), Master of Physician Assistant Studies (MPAS, n = 44), and Doctor of Occupational Therapy (OTD, n = 35) students to the basic human anatomical concepts and structures in a medical context. Students progressed through the anatomy of the human using a regional approach organized into four blocks: (1) Back and Upper Limb, (2) Thorax and Abdomen, (3) Pelvis and Lower Limb, and (4) Head and Neck. Each in‐person laboratory session tasked interprofessional laboratory groups ( n = 6 students/group, 21 groups total) with dissecting a specified region of the human donor. Typically, two students from each program (i.e., DPT, MPAS, and OTD) were present in each laboratory group. During the laboratory sessions, each group used a digital dissection guide that included detailed dissection instructions, cadaveric images, and short answer and multiple‐choice questions, to guide their dissections. Summative assessments were administered following the completion of each course block and included both lecture and laboratory components. Anatomy Catch‐Phrase game Development First, an anatomical terms list was curated from the highest‐yield topics from the course by the course director (M.A.M.) and an instructor (A.S.C.). Emphasis was placed on structures that were frequently tested on exams and clinically relevant to the health professional students. Thirty terms were ultimately selected for each of the four blocks for a total of 120 possible terms (Appendix ). The learning objectives for the Anatomy Catch‐Phrase game are given in Table . Afterward, the physical materials needed for the game were purchased, including a Catch‐Phrase game device and 120 two‐inch cardboard coasters (Figure ). All anatomical terms were then printed in large font and glued to one side of the cardboard coasters. Gameplay Anatomy Catch‐Phrase is played in three‐person teams with one player in the clue‐giver role and two players in the guesser role. To start the game, the clue‐giver starts the 60 s timer and pulls a card from the stack. The clue‐giver must then get their teammates to identify the anatomical term on the card by describing its features, functions, or relationships or by physically gesturing. However, the clue‐giver cannot say the term itself or any key parts of the term. Non‐identifying words such as “nerve,” “artery,” “vein,” or “muscle” that are necessary for description, but do not give away the specific structure are allowed. Once the term is identified, the players rotate roles and continue playing with a new anatomical term. Skipping terms is not allowed. Gameplay continues until the 60‐s timer runs out. The team's score is then based on the number of anatomical terms the team successfully identified within the time limit. Implementation The Anatomy Catch‐Phrase game was piloted in the summer 2022 iteration of the “Anatomy for Healthcare Professionals” course. Students had the option to play the game during the dedicated laboratory review sessions held prior to each Block exam. To play, students needed to create three‐person teams comprised of players from their dissection table and sign up their team using a Google Sheets page open on easily accessible laptop or tablet in the lab. This Google Sheet was updated in real‐time by the facilitator and served as both a waiting list and a leaderboard (Appendix ). When it was a team's turn to play the game, the facilitator called the team using the laboratory‐wide speaker system. Students then proceeded to an office space adjacent to the cadaver anatomy laboratory in which the game would be played. Once all students were ready, the facilitator informed them of the rules of Anatomy Catch‐Phrase (Appendix ). Students were free to ask for any clarification on any of the rules if they still had questions. Students were also offered the opportunity to play a practice round that would not count toward their official score to allow them to familiarize themselves with the gameplay and rules. Students then played a single round of Anatomy Catch‐Phrase that counted toward their official score. Meanwhile, the facilitator paid close attention to make sure that all rules were followed and made note of any anatomical terms that students struggled with. Once gameplay ended, the facilitator briefly discussed the features, functions, and relationships of any terms that students struggled to identify to address that gap in knowledge. The Google Sheet page was then updated with the team's official score and the waiting list was advanced. If any teams wanted to play another round to improve their high score, they had the option to sign up again at the end of the waiting list. Teams could also swap out players from their dissection table if they so choose. The competition ended when the waiting list was cleared or the laboratory review session ended, whichever came first. If any teams tied for the top score, a tiebreaker would be played. The competing teams would each play an additional round of Anatomy Catch‐Phrase and the team with the highest score from that round would be crowned the winners. As the winners of Anatomy Catch‐Phrase, the team would have the option to choose from two extra credit prizes: (1) each member of the winning team's dissection table would receive three extra points on their exam or (2) every student in the class would receive one extra credit point on their exam. Evaluation Prior to the first Block exam, all students were given an anonymous article evaluation for the Anatomy Catch‐Phrase game (Appendix ). The evaluation included seven Likert‐style items rated on a 5‐point scale (1 = strongly disagree, 5 = strongly agree). The evaluation items included: (1) I enjoyed the Catch‐Phrase game, (2) I found the Catch‐Phrase game useful for reviewing anatomy, (3) the Catch‐Phrase game helped reinforce my knowledge of anatomical relationships, (4) the Catch‐Phrase game content was relevant to the course material, (5) I would recommend the Catch‐Phrase game to my peers, (6) I would like to play the Catch‐Phrase game again in future Blocks of the course, and (7) overall, how would you rate the Catch‐Phrase game? These items represent Level 1 of Kirkpatrick's Four Levels framework, which is widely used to evaluate educational programs. Level 1: Reaction determines how participants react to an educational program and serves as a foundation for subsequent investigation into the impact of the program on participant knowledge (Level 2), behaviors (Level 3), and results (Level 4). Completed article evaluations were collected from students by one author (M.A.M) at the end of the exam period and given to a second author (A.S.C.) for analysis to maintain participant anonymity. Statistical analysis All evaluation responses were transcribed from their original article format into SPSS (IBM Co., Armonk, NY) for statistical analysis. Means and standard deviations were calculated for each evaluation item. All unsolicited comments were also recorded verbatim and reviewed for significant insights into how students perceived the Anatomy Catch‐Phrase game. Course overview The Anatomy Catch‐Phrase game was implemented in “Anatomy for Healthcare Professionals,” a graduate‐level interprofessional gross anatomy course in 2022. This course was designed to introduce Doctor of Physical Therapy (DPT, n = 41), Master of Physician Assistant Studies (MPAS, n = 44), and Doctor of Occupational Therapy (OTD, n = 35) students to the basic human anatomical concepts and structures in a medical context. Students progressed through the anatomy of the human using a regional approach organized into four blocks: (1) Back and Upper Limb, (2) Thorax and Abdomen, (3) Pelvis and Lower Limb, and (4) Head and Neck. Each in‐person laboratory session tasked interprofessional laboratory groups ( n = 6 students/group, 21 groups total) with dissecting a specified region of the human donor. Typically, two students from each program (i.e., DPT, MPAS, and OTD) were present in each laboratory group. During the laboratory sessions, each group used a digital dissection guide that included detailed dissection instructions, cadaveric images, and short answer and multiple‐choice questions, to guide their dissections. Summative assessments were administered following the completion of each course block and included both lecture and laboratory components. The Anatomy Catch‐Phrase game was implemented in “Anatomy for Healthcare Professionals,” a graduate‐level interprofessional gross anatomy course in 2022. This course was designed to introduce Doctor of Physical Therapy (DPT, n = 41), Master of Physician Assistant Studies (MPAS, n = 44), and Doctor of Occupational Therapy (OTD, n = 35) students to the basic human anatomical concepts and structures in a medical context. Students progressed through the anatomy of the human using a regional approach organized into four blocks: (1) Back and Upper Limb, (2) Thorax and Abdomen, (3) Pelvis and Lower Limb, and (4) Head and Neck. Each in‐person laboratory session tasked interprofessional laboratory groups ( n = 6 students/group, 21 groups total) with dissecting a specified region of the human donor. Typically, two students from each program (i.e., DPT, MPAS, and OTD) were present in each laboratory group. During the laboratory sessions, each group used a digital dissection guide that included detailed dissection instructions, cadaveric images, and short answer and multiple‐choice questions, to guide their dissections. Summative assessments were administered following the completion of each course block and included both lecture and laboratory components. Development First, an anatomical terms list was curated from the highest‐yield topics from the course by the course director (M.A.M.) and an instructor (A.S.C.). Emphasis was placed on structures that were frequently tested on exams and clinically relevant to the health professional students. Thirty terms were ultimately selected for each of the four blocks for a total of 120 possible terms (Appendix ). The learning objectives for the Anatomy Catch‐Phrase game are given in Table . Afterward, the physical materials needed for the game were purchased, including a Catch‐Phrase game device and 120 two‐inch cardboard coasters (Figure ). All anatomical terms were then printed in large font and glued to one side of the cardboard coasters. Gameplay Anatomy Catch‐Phrase is played in three‐person teams with one player in the clue‐giver role and two players in the guesser role. To start the game, the clue‐giver starts the 60 s timer and pulls a card from the stack. The clue‐giver must then get their teammates to identify the anatomical term on the card by describing its features, functions, or relationships or by physically gesturing. However, the clue‐giver cannot say the term itself or any key parts of the term. Non‐identifying words such as “nerve,” “artery,” “vein,” or “muscle” that are necessary for description, but do not give away the specific structure are allowed. Once the term is identified, the players rotate roles and continue playing with a new anatomical term. Skipping terms is not allowed. Gameplay continues until the 60‐s timer runs out. The team's score is then based on the number of anatomical terms the team successfully identified within the time limit. Implementation The Anatomy Catch‐Phrase game was piloted in the summer 2022 iteration of the “Anatomy for Healthcare Professionals” course. Students had the option to play the game during the dedicated laboratory review sessions held prior to each Block exam. To play, students needed to create three‐person teams comprised of players from their dissection table and sign up their team using a Google Sheets page open on easily accessible laptop or tablet in the lab. This Google Sheet was updated in real‐time by the facilitator and served as both a waiting list and a leaderboard (Appendix ). When it was a team's turn to play the game, the facilitator called the team using the laboratory‐wide speaker system. Students then proceeded to an office space adjacent to the cadaver anatomy laboratory in which the game would be played. Once all students were ready, the facilitator informed them of the rules of Anatomy Catch‐Phrase (Appendix ). Students were free to ask for any clarification on any of the rules if they still had questions. Students were also offered the opportunity to play a practice round that would not count toward their official score to allow them to familiarize themselves with the gameplay and rules. Students then played a single round of Anatomy Catch‐Phrase that counted toward their official score. Meanwhile, the facilitator paid close attention to make sure that all rules were followed and made note of any anatomical terms that students struggled with. Once gameplay ended, the facilitator briefly discussed the features, functions, and relationships of any terms that students struggled to identify to address that gap in knowledge. The Google Sheet page was then updated with the team's official score and the waiting list was advanced. If any teams wanted to play another round to improve their high score, they had the option to sign up again at the end of the waiting list. Teams could also swap out players from their dissection table if they so choose. The competition ended when the waiting list was cleared or the laboratory review session ended, whichever came first. If any teams tied for the top score, a tiebreaker would be played. The competing teams would each play an additional round of Anatomy Catch‐Phrase and the team with the highest score from that round would be crowned the winners. As the winners of Anatomy Catch‐Phrase, the team would have the option to choose from two extra credit prizes: (1) each member of the winning team's dissection table would receive three extra points on their exam or (2) every student in the class would receive one extra credit point on their exam. Evaluation Prior to the first Block exam, all students were given an anonymous article evaluation for the Anatomy Catch‐Phrase game (Appendix ). The evaluation included seven Likert‐style items rated on a 5‐point scale (1 = strongly disagree, 5 = strongly agree). The evaluation items included: (1) I enjoyed the Catch‐Phrase game, (2) I found the Catch‐Phrase game useful for reviewing anatomy, (3) the Catch‐Phrase game helped reinforce my knowledge of anatomical relationships, (4) the Catch‐Phrase game content was relevant to the course material, (5) I would recommend the Catch‐Phrase game to my peers, (6) I would like to play the Catch‐Phrase game again in future Blocks of the course, and (7) overall, how would you rate the Catch‐Phrase game? These items represent Level 1 of Kirkpatrick's Four Levels framework, which is widely used to evaluate educational programs. Level 1: Reaction determines how participants react to an educational program and serves as a foundation for subsequent investigation into the impact of the program on participant knowledge (Level 2), behaviors (Level 3), and results (Level 4). Completed article evaluations were collected from students by one author (M.A.M) at the end of the exam period and given to a second author (A.S.C.) for analysis to maintain participant anonymity. First, an anatomical terms list was curated from the highest‐yield topics from the course by the course director (M.A.M.) and an instructor (A.S.C.). Emphasis was placed on structures that were frequently tested on exams and clinically relevant to the health professional students. Thirty terms were ultimately selected for each of the four blocks for a total of 120 possible terms (Appendix ). The learning objectives for the Anatomy Catch‐Phrase game are given in Table . Afterward, the physical materials needed for the game were purchased, including a Catch‐Phrase game device and 120 two‐inch cardboard coasters (Figure ). All anatomical terms were then printed in large font and glued to one side of the cardboard coasters. Anatomy Catch‐Phrase is played in three‐person teams with one player in the clue‐giver role and two players in the guesser role. To start the game, the clue‐giver starts the 60 s timer and pulls a card from the stack. The clue‐giver must then get their teammates to identify the anatomical term on the card by describing its features, functions, or relationships or by physically gesturing. However, the clue‐giver cannot say the term itself or any key parts of the term. Non‐identifying words such as “nerve,” “artery,” “vein,” or “muscle” that are necessary for description, but do not give away the specific structure are allowed. Once the term is identified, the players rotate roles and continue playing with a new anatomical term. Skipping terms is not allowed. Gameplay continues until the 60‐s timer runs out. The team's score is then based on the number of anatomical terms the team successfully identified within the time limit. The Anatomy Catch‐Phrase game was piloted in the summer 2022 iteration of the “Anatomy for Healthcare Professionals” course. Students had the option to play the game during the dedicated laboratory review sessions held prior to each Block exam. To play, students needed to create three‐person teams comprised of players from their dissection table and sign up their team using a Google Sheets page open on easily accessible laptop or tablet in the lab. This Google Sheet was updated in real‐time by the facilitator and served as both a waiting list and a leaderboard (Appendix ). When it was a team's turn to play the game, the facilitator called the team using the laboratory‐wide speaker system. Students then proceeded to an office space adjacent to the cadaver anatomy laboratory in which the game would be played. Once all students were ready, the facilitator informed them of the rules of Anatomy Catch‐Phrase (Appendix ). Students were free to ask for any clarification on any of the rules if they still had questions. Students were also offered the opportunity to play a practice round that would not count toward their official score to allow them to familiarize themselves with the gameplay and rules. Students then played a single round of Anatomy Catch‐Phrase that counted toward their official score. Meanwhile, the facilitator paid close attention to make sure that all rules were followed and made note of any anatomical terms that students struggled with. Once gameplay ended, the facilitator briefly discussed the features, functions, and relationships of any terms that students struggled to identify to address that gap in knowledge. The Google Sheet page was then updated with the team's official score and the waiting list was advanced. If any teams wanted to play another round to improve their high score, they had the option to sign up again at the end of the waiting list. Teams could also swap out players from their dissection table if they so choose. The competition ended when the waiting list was cleared or the laboratory review session ended, whichever came first. If any teams tied for the top score, a tiebreaker would be played. The competing teams would each play an additional round of Anatomy Catch‐Phrase and the team with the highest score from that round would be crowned the winners. As the winners of Anatomy Catch‐Phrase, the team would have the option to choose from two extra credit prizes: (1) each member of the winning team's dissection table would receive three extra points on their exam or (2) every student in the class would receive one extra credit point on their exam. Prior to the first Block exam, all students were given an anonymous article evaluation for the Anatomy Catch‐Phrase game (Appendix ). The evaluation included seven Likert‐style items rated on a 5‐point scale (1 = strongly disagree, 5 = strongly agree). The evaluation items included: (1) I enjoyed the Catch‐Phrase game, (2) I found the Catch‐Phrase game useful for reviewing anatomy, (3) the Catch‐Phrase game helped reinforce my knowledge of anatomical relationships, (4) the Catch‐Phrase game content was relevant to the course material, (5) I would recommend the Catch‐Phrase game to my peers, (6) I would like to play the Catch‐Phrase game again in future Blocks of the course, and (7) overall, how would you rate the Catch‐Phrase game? These items represent Level 1 of Kirkpatrick's Four Levels framework, which is widely used to evaluate educational programs. Level 1: Reaction determines how participants react to an educational program and serves as a foundation for subsequent investigation into the impact of the program on participant knowledge (Level 2), behaviors (Level 3), and results (Level 4). Completed article evaluations were collected from students by one author (M.A.M) at the end of the exam period and given to a second author (A.S.C.) for analysis to maintain participant anonymity. All evaluation responses were transcribed from their original article format into SPSS (IBM Co., Armonk, NY) for statistical analysis. Means and standard deviations were calculated for each evaluation item. All unsolicited comments were also recorded verbatim and reviewed for significant insights into how students perceived the Anatomy Catch‐Phrase game. A total of 18 dissection groups (86%) formed three‐person teams to represent them in the Anatomy Catch‐Phrase game in Block 1, followed by 13 groups (62%) in Block 2, 7 groups (33%) in Block 3, and 5 groups (28%) in Block 4. In each Block, multiple groups who played the game opted to return and play multiple rounds of the game to further improve their high score. The high scores were 10 points in Block 1, 7 points in Block 2, 7 points in Block 3, and 10 points in Block 4. For every Block, the winning team opted to give one point to every student in the class rather than keep three points for their group only. After the Block 1 exam, 73 students (61% of the class) completed the evaluation questionnaire. Overall, the responding students viewed the Anatomy Catch‐Phrase game positively and found it to be an effective activity for studying anatomy. On a 5‐point scale (1 = strongly disagree, 5 = strongly agree), the majority of responding students indicated that they enjoyed the Anatomy Catch‐Phrase game with an average rating of 4.3 ± 0.9. The majority of students also found the game to be relevant to the course material (4.5 ± 0.8), useful for reviewing anatomy (3.9 ± 0.9), and helped reinforce their knowledge of anatomical relationships (3.9 ± 0.9). Students also highly recommended the Anatomy Catch‐Phrase game to their peers (4.2 ± 0.9) and wanted to play the game in future Blocks of the course (4.3 ± 1.0). Ultimately, the students rated the Anatomy Catch‐Phrase game very highly, giving it a 4.3 ± 0.9 score. This high satisfaction with the game suggests successful fulfillment of Level 1: Reaction of Kirkpatrick's Four Levels framework. However, additional data are required to determine if the Anatomy Catch‐Phrase fulfills the remaining levels of the Kirkpatrick framework. Several students also left unsolicited comments on their evaluation questionnaires, the majority of which were incredibly positive. Some of the positive comments were incredibly enthusiastic, such as “ LOVED IT! A fun way to study anatomy! :)” and “ S o fun! I wish we tried more times! ” A few students also left constructive criticisms such as: “ I genuinely enjoyed it, but I felt like I didn't get enough time to interact with it. I feel like it would be better if it wasn't on mock‐practical or maybe done in a way that we play it on our own time as an additional study aide .” Multiple students also indicated that one of the major drawbacks of the game was that only a few players could participate at one time: “ Bummer only 3 teammates could play—I did not. Would enjoy whole group teams. I like the concept ” and “ It would be nice if multiple teams could go at one time .” Anatomy Catch‐Phrase, a fast‐paced, team‐based word guessing game, was well‐received by health professional students in a doctoral‐level, clinically oriented gross anatomy course. Students viewed the game as relevant to the course and useful for reviewing and reinforcing their anatomical knowledge. Students also rated the game highly and would recommend the game to their peers. Based on the positive student response, Anatomy Catch‐Phrase successfully fulfilled Level 1: Reaction of Kirkpatrick's Four Levels framework. Although most students viewed the game positively, several provided constructive criticisms related to the timing and duration of the game, the size of teams, and the number of concurrent players. Overall, the Anatomy Catch‐Phrase game was perceived as an enjoyable and useful activity for health professional students to reviewing gross anatomy prior to high‐stakes summative assessments. One of the primary benefits of the Anatomy Catch‐Phrase game is that it provided participating students with an opportunity for active recall. To play either role in the game, students needed to quickly retrieve from their memory the features, functions, or relationships associated with a particular anatomical term. This regular, purposeful retrieval of previously consolidated anatomical content information encourages re‐encoding and re‐consolidation of information, resulting in easier and longer‐lasting access to that information in the future. As described previously, swift and accurate recall of medical knowledge is essential in both academic and clinical settings. , Increased active recall is considered one of the key benefits of gamification of educational activities. Since traditional methods of active recall are perceived as dull and requiring greater mental effort, students often prefer to use more passive, less effective methods of studying. , Moreover, a positive emotional context has been shown to enhance memorization and recall, whereas stress and anxiety can be detrimental. Novel game‐based learning activities address this perceived barrier by placing the active recall activity within the context of fun, engaging games, thereby reducing the monotony of traditional methods of active recall. The Anatomy Catch‐Phrase game also provided participating students with immediate feedback they could use to identify their weaknesses or gaps in knowledge. Whenever students were unable to quickly recall enough details to accurately describe or identify an anatomical term, it suggested that they had a gap in their knowledge regarding that specific topic. Once identified, the students could then take steps to address the gaps. The placement of the Anatomy Catch‐Phrase game during the laboratory review session allowed students to identify gaps in their knowledge by playing the game and then immediately address those gaps by visualizing them in a cadaveric donor or asking an instructor for assistance. This immediate feedback is well‐documented as one of the key benefits of gamification. Increased frequency and immediacy of feedback is positively associated with learning effectiveness and enhances immersion in the gameplay. Facilitation of the Anatomy Catch‐Phrase game also provided instructors with an opportunity to identify structures or concepts that students were still struggling with. As the facilitator, the instructor was present for all rounds of the game and could keep track of which terms most frequently stumped the students. For example, in Block 3, the facilitator noted that multiple teams struggled with describing and/or guessing the term “Pelvic Diaphragm.” This indicated to the facilitator that something was amiss with the students' knowledge regarding the topic. After the conclusion of the competition, instructors could subsequently use this insight to address common weaknesses across students such as re‐explaining a topic or demonstrating a structure again on a cadaveric donor. Therefore, Anatomy Catch‐Phrase can serve as a last‐minute method of identifying and addressing student weaknesses shortly before high‐stakes summative assessments. Given its timing prior to high‐stakes summative assessments, the Anatomy Catch‐Phrase game seemed to have also provided students with much‐needed levity and camaraderie. As noted in the evaluation comments, students thought the game was “fun” and “genuinely enjoyed” the game. The facilitator also frequently noted that the teams were incredibly animated and motivated to earn the highest score. Several teams even devised strategies for efficient teamwork and communication to boost their performance. This added lightheartedness and sociability are hallmarks of gamification and particularly beneficial for medical professional students. These students often experience high stress and anxiety related to the pressures of their studies, which can adversely affect their learning and performance. , Gamification can help mitigate this effect by promoting a low‐stakes learning environment. Moreover, the team‐based nature of games encourages students to work together with their peers to achieve a common goal, a skill that is essential in the clinical setting. As such, the Anatomy Catch‐Phrase game can provide educational benefits beyond active recall practice and identification of knowledge gaps as well. Several games that use mechanics similar to Anatomy Catch‐Phrase have also been described in the medical education literature. , , These games are based around limiting either verbal, written, or gestural communication between the participants. For example, the Neurological Hat Game created by Garcin et al. tasked medical students with getting their peers to guess as many terms as possible within 90 s using any verbal description (round 1), one word (round 2), and only physical gestures (round 3). Similarly, Burney et al. adapted the game Taboo! by Hasbro to create the Patient Communication Challenge, which tasked medical students to get their teammates to identify a patient communication‐related term within a set timeframe without saying the term itself. The MediLex game by Smith et al. also adapted Taboo! and its rules to support learning of basic sciences, signs and symptoms, medical procedures, diseases, and drugs. Much like Anatomy Catch‐Phrase, these games were well‐received by their respective student populations and were touted as fun and engaging ways to promote retention of material through repetition and retrieval practice. These studies also noted how this game style allows students to identify and address gaps in their knowledge. , As one study stated, “If you can't explain it simply enough, you don't understand it well enough”. Much like Anatomy Catch‐Phrase though, these studies do not objectively evaluate the impact of the games on Levels 2–4 of the Kirkpatrick Four Levels framework. , , , Limitations This research study is not without limitations. First, this study only evaluated Anatomy Catch‐Phrase to the first level of the Kirkpatrick Four Levels framework, which focuses on the reaction of participants to the educational intervention. Although the participating students indicated that helped them review and reinforce their anatomical knowledge, the evaluation methods used in this study did not directly assess change in knowledge or impact on exam performance (Level 2: Knowledge). Therefore, it was not possible to determine how the game impacted the levels above Level 1. The exact game experience was also highly variable between game rounds. Due to the short game duration and shuffling of terms prior to each round, it was not possible for every team to encounter terms in the same order or all the terms overall. As a result, some teams may have had more exposure and practice with certain anatomical terms compared with other teams. Students with certain characteristics may have been more inclined to participate in the optional Anatomy Catch‐Phrase game, leading to a self‐selection bias. Students who are more competitive, social, and/or confident in their anatomical knowledge may have found the game more appealing and may disproportionately represent more of the participants or evaluations. Moreover, the reward structure of the game (i.e., three extra points for the winning team only or one point for the whole class) may have influenced game participation. Implicit peer pressure may have pushed the winning teams to always pick the more altruistic option. Once this became the known trend, some students may have anticipated the winning teams' altruistic choice and opted not to compete themselves, leading to the decline in participation. Fatigue may have also played a role in the declining participation rate. As the semester progressed, students tend to become overwhelmed and fatigued by their academic and professional commitments, leading them to focus their time on tried‐and‐true study strategies (e.g., lab study, lecture review, practice questions, flashcards, etc.) rather than trying novel strategies such as the Anatomy Catch‐Phrase game. Future directions In the future, this study could be expanded upon through several different areas. First, the structure and organization of the Anatomy Catch‐Phrase game should be revised based on the feedback provided by the students. These modifications could address the timing and duration of the game, size of teams, and number of concurrent players. Additionally, the evaluation method should be expanded to assess the impact of the game on recall of anatomical knowledge and exam performance (Level 2: Knowledge of the Kirkpatrick Four Levels framework). Given that the game seemed to positively affect the disposition of the students, it may also be interesting to explore the impact of the game on pre‐exam stress and team camaraderie. This research study is not without limitations. First, this study only evaluated Anatomy Catch‐Phrase to the first level of the Kirkpatrick Four Levels framework, which focuses on the reaction of participants to the educational intervention. Although the participating students indicated that helped them review and reinforce their anatomical knowledge, the evaluation methods used in this study did not directly assess change in knowledge or impact on exam performance (Level 2: Knowledge). Therefore, it was not possible to determine how the game impacted the levels above Level 1. The exact game experience was also highly variable between game rounds. Due to the short game duration and shuffling of terms prior to each round, it was not possible for every team to encounter terms in the same order or all the terms overall. As a result, some teams may have had more exposure and practice with certain anatomical terms compared with other teams. Students with certain characteristics may have been more inclined to participate in the optional Anatomy Catch‐Phrase game, leading to a self‐selection bias. Students who are more competitive, social, and/or confident in their anatomical knowledge may have found the game more appealing and may disproportionately represent more of the participants or evaluations. Moreover, the reward structure of the game (i.e., three extra points for the winning team only or one point for the whole class) may have influenced game participation. Implicit peer pressure may have pushed the winning teams to always pick the more altruistic option. Once this became the known trend, some students may have anticipated the winning teams' altruistic choice and opted not to compete themselves, leading to the decline in participation. Fatigue may have also played a role in the declining participation rate. As the semester progressed, students tend to become overwhelmed and fatigued by their academic and professional commitments, leading them to focus their time on tried‐and‐true study strategies (e.g., lab study, lecture review, practice questions, flashcards, etc.) rather than trying novel strategies such as the Anatomy Catch‐Phrase game. In the future, this study could be expanded upon through several different areas. First, the structure and organization of the Anatomy Catch‐Phrase game should be revised based on the feedback provided by the students. These modifications could address the timing and duration of the game, size of teams, and number of concurrent players. Additionally, the evaluation method should be expanded to assess the impact of the game on recall of anatomical knowledge and exam performance (Level 2: Knowledge of the Kirkpatrick Four Levels framework). Given that the game seemed to positively affect the disposition of the students, it may also be interesting to explore the impact of the game on pre‐exam stress and team camaraderie. Overall, Anatomy Catch‐Phrase was well‐received by health professional students learning gross anatomy and perceived as a fun, effective activity for reviewing anatomical structures and relationships relevant to the course, thereby achieving the first level (Reaction) of the Kirkpatrick framework. The novel game encouraged deeper learning and longer retention of knowledge by providing students with an opportunity to actively recall their anatomical knowledge and practice describing anatomical structures to their peers. Moreover, the game provided instructors with an opportunity to identify and address gaps in student knowledge. Additionally, the Anatomy Catch‐Phrase game seemed to bring some levity to a high‐stress course and foster camaraderie between interprofessional peers. Although more work needs to be completed to determine the full impact of Anatomy Catch‐Phrase on knowledge, behaviors, and results of the students, the findings of this pilot study suggest that it can serve as a useful activity for reviewing gross anatomy. Andrew S. Cale: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; resources; writing – original draft; writing – review and editing. Margaret A. McNulty: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; resources; supervision; writing – original draft; writing – review and editing. This study was given exempt‐status by the Indiana University Institutional Review Board (Protocol # 1804885093). |
A 15-year experience in pediatric palliative care: a retrospective hospital-based study | ec7eda40-6a6d-4e3f-8a8e-213cfc44fbd0 | 11301942 | Pediatrics[mh] | Pediatric palliative care (PPC) globally addresses the physical and psychological needs of children with life-limiting and/or life-threatening conditions and their families, with the ultimate aim of improving their quality of life . Several conditions can make a child eligible for PPC, including advanced chronic non-communicable diseases, acute life-threatening conditions, severe neurodegenerative and neurological conditions, extreme prematurity, and congenital abnormalities . Remarkably, patients with non-oncological diseases represent most of those referring to PPC services . According to the latest estimates, in Italy, approximately 10,600 children require specialized PPC . However, the recent nationwide PalliPed project showed that less than two children out of ten have access to PPC service, suggesting that improvements in its delivery of service and better allocation of resources are eagerly required . Furthermore, the Italian Law 38/2010 set off a customized response for children requiring PPC and their families to implement a network of PPC-specific services at a regional level under the coordination of a referral center. Italy is divided into 20 regions, and each one must have at least one PPC reference center and one pediatric hospice . According to this model, there is a specialized and interdisciplinary team in the referral center that is responsible for coordinating 24/7 the care activity of the entire network in all settings of care, including home, hospital, general PPC, or pediatric hospice, namely specialized PPC. The identification of the most adequate setting of care to which refer patients can be given by the use of appropriate tools to evaluate the PPC needs, such as the ACCAPED (Accertamento dei bisogni Clinico-Assistenziali Complessi in PEDiatria) scale . To achieve these goals and to allow accurate planning of services and allocation of resources, the accurate evaluation of available epidemiological data and the characterization of children in PPC are necessary . In particular, information on the current number of referrals and length of follow-up of patients at specialized PPC services is lacking. It also appears fundamental to differentiate between patients with oncological and non-oncological diseases since their trajectories in the PPC service are markedly different . In the Italian scenario, one of the oldest centers is the PPC Referral Center of the University of Padua (Padua, Italy), which is the Reference Center of the PPC Network within the Veneto Region. The center started its activities in the first half of the 1980s and is based at the Department of Women’s and Children’s Health in Padua (University of Padua). It organizes, coordinates, and supports the activities of the PPC network of the Veneto region, which is constituted by all social and healthcare services, including hospital and local facilities, aimed at pediatric patients. The PPC Referral Center of the University of Padua coordinates integrated care responses 24/7 to the needs of children in all settings of their lives through the work of a multi-disciplinary team. While the center is the point of reference for the training and qualification of caregivers, more than 90% of the activities are carried out at home, which is the preferred place of care. In the case of special needs, the same team assists the child and his family at the Pediatric Hospice of Padua, which comprises social and health services. This pathway ensures the continuity of care, the strengthening of relationships, and the pursuit of the objectives. Since more than 1,000 patients have been followed at the Pediatric Hospice of Padua over more than 30 years of activity, analyzing data from this specialized PPC service can provide important information on the characteristics of patients, the number of correct and inappropriate referrals, and the length of their follow-up. Here, we aim to extensively present and analyze the data of these patients over a 15-year period. Study design This retrospective chart review was conducted on the patients who were referred to the Pediatric Hospice of Padua (Padua, Italy) or to the PPC network of the Veneto Region from 1 January 2008 to 31 December 2022. For the sake of brevity, from now on, the term ‘Padua Hospice’ will also include the PPC network of the Veneto region. Eligibility criteria for specialized PPC were as follows (see Benini et al. for more details): (i) being affected by a life-threatening or life-limiting disease together with complex clinical and/or psycho-social needs; (ii) risk of premature death (iii) serious episodes of hospitalization; (iv) use of invasive medical devices for life support. For patients referring to the hospice from 2020 onwards, a score > 50 on the ACCAPED scale was used as an inclusion criterion for specialized PPC . The Local Ethical Committee approved the study design and all Legal Guardians who could be contacted signed an informed consent to the use of their child’s data for research purposes. Data collection and variables considered in the analysis Data were extracted from clinical charts and included in a dedicated spreadsheet specifically designed for this analysis and prepared with the assistance of a professional statistician. The following variables were included in the analysis: (i) the number of patients who were referred to the hospice, i.e., all patients who were referred to the hospice, whether taken in charge or not; (ii) the number of patients taken in charge by the hospice service, i.e. the proportion of patients referred to the service who were actually taken in charge; (iii) the number of patients followed at the hospice, in total and per year, i.e., patients taken in charge during the analyzed year, or during the previous years but not yet discharged; (iv) the mortality during the observation period; (v) the number of patients discharged during the observation period; and (vi) the length of follow-up per patient, i.e. the total time in which the patient was followed up by the hospice. The above variables were analyzed over the entire study period (15 years) or per year, as appropriate. When possible, results were stratified by oncological and non-oncological conditions. Statistical analysis Data were analyzed by descriptive statistics using mean and standard deviation or number and percentage. The significance level was set at p < 0.05. The MedCalc ® Statistical Software version 22.016 software was used for the analysis. This retrospective chart review was conducted on the patients who were referred to the Pediatric Hospice of Padua (Padua, Italy) or to the PPC network of the Veneto Region from 1 January 2008 to 31 December 2022. For the sake of brevity, from now on, the term ‘Padua Hospice’ will also include the PPC network of the Veneto region. Eligibility criteria for specialized PPC were as follows (see Benini et al. for more details): (i) being affected by a life-threatening or life-limiting disease together with complex clinical and/or psycho-social needs; (ii) risk of premature death (iii) serious episodes of hospitalization; (iv) use of invasive medical devices for life support. For patients referring to the hospice from 2020 onwards, a score > 50 on the ACCAPED scale was used as an inclusion criterion for specialized PPC . The Local Ethical Committee approved the study design and all Legal Guardians who could be contacted signed an informed consent to the use of their child’s data for research purposes. Data were extracted from clinical charts and included in a dedicated spreadsheet specifically designed for this analysis and prepared with the assistance of a professional statistician. The following variables were included in the analysis: (i) the number of patients who were referred to the hospice, i.e., all patients who were referred to the hospice, whether taken in charge or not; (ii) the number of patients taken in charge by the hospice service, i.e. the proportion of patients referred to the service who were actually taken in charge; (iii) the number of patients followed at the hospice, in total and per year, i.e., patients taken in charge during the analyzed year, or during the previous years but not yet discharged; (iv) the mortality during the observation period; (v) the number of patients discharged during the observation period; and (vi) the length of follow-up per patient, i.e. the total time in which the patient was followed up by the hospice. The above variables were analyzed over the entire study period (15 years) or per year, as appropriate. When possible, results were stratified by oncological and non-oncological conditions. Data were analyzed by descriptive statistics using mean and standard deviation or number and percentage. The significance level was set at p < 0.05. The MedCalc ® Statistical Software version 22.016 software was used for the analysis. Study population In total, 870 patients were referred to the Padua Hospice between 2008 and 2022. The mean age of children was 6.8 ± 6.3 years. Approximately three out of four patients (658/870, 76%) were affected by non-oncological conditions of which neurologic diseases were present in 152 (22%), neuromuscular disease in 130 (20%), cardiological disease in 43 (7%), lung disease in 14 (2%), adverse perinatal outcomes in 72 (11%), infectious-inflammatory disease in 22 (3%), chromosomal alterations in 35 (5%), congenital anomalies in 41 (6%) and other genetic conditions in 149 (23%) patients. The remaining 212/870 (24%) patients were affected by oncological disease, including solid tumors in 80 (38%), brain tumors in 84 (40%), and liquid tumors in 38 (18%), while definitive diagnoses were not available for 10 patients (5%). The ACCAPED median score was 62 (clinical range: 52–88) in patients with non-oncological conditions and 60 (55–93) for those with oncological diseases. New referrals and patients taken into charge Details on the total number of newly referred patients per year, based on the categorization in oncological or non-oncological conditions, are presented in Table . Overall, a steadily increasing trend was observed over time, with 109 patients referred to the hospice in 2022 compared to 37 patients referred in 2008 (195% increase). During the entire observation period, most new referrals concerned patients with non-oncological diagnoses. In total, 716/870 (82%) patients were taken in charge by the hospice service, with a trend towards an increasing proportion over time (Fig. ). In most cases, patients were not taken in charge due to inappropriate referrals (Table ), happening more frequently for non-oncological diagnoses than oncological. 24/870 (2.8%) patients were referred more than once (21/658, 3.2%, patients with non-oncological disease and 3/212, 1.4%, with oncological disease) due to requests for reassessments as the disease progressed or due to changes in the course of the pathology or given the increase in the needs of the patient and his family. Patients followed at the hospice The total number of patients followed each year at the hospice increased each year, from 58 in 2008 to 291 in 2022, while the proportion of patients with oncological disease decreased (Fig. ). Mortality during the observation period Over the observation period, 383 out of the 716 patients taken in charge died, resulting in an overall mortality rate of 53%. As presumable, this rate was higher in oncological conditions (165/183, 90%) compared to non-oncological disorders (218/533, 41%). Interestingly, a decreasing trend in the mortality rate was observed over time (Table ). The mean age at death was 7.1 ± 6.0 years, considering that patients affected by non-oncological conditions died at a younger age (Table ). Number of patients discharged during the observation period Over the observation period, 108/716 patients were discharged since they showed lower complexity of needs (15%), with a mean age of 9.1 ± 7.4 years (Table ). This occurred in 18% of the non-oncological cases (98/533) and 5% of the oncological ones (9/183). A decreasing trend in the discharge rate per year was observed over time (Table ). Length of follow-up Table depicts the distribution of follow-up length over the observation period. In total, 39% of patients (277/716) were followed for > 12 months. This percentage rose to 52% for non-oncological diseases (275/533). Of the remaining 48% of children with non-oncological disease (258/533) who had a follow-up < 12 months, a good proportion (93, 34%) were referred to the hospice in 2022, and, therefore, their follow-up was not concluded. Considering the non-oncological population followed for > 12 months, the median duration of the follow-up was 43 months (95% CI: 37–47 months). Within the oncological population, 91% of children had a follow-up < 12 months (167/183) with a median of 1.8 months (95% CI: 1.4–2.7 months). In a 5-year timeframe, the proportion of patients with ongoing follow-up increased over time (Table ). Remarkably, among the patients still receiving care in 2022, 13% (37/291) and 28% (81/291) were taken in charge more than 10 or more than 5 years earlier, respectively. In total, 870 patients were referred to the Padua Hospice between 2008 and 2022. The mean age of children was 6.8 ± 6.3 years. Approximately three out of four patients (658/870, 76%) were affected by non-oncological conditions of which neurologic diseases were present in 152 (22%), neuromuscular disease in 130 (20%), cardiological disease in 43 (7%), lung disease in 14 (2%), adverse perinatal outcomes in 72 (11%), infectious-inflammatory disease in 22 (3%), chromosomal alterations in 35 (5%), congenital anomalies in 41 (6%) and other genetic conditions in 149 (23%) patients. The remaining 212/870 (24%) patients were affected by oncological disease, including solid tumors in 80 (38%), brain tumors in 84 (40%), and liquid tumors in 38 (18%), while definitive diagnoses were not available for 10 patients (5%). The ACCAPED median score was 62 (clinical range: 52–88) in patients with non-oncological conditions and 60 (55–93) for those with oncological diseases. Details on the total number of newly referred patients per year, based on the categorization in oncological or non-oncological conditions, are presented in Table . Overall, a steadily increasing trend was observed over time, with 109 patients referred to the hospice in 2022 compared to 37 patients referred in 2008 (195% increase). During the entire observation period, most new referrals concerned patients with non-oncological diagnoses. In total, 716/870 (82%) patients were taken in charge by the hospice service, with a trend towards an increasing proportion over time (Fig. ). In most cases, patients were not taken in charge due to inappropriate referrals (Table ), happening more frequently for non-oncological diagnoses than oncological. 24/870 (2.8%) patients were referred more than once (21/658, 3.2%, patients with non-oncological disease and 3/212, 1.4%, with oncological disease) due to requests for reassessments as the disease progressed or due to changes in the course of the pathology or given the increase in the needs of the patient and his family. The total number of patients followed each year at the hospice increased each year, from 58 in 2008 to 291 in 2022, while the proportion of patients with oncological disease decreased (Fig. ). Over the observation period, 383 out of the 716 patients taken in charge died, resulting in an overall mortality rate of 53%. As presumable, this rate was higher in oncological conditions (165/183, 90%) compared to non-oncological disorders (218/533, 41%). Interestingly, a decreasing trend in the mortality rate was observed over time (Table ). The mean age at death was 7.1 ± 6.0 years, considering that patients affected by non-oncological conditions died at a younger age (Table ). Over the observation period, 108/716 patients were discharged since they showed lower complexity of needs (15%), with a mean age of 9.1 ± 7.4 years (Table ). This occurred in 18% of the non-oncological cases (98/533) and 5% of the oncological ones (9/183). A decreasing trend in the discharge rate per year was observed over time (Table ). Table depicts the distribution of follow-up length over the observation period. In total, 39% of patients (277/716) were followed for > 12 months. This percentage rose to 52% for non-oncological diseases (275/533). Of the remaining 48% of children with non-oncological disease (258/533) who had a follow-up < 12 months, a good proportion (93, 34%) were referred to the hospice in 2022, and, therefore, their follow-up was not concluded. Considering the non-oncological population followed for > 12 months, the median duration of the follow-up was 43 months (95% CI: 37–47 months). Within the oncological population, 91% of children had a follow-up < 12 months (167/183) with a median of 1.8 months (95% CI: 1.4–2.7 months). In a 5-year timeframe, the proportion of patients with ongoing follow-up increased over time (Table ). Remarkably, among the patients still receiving care in 2022, 13% (37/291) and 28% (81/291) were taken in charge more than 10 or more than 5 years earlier, respectively. Given the overall poor coverage on the Italian territory, the PPC service in Italy needs to be further implemented through the establishment of PPC networks . Hence, a proper allocation of resources on the base of an accurate estimation of the actual burden of needs and the characterization of children requiring PPC in Italy is needed . Intending to gather information on the number of referrals and the follow-up length of patients in PPC, we retrospectively analyzed the information of the patients who were referred to the Padua Hospice over a 15-year period. Among the 870 patients who were referred to the Padua Hospice between 2008 and 2022, the majority (76%) were affected by non-oncological conditions. This finding is not unexpected since it is in line with previous reports in the PPC setting and contradicts, at least in part, the common notion that PPC focuses on the ‘care of children with oncological disease at a terminal stage’ . Notably, PPC patients affected by non-oncological conditions showed better survival rates than those with oncological disease (41% vs. 90%) since non-oncological conditions do not cause deaths as much as cancer does. For this reason, their follow-up in PPC was much longer (43 months vs. 1.8 months). Survival rates also increased over time. Indeed, approximately 40% of patients taken in charge are followed for more than 1 year and a remarkable proportion of children (28%) for more than five. Such a long period of follow-up translates into a continuous change in the needs of the patients due to the unpredictable course of some pathologies, the different phases of the condition or disease progression and due to the transition into adulthood . Another expected finding is the short follow-up for patients in PPC with oncological conditions, whose mortality rate was also very high (90%). Since the overall survival rate for children with oncological disease not in PPC is increasing worldwide , it is possible to speculate that, in our setting of care, children with cancer are referred to PPC only at a very late stage of the disease. It is possible to suggest that earlier referral would translate into a better quality of life for the patient and his/her family without any adverse impact on survival . The analysis of the referrals per year shows a steady increase over the study period: in 2022, more than 100 patients were referred to our network, a 195% increase compared to 2008. The same increasing trend could be observed, at least in part, for the number of patients actually taken in charge (91% in 2022). These findings disclose an improvement in the overall implementation of the PPC in the Veneto Region, with higher coverage and more accurate referral of patients to be followed by a specialized service. Indeed, it is worth noting that in recent years, in the Veneto region, training has been given to PPC providers to support the identification of eligible patients and the activation of the PPC network and to educate them for integrated working. This certainly had a good impact on the activity of the network and the quality of the assistance provided. Also, the increased number of patients taken in charge of the Padua Hospice and their increasing survival account for the increasing number of patients followed each year (291 in 2022). It has to be considered that the longer survival of patients observed implies the necessity for the healthcare system to monitor the patients’ needs and dynamically and effectively adapt its response. This is the pathway followed until an eventual discharge if the patient does not require specialist intervention anymore, which in our data occurred in 10% of cases per year. These data highlight the importance of ensuring the enrollment and discharge of patients from specialized PPC by a shared and validated assessment (ACCAPED) of the individual patient’s needs . Furthermore, there is an increase in the number of long-surviving pediatric patients who reach adulthood. Indeed, 16% of patients in charge of the Italian network are over 16 years old . This emerging problem triggers the need for the establishment of an appropriate care model for the transition to adult services after having appropriately evaluated data, needs, resources, and available healthcare models. Currently, there is no determination of the age at which start the transition to adult palliative care . In the UK patients are referred to specialist pediatric care from age 16 to 19 years before transitioning , and the transition is planned to occur around 18 years in the USA . However, older patients with very complex syndromes can still be referred to pediatric palliative care services . Collectively, these findings highlight a major burden for the PPC network in the Veneto Region, also increasing over time (a preliminary analysis of patients followed in 2023, still immature, seems to confirm the overall results). Data collected in a nationwide cross-sectional study also confirm the high number of patients followed by PPC services in Italy . This prompts an adequate allocation of resources and, at the same time, the establishment of dedicated educational curricula for healthcare providers potentially involved in PPC . Moreover, educative and awareness campaigns should be implemented since parents/patients are still afraid of accessing services because they believe that palliative care is treatment aimed only at dying children. In addition, appropriate communication focused on the role of PPCs in providing treatments to manage incurable diseases and on the rights that each child and his/her family have in this area could help address the cultural challenge around PPC services. Although our data were collected retrospectively, the present study can represent the basis for the prospective collection of data that will help keep track of the burden of PPC and monitor the epidemiology of patients. Indeed, further studies are needed to collect data on patients who have been recently taken into charge by PPC services. It could also be helpful to extend this study design to national and international levels to collect comprehensive data on this often-neglected research field and to consolidate PPC networks. Below is the link to the electronic supplementary material. Supplementary Material 1 |
Korean Bureaucrats Underestimate the Medical School Curriculum, Taking Anatomy Education as an Example | e33b81d1-c51c-4a33-8c1f-b6e7065f251b | 11136679 | Anatomy[mh] | No one will dispute the fact that medicine is a science. Therefore, the curriculum of medical education should include teaching principles based on scientific grounds as well as teaching how to treat patients. This principle - basic medical science - is the first gateway for freshmen at medical schools. Courses that teach this principle include anatomy, physiology, biochemistry, microbiology, pathology, and so on. Only when mastering these subjects the next step is to start with clinical medicine that deals with real human patients. This is the outline of medical education. The Korean government unilaterally announced that it would implement a total of 5,000 students each year for five years by rapidly adding 2,000 to the current 3,000 students, starting in 2025. The Korean medical community strongly protested. Under the current infrastructure, it is very natural that a rapid increase will cause great chaos in the medical school education field. For example, as many as 200 students have been assigned to a medical college with a current quota of 50. As classrooms and practice laboratories are geared to just 50 students in that college, it is clear what will happen if there are 200 new students every year. A typical example is the disruption of anatomy cadaver dissection. If there were previously 5 students assigned per cadaver, now 20 students should be assigned per cadaver. Most professors of all medical schools throughout this country have criticized government policy, but the Government remains stubborn. Even a vice minister of Korean Ministry of Health and Welfare refuted this: "Cadavers can be shared with each other in medical schools across the country. If this is not enough, we can also consider importing as some foreign countries do." To the best of my knowledge, I've never heard of a country exporting cadavers in toto . Even if there is, it's probably a felony. The vice minister is not a medical school graduate. Therefore, he is insulting not only the anatomy education but also the people who donated the cadaver because he has no idea about the medical school education and its ethics. In other words, Korean bureaucrats underestimate the medical school education. Let me be clear, the medical school curriculum is not that easy, as you think of it. Speaking of which, let's talk about anatomy. Modern medicine began with the establishment of anatomy. The ultimate goal of all medicine is to treat patients, so grasping the normal body structure of humans is more important and fundamental than any other knowledge. Therefore, anatomy is the first subject that a new medical student meets and must master. The first time I learned anatomy was in 1981. A total of 25 groups of 100 students - 4 students per cadaver were arranged. After we all gave a sincere Mass to those who were willing to donate their bodies to honor their souls, we finally got to meet cadaver. With the disgusting smell of formalin filling the dissection room, various mummified bodies were placed per table. Here and there, our co-eds were shocked, terrified, and were weeping. Some fainted while hyperventilating and were carried away by an assistant. We men pretended not to be terrified because “we are men!” But, indeed, we were also shocked and terrified. I thought 'Ah, this is only one thing I have to do for a year, right?' However, the adaptability of human was so great. It took only a week for our co-eds, who cried and even fainted on the first day, to work most enthusiastically on cadaver dissection and to study Grant's Atlas of Anatomy simultaneously. I didn't know at that time till I reflect it now that I had a really good educational environment. From the standpoint of my own experience, cadaver dissection is not that simple. Learning the structure of the human body seems to be easy to master only if you read and memorize textbooks and atlas, but that is not the case at all. In fact, you must touch the cadaver directly, make an incision, identify the arteries, veins, nerves and organs by comparing them with illustrations in atlas one by one, and make each knowledge implemented to your hands as well as to your hippocampus. These collections of knowledge are established inside each person as a concept of human beings, and act as fundamental knowledge unconsciously when actually treating a patient in the future. By the way, if 20 students are assigned per cadaver, will they be able to study properly? In this issue, the review by Kim et al. timely presents important subjects and data about the current status and anticipation of anatomy education in Korea. They conducted a survey targeting 40 medical colleges nationwide and have shown that the current nationwide student-to-anatomy professor ratio is 24.4 students per professor, and the ratio of students per cadaver is 7.4 (3,246 students/450 cadavers). They anticipated that approximately 20 more anatomy professors and 68 additional cadavers would be required for an increase of 500 students. About 41 more professors and 135 additional cadavers would be needed for an increase of 1,000 students. If the Korean government stubbornly insists on increasing the number of students by 2,000, roughly 82 more professors and 270 additional cadavers would be necessary. No one will think these estimates are feasible, unless one is insane. It is anticipated that anatomy education in Korea, which had been barely able to withstand poor infrastructure, could end up ruined. The real problem is that it is not limited to anatomy education. Anything messy can happen if each medical school has an excess of students beyond their capacities. Medical school education does not mean that students are crammed into a lecture hall and only lectures are given in an infused manner. Do you train doctors who deal with patients' lives through education consisting of only students, professors, and textbooks? Medical schools that are not supported by infrastructure and various resources are nothing more than poor training centers. Until the Flexner report was published in 1910, there were a number of substandard medical schools and doctor training centers in the United States. By undergoing strict self-purification on scientific grounds, America is boasting the world's best quality of medicine today. Why does the Korean government bother to recreate this precedent when this has already happened in the past more than 100 years ago? |
A study on the effect of school and family environments and self-efficacy on health literacy of college students | f52172ae-8357-4c83-9873-2ff5e8ec3877 | 11358104 | Health Literacy[mh] | Health literacy refers to an individual ability to acquire, understand and use health information to maintain and promote their own health . Health literacy, which is a midstream determinant of health, can improve health outcomes, reduce health disparities and promote various health-related behaviors of individuals . College students are in a period of transition to independent living and need to face new challenges, such as academic responsibilities, financial worries and adaptation to new life circumstances . These challenges may be reasons why poor values or limited health literacy were reported among college students in many countries, such as Jordan , Australia , Nepal , and America . Moreover, a systematic review indicated that limited health literacy was a common issue among individuals , which included Chinese college students . Therefore, we must make efforts to enhance the health literacy of college students, improve their physical quality and raise the national health level. Ecological systems theory (EST), which was developed by Bronfenbrenner, is a widely accepted model that explains the influence of social environments on the human experience, including public health . EST subdivides environmental influences into multiple levels (microsystem, mesosystem, exosystem, macrosystem, and choronosystem) reflecting the relative size, immediacy of interaction and degree of formality/informality of the environmental setting . EST proposes that behavior both affects and is affected by the interactions between individuals and their surrounding environments. Family systems theory (FST), which was developed by Bowen, consists of a system of eight interlocking states that describe the inevitable chronic emotional anxiety present in family relationships . FST suggests that a family is an interactive whole system formed by the interaction and mutual influence among family members . The research conducted among 600 high school students in China has shown that family atmosphere can directly or indirectly affect mental health literacy through self-efficacy . In addition, family functioning, which affects the psychological development of family members, is the main reason for family systems to maintain dynamic stability . Olson’s family functioning theory (FFT) states that the effective functioning of a family depends on family closeness . Based on EST, family environment, which was regarded as a microsystem, had the greatest impact on college students. Hence, we have selected family closeness and family conflict to measure the family environment and explore its direct or indirect effects on health literacy. For student populations, school environment is the microsystem that has the greatest impact on them beyond their family environment. Input-environment-output (IEO) theory, developed by Astin, is one of the most frequently used frameworks for understanding the effects of college on a range of outcomes of education . The theory holds that educational attainment (output) is a function of the entering students’ characteristics (input), which influences the students’ interaction with their educational environment (environment) . School environment is one of the educational environments, such as physical environment, which includes courses, hardware facilities and spiritual environment. Evidence has shown that school climate is significantly and positively correlated with mental health literacy among high school students in China . Ecological factors, including campus health education and campus tobacco culture, can directly predict health literacy in black college students in the Southeastern United States . Other educational characteristics, such as year of study and field of study, may also affect the health literacy of college students . Moreover, taking elective courses affects students’ health literacy level . The physical environment, such as health education courses and medical services in universities, may lay a foundation for students to develop health literacy. Therefore, we explore the effect of the school environment on health literacy from the perspective of the physical environment. Social cognitive theory (SCT) holds that learning, functioning and actions result from a dynamic and reciprocal triadic interaction among personal, environmental and behavioral factors. SCT is mainly used to explain the acquisition process of complex human behavior and regard self-efficacy as a central tenet . SCT posits that if individuals have the behavioral capability (knowledge and/or skills) to perform the specific act, their self-efficacy can drive healthy behaviors . This drive can be understood that individuals have self-efficacy to learn health knowledge and/or skills through the interaction with the health information environment. Self-efficacy refers to the perceived ability or belief of individuals to complete specific tasks . It explains individual information-seeking motivation and has a positive effect on maintaining and stimulating health promotion behaviors . Lack of self-efficacy is not conducive to improving health literacy . A study conducted in Germany identified that respondents with better self-efficacy had better health literacy scores within the general population . In addition, studies have also found that self-efficacy plays a partial mediating role between school environment and learning effectiveness in adult learners and may be the mediating variable between patients’ cognition and self-management behavior in China . Furthermore, high school students’ self-efficacy in dealing with psychological problems partially mediates the relationship between school climate and mental health literacy . Consequently, it is imperative to investigate whether the environment can influence health literacy through the mediating role of self-efficacy. Sociodemographic characteristics (gender and age, etc.) and family-related factors (residential area, family income, father’s education level, etc.) were found to be associated with health literacy in other populations [including nurses , Iranian populations , residents in Kingdom of Saudi Arabia , Indonesian adolescents , etc.]. In addition, previous studies on the influencing factors of health literacy among college students mostly focused on these relatively unchangeable factors, such as sociodemographic, school characteristics and family-related factors . However, insufficient research has been conducted on the role of factors that can be intervened on health literacy. EST holds that the most direct and close relationship for individual development is microsystem in ecological model. Therefore, we screened two microsystems—school environment and family environment to explore the relationship with health literacy on the basis of FST, FFT, and IEO. Based on EST and the central role of self-efficacy in SCT, the current study aims to explore how school environment and family environment affect students’ health literacy directly or through self-efficacy. Previous studies have confirmed the direct impact of self-efficacy on health literacy . Nevertheless, whether school and family environments can indirectly affect health literacy through enhancing self-efficacy among college students remains to be explored. This article proposes the following hypotheses: School and family environments can directly affect health literacy. School and family environments can indirectly affect health literacy through self-efficacy.
Participants The present study was an observational cross-sectional study. This study selected college students in one university in Wuhan using a stratified random sampling method. We stratified 43 schools of the university into humanities and social sciences, science and engineering, and medical categories based on their respective disciplines. Then, we randomly selected one or two major college students in each discipline according to disciplinary attributes. And then all students from five majors were selected to participate in the survey. An individual was included in the survey if he or she was willing to participate in it, but was excluded if he or she was unwilling to do it. The staff from teaching offices used WeChat tools to distribute the questionnaire via the Wenjuanxing platform from November to December 2023. Before the investigation, the research objectives were explained to respondents and their informed consent was obtained. Participants were also told that all data would be presented in statistical form, with no disclosure of personal information, to assure the anonymity and confidentiality of the survey. In addition, the sample size was determined to be a minimum of 317 (α = 0.05, 1−β = 0.9, dropout rate, DR = 0.2). A total of 500 questionnaires were distributed, of which 452 were collected in this study. However, a total of 5 invalid data were excluded, resulting in 447 valid samples. Therefore, the sample efficiency was 89.4%. Instruments The self-administered questionnaire was divided into 5 parts. Part 1: Sociodemographic characteristics Part 1 consisted of general personal and family-related characteristics. General personal characteristics include gender, age, living costs (in CNY), time spent browsing health education information online, and disciplines. Family-related characteristics include residential area, average annual household income (in CNY), parents’ educational level, and parents’ occupation type. Part 2: School environment questionnaire SEQ was adapted based on student environment perception questionnaire (SEPQ), which was designed by a research on college student development according to IEO model, to measure students’ satisfaction with the school’s health education environment . SEPQ contains 4 dimensions (course construction, teacher instruction, service support, and facility environment) and 21 items. Service support and facility environment dimensions were merged into hardware resources dimension. Course construction and teacher instruction dimensions were revised to the dimensions of health education courses and teachers. In addition, a total of 6 items (e.g., “the setting of course practice is reasonable”) were excluded and the others were modified (e.g., changing the following “The course can stimulate my interest in learning” to “Health education curriculum can stimulate my interest in learning”, changing the following “The course emphasizes the cultivation of my ability to analyze and solve problems” to “Health education curriculum emphasizes the cultivation of my ability to analyze and solve problems”) according to our research topic. SEQ consists of 15 items covering 3 domains of the school environment: health education courses (5 items), teachers (4 items) and hardware resources (6 items). Items were scored on a 5-point Likert scale ranging from 1 (totally disagree) to 5 (totally agree). The score range of this scale was 15 to 75 points. A higher score entailed that the health education environment was better in the school. SEPQ had good reliability and validity . The Cronbach’s alpha of SEQ was 0.977 in this article. The English language version of SEQ is shown in Additional 1. Part 3: Family environment questionnaire The FEQ was adapted by Chinese researchers on the basis of inventories used in a national survey of families across the family life cycle and family environment scale manual, which was developed by Moss and Wang . The questionnaire contains 10 subscales that evaluate 10 different family social and environmental characteristics. We selected the Family Intimacy Scale (FIS) and the Family Contradiction Scale (FCS) to measure the family environment based on the research topic. We use FIS to measure the degree of mutual commitment, help and support (such as “Family members always sincerely support each other”, “There is a harmonious and consistent atmosphere in our home”), and FCS to measure the degree of public expression of anger, aggression and conflict (such as “We often argue in our home”, “Family members often blame and criticize each other”) among family members. Items were scored by yes (1 point) and no (2 points) ranging from 12 to 30 points. A higher FIS score resulted in a lower FCS score and entailed a better family environment. The Cronbach alphas of the FIS and FCS were 0.829 and 0.726, respectively, in this article. Part 4: General self-efficacy questionnaire The GSEQ was developed by Wang et al. on the basis of Schwarzer’s General Self-efficacy Scale . The questionnaire, which contains 10 items, is used to measure self-confidence in solving problems (such as “I am able to solve most problems on my own”), facing difficulties (such as “I can rely on my own abilities in difficult situations”), achieving goals (such as “It is easy for me to stick to my aims and accomplish my goals”), etc. Items were scored on a 4-point Likert scale ranging from 1 (totally incorrect) to 4 (totally correct). The score range of this scale was 10 to 40 points. A higher score entailed better self-efficacy. The Chinese version of the scale had good reliability and validity and was widely used . The Cronbach’s alpha of the scale was 0.955 in this article. Part 5: Health literacy questionnaire This questionnaire was developed by the Asian Health Literacy Research Association and has been validated among residents in six Asian countries . It covers 3 dimensions of health care (such as “Can you search for disease treatment information related to you?”), disease prevention (such as “Can you identify which vaccines you may need to receive?”) and health promotion (such as “Do you want to participate in sports or go to the gym for exercise?”) with a total of 12 items. Items were scored on a 4-point Likert scale ranging from 1 (very difficult) to 4 (very easy) with a score range of 12 to 48 points. A higher score entailed a higher level of health literacy. The Cronbach’s alpha of the scale was 0.932 in this article. Data analysis The SPSS and PASS (version 21.0) software were used for statistical analysis and calculating the sample size. Quantitative data were expressed as mean (standard deviation, SE) or median (interquartile range, IQR). Qualitative data were expressed as frequency and percentage. Categorical variables were compared with chi-square tests and continuous variables with Student’s t -tests, or analyses of variance, or nonparametric tests. In addition, the Pearson or Spearman correlation coefficient was applied to describe the correlation between continuous variables. The mechanisms of school and family environments and self-efficacy on health literacy were explored using linear regression analysis and the nonparametric percentile Bootstrap method of deviation correction in the PROCESS plug-in model with 5,000 repeated samplings. If 0 was not included in the 95% confidence interval, then the mediating effect was significant . Due to the fact that the scores of SEQ, FEQ, GSEQ, and HLQ did not follow a normal distribution, median (IQR) and Spearman correlation were used in the descriptive and correlation analyses. In addition, Student’s t tests or analyses of variance were employed to test the difference in health literacy, because the scores of different groups were in accordance with a normal distribution. Moreover, the assumptions of the linear regression analysis were investigated before performing the analysis. Variance inflation factor (VIF) was used to examine the multicollinearity of the regression analysis. Durbin-Watson (DW) autocorrelation statistic was generated to identify models with serial autocorrelation. A value of VIF higher than 5 was considered to have the multicollinearity . A value of DW between 1.5 and 2.5 was regarded to exhibit no autocorrelation . p < 0.05 indicated a statistically significant difference. In this study, the values of VIF (1.016–2.741) and DW (1.942–2.038) were both in the allowable range.
The present study was an observational cross-sectional study. This study selected college students in one university in Wuhan using a stratified random sampling method. We stratified 43 schools of the university into humanities and social sciences, science and engineering, and medical categories based on their respective disciplines. Then, we randomly selected one or two major college students in each discipline according to disciplinary attributes. And then all students from five majors were selected to participate in the survey. An individual was included in the survey if he or she was willing to participate in it, but was excluded if he or she was unwilling to do it. The staff from teaching offices used WeChat tools to distribute the questionnaire via the Wenjuanxing platform from November to December 2023. Before the investigation, the research objectives were explained to respondents and their informed consent was obtained. Participants were also told that all data would be presented in statistical form, with no disclosure of personal information, to assure the anonymity and confidentiality of the survey. In addition, the sample size was determined to be a minimum of 317 (α = 0.05, 1−β = 0.9, dropout rate, DR = 0.2). A total of 500 questionnaires were distributed, of which 452 were collected in this study. However, a total of 5 invalid data were excluded, resulting in 447 valid samples. Therefore, the sample efficiency was 89.4%.
The self-administered questionnaire was divided into 5 parts. Part 1: Sociodemographic characteristics Part 1 consisted of general personal and family-related characteristics. General personal characteristics include gender, age, living costs (in CNY), time spent browsing health education information online, and disciplines. Family-related characteristics include residential area, average annual household income (in CNY), parents’ educational level, and parents’ occupation type. Part 2: School environment questionnaire SEQ was adapted based on student environment perception questionnaire (SEPQ), which was designed by a research on college student development according to IEO model, to measure students’ satisfaction with the school’s health education environment . SEPQ contains 4 dimensions (course construction, teacher instruction, service support, and facility environment) and 21 items. Service support and facility environment dimensions were merged into hardware resources dimension. Course construction and teacher instruction dimensions were revised to the dimensions of health education courses and teachers. In addition, a total of 6 items (e.g., “the setting of course practice is reasonable”) were excluded and the others were modified (e.g., changing the following “The course can stimulate my interest in learning” to “Health education curriculum can stimulate my interest in learning”, changing the following “The course emphasizes the cultivation of my ability to analyze and solve problems” to “Health education curriculum emphasizes the cultivation of my ability to analyze and solve problems”) according to our research topic. SEQ consists of 15 items covering 3 domains of the school environment: health education courses (5 items), teachers (4 items) and hardware resources (6 items). Items were scored on a 5-point Likert scale ranging from 1 (totally disagree) to 5 (totally agree). The score range of this scale was 15 to 75 points. A higher score entailed that the health education environment was better in the school. SEPQ had good reliability and validity . The Cronbach’s alpha of SEQ was 0.977 in this article. The English language version of SEQ is shown in Additional 1. Part 3: Family environment questionnaire The FEQ was adapted by Chinese researchers on the basis of inventories used in a national survey of families across the family life cycle and family environment scale manual, which was developed by Moss and Wang . The questionnaire contains 10 subscales that evaluate 10 different family social and environmental characteristics. We selected the Family Intimacy Scale (FIS) and the Family Contradiction Scale (FCS) to measure the family environment based on the research topic. We use FIS to measure the degree of mutual commitment, help and support (such as “Family members always sincerely support each other”, “There is a harmonious and consistent atmosphere in our home”), and FCS to measure the degree of public expression of anger, aggression and conflict (such as “We often argue in our home”, “Family members often blame and criticize each other”) among family members. Items were scored by yes (1 point) and no (2 points) ranging from 12 to 30 points. A higher FIS score resulted in a lower FCS score and entailed a better family environment. The Cronbach alphas of the FIS and FCS were 0.829 and 0.726, respectively, in this article. Part 4: General self-efficacy questionnaire The GSEQ was developed by Wang et al. on the basis of Schwarzer’s General Self-efficacy Scale . The questionnaire, which contains 10 items, is used to measure self-confidence in solving problems (such as “I am able to solve most problems on my own”), facing difficulties (such as “I can rely on my own abilities in difficult situations”), achieving goals (such as “It is easy for me to stick to my aims and accomplish my goals”), etc. Items were scored on a 4-point Likert scale ranging from 1 (totally incorrect) to 4 (totally correct). The score range of this scale was 10 to 40 points. A higher score entailed better self-efficacy. The Chinese version of the scale had good reliability and validity and was widely used . The Cronbach’s alpha of the scale was 0.955 in this article. Part 5: Health literacy questionnaire This questionnaire was developed by the Asian Health Literacy Research Association and has been validated among residents in six Asian countries . It covers 3 dimensions of health care (such as “Can you search for disease treatment information related to you?”), disease prevention (such as “Can you identify which vaccines you may need to receive?”) and health promotion (such as “Do you want to participate in sports or go to the gym for exercise?”) with a total of 12 items. Items were scored on a 4-point Likert scale ranging from 1 (very difficult) to 4 (very easy) with a score range of 12 to 48 points. A higher score entailed a higher level of health literacy. The Cronbach’s alpha of the scale was 0.932 in this article.
Part 1 consisted of general personal and family-related characteristics. General personal characteristics include gender, age, living costs (in CNY), time spent browsing health education information online, and disciplines. Family-related characteristics include residential area, average annual household income (in CNY), parents’ educational level, and parents’ occupation type.
SEQ was adapted based on student environment perception questionnaire (SEPQ), which was designed by a research on college student development according to IEO model, to measure students’ satisfaction with the school’s health education environment . SEPQ contains 4 dimensions (course construction, teacher instruction, service support, and facility environment) and 21 items. Service support and facility environment dimensions were merged into hardware resources dimension. Course construction and teacher instruction dimensions were revised to the dimensions of health education courses and teachers. In addition, a total of 6 items (e.g., “the setting of course practice is reasonable”) were excluded and the others were modified (e.g., changing the following “The course can stimulate my interest in learning” to “Health education curriculum can stimulate my interest in learning”, changing the following “The course emphasizes the cultivation of my ability to analyze and solve problems” to “Health education curriculum emphasizes the cultivation of my ability to analyze and solve problems”) according to our research topic. SEQ consists of 15 items covering 3 domains of the school environment: health education courses (5 items), teachers (4 items) and hardware resources (6 items). Items were scored on a 5-point Likert scale ranging from 1 (totally disagree) to 5 (totally agree). The score range of this scale was 15 to 75 points. A higher score entailed that the health education environment was better in the school. SEPQ had good reliability and validity . The Cronbach’s alpha of SEQ was 0.977 in this article. The English language version of SEQ is shown in Additional 1.
The FEQ was adapted by Chinese researchers on the basis of inventories used in a national survey of families across the family life cycle and family environment scale manual, which was developed by Moss and Wang . The questionnaire contains 10 subscales that evaluate 10 different family social and environmental characteristics. We selected the Family Intimacy Scale (FIS) and the Family Contradiction Scale (FCS) to measure the family environment based on the research topic. We use FIS to measure the degree of mutual commitment, help and support (such as “Family members always sincerely support each other”, “There is a harmonious and consistent atmosphere in our home”), and FCS to measure the degree of public expression of anger, aggression and conflict (such as “We often argue in our home”, “Family members often blame and criticize each other”) among family members. Items were scored by yes (1 point) and no (2 points) ranging from 12 to 30 points. A higher FIS score resulted in a lower FCS score and entailed a better family environment. The Cronbach alphas of the FIS and FCS were 0.829 and 0.726, respectively, in this article.
The GSEQ was developed by Wang et al. on the basis of Schwarzer’s General Self-efficacy Scale . The questionnaire, which contains 10 items, is used to measure self-confidence in solving problems (such as “I am able to solve most problems on my own”), facing difficulties (such as “I can rely on my own abilities in difficult situations”), achieving goals (such as “It is easy for me to stick to my aims and accomplish my goals”), etc. Items were scored on a 4-point Likert scale ranging from 1 (totally incorrect) to 4 (totally correct). The score range of this scale was 10 to 40 points. A higher score entailed better self-efficacy. The Chinese version of the scale had good reliability and validity and was widely used . The Cronbach’s alpha of the scale was 0.955 in this article.
This questionnaire was developed by the Asian Health Literacy Research Association and has been validated among residents in six Asian countries . It covers 3 dimensions of health care (such as “Can you search for disease treatment information related to you?”), disease prevention (such as “Can you identify which vaccines you may need to receive?”) and health promotion (such as “Do you want to participate in sports or go to the gym for exercise?”) with a total of 12 items. Items were scored on a 4-point Likert scale ranging from 1 (very difficult) to 4 (very easy) with a score range of 12 to 48 points. A higher score entailed a higher level of health literacy. The Cronbach’s alpha of the scale was 0.932 in this article.
The SPSS and PASS (version 21.0) software were used for statistical analysis and calculating the sample size. Quantitative data were expressed as mean (standard deviation, SE) or median (interquartile range, IQR). Qualitative data were expressed as frequency and percentage. Categorical variables were compared with chi-square tests and continuous variables with Student’s t -tests, or analyses of variance, or nonparametric tests. In addition, the Pearson or Spearman correlation coefficient was applied to describe the correlation between continuous variables. The mechanisms of school and family environments and self-efficacy on health literacy were explored using linear regression analysis and the nonparametric percentile Bootstrap method of deviation correction in the PROCESS plug-in model with 5,000 repeated samplings. If 0 was not included in the 95% confidence interval, then the mediating effect was significant . Due to the fact that the scores of SEQ, FEQ, GSEQ, and HLQ did not follow a normal distribution, median (IQR) and Spearman correlation were used in the descriptive and correlation analyses. In addition, Student’s t tests or analyses of variance were employed to test the difference in health literacy, because the scores of different groups were in accordance with a normal distribution. Moreover, the assumptions of the linear regression analysis were investigated before performing the analysis. Variance inflation factor (VIF) was used to examine the multicollinearity of the regression analysis. Durbin-Watson (DW) autocorrelation statistic was generated to identify models with serial autocorrelation. A value of VIF higher than 5 was considered to have the multicollinearity . A value of DW between 1.5 and 2.5 was regarded to exhibit no autocorrelation . p < 0.05 indicated a statistically significant difference. In this study, the values of VIF (1.016–2.741) and DW (1.942–2.038) were both in the allowable range.
Subject characteristics presents the different characteristics of the included students. Regarding the personal characteristics, of the 447 participants, the average age was 18.92 years (SD = 1.13) with 55.93% males and 44.07% females. Most college students spent less than 1 hour browsing health literacy information online ( N = 309). Nearly half of the students were in their freshman year (213, 47.65%) and the majority of the students’ disciplines were science and engineering (276, 61.74%). In family-related characteristics, students living in a county-level city and with an average annual household income of ¥50,000 to ¥100,000 accounted for 27.07 and 35.57%, respectively. Their parents’ education levels were similar and most of them were in junior high school. Compared with fathers’ occupational types, the proportion of mothers who were unemployed, semi-unemployed, or agricultural workers was higher. Analysis of differences in health literacy according to sociodemographic factors also presents the results of univariate analysis on students’ health literacy. Among these factors, living costs ( p = 0.011), residential area ( p = 0.003), annual household income ( p = 0.001), and parents’ education level (fathers: p = 0.001; mothers: p = 0.01) and occupation type (fathers: p < 0.001; mothers: p = 0.044) had close correlations with health literacy. However, no differences existed in grade, disciplines, and online browsing health education information. Meanwhile, compared to females, males got a higher score in health literacy (T = 2.684, p = 0.008). Relationship among school environment, family environment, self-efficacy, and health literacy The median (IQR) scores of SEQ, FEQ, GSEQ, and HLQ were 16(4), 36(6), 28(8), and 60(10), respectively. In terms of the correlation with health literacy, positive relationships were found among school environment, family environment and self-efficacy ( r s = 0.469, 0.271 and 0.531, respectively). Results have also revealed that self-efficacy had positive correlations with school environment and family environment ( r s = 0.344 and 0.225, respectively). Moreover, a correlation existed between school environment and family environment ( r s = 0.255). p values were all <0.001 in Spearman’s tests. Direct and indirect effects of school and family environments on health literacy Several factors, such as gender, living costs and average annual household income, which might affect health literacy or self-efficacy, were regarded as control variables in the stepwise regression analysis. In addition, males were used as the reference group in the gender variable. In model 1, self-efficacy had a direct positive effect on health literacy ( β = 0.489, p < 0.001). In model 2, school environment had a direct positive effect on self-efficacy ( β = 0.197, p < 0.001). In model 3, family environment had a direct positive effect on self-efficacy ( β = 0.282, p < 0.01). In model 4, school environment had a direct positive effect on health literacy ( β = 0.235, p < 0.001). In model 5, family environment had a direct positive effect on health literacy ( β = 0.323, p < 0.001). In model 6, school environment could impact health literacy through self-efficacy ( β = 0.395, p < 0.001). In model 7, family environment could impact health literacy through self-efficacy ( β = 0.475, p < 0.001). In model 8, school and family environments could impact health literacy through self-efficacy ( β = 0.393, p < 0.001). The specific data are shown in . shows that the mediating effect size of self-efficacy between school environment and health literacy was 0.078 (95% CI = [0.119, 0.195]). shows that the mediating effect size of self-efficacy between family environment and health literacy was 0.134 (95% CI = [0.048, 0.236]). A mediating model path diagram was shown in based on the mediating role of self-efficacy between school and family environments and health literacy.
presents the different characteristics of the included students. Regarding the personal characteristics, of the 447 participants, the average age was 18.92 years (SD = 1.13) with 55.93% males and 44.07% females. Most college students spent less than 1 hour browsing health literacy information online ( N = 309). Nearly half of the students were in their freshman year (213, 47.65%) and the majority of the students’ disciplines were science and engineering (276, 61.74%). In family-related characteristics, students living in a county-level city and with an average annual household income of ¥50,000 to ¥100,000 accounted for 27.07 and 35.57%, respectively. Their parents’ education levels were similar and most of them were in junior high school. Compared with fathers’ occupational types, the proportion of mothers who were unemployed, semi-unemployed, or agricultural workers was higher.
also presents the results of univariate analysis on students’ health literacy. Among these factors, living costs ( p = 0.011), residential area ( p = 0.003), annual household income ( p = 0.001), and parents’ education level (fathers: p = 0.001; mothers: p = 0.01) and occupation type (fathers: p < 0.001; mothers: p = 0.044) had close correlations with health literacy. However, no differences existed in grade, disciplines, and online browsing health education information. Meanwhile, compared to females, males got a higher score in health literacy (T = 2.684, p = 0.008).
The median (IQR) scores of SEQ, FEQ, GSEQ, and HLQ were 16(4), 36(6), 28(8), and 60(10), respectively. In terms of the correlation with health literacy, positive relationships were found among school environment, family environment and self-efficacy ( r s = 0.469, 0.271 and 0.531, respectively). Results have also revealed that self-efficacy had positive correlations with school environment and family environment ( r s = 0.344 and 0.225, respectively). Moreover, a correlation existed between school environment and family environment ( r s = 0.255). p values were all <0.001 in Spearman’s tests.
Several factors, such as gender, living costs and average annual household income, which might affect health literacy or self-efficacy, were regarded as control variables in the stepwise regression analysis. In addition, males were used as the reference group in the gender variable. In model 1, self-efficacy had a direct positive effect on health literacy ( β = 0.489, p < 0.001). In model 2, school environment had a direct positive effect on self-efficacy ( β = 0.197, p < 0.001). In model 3, family environment had a direct positive effect on self-efficacy ( β = 0.282, p < 0.01). In model 4, school environment had a direct positive effect on health literacy ( β = 0.235, p < 0.001). In model 5, family environment had a direct positive effect on health literacy ( β = 0.323, p < 0.001). In model 6, school environment could impact health literacy through self-efficacy ( β = 0.395, p < 0.001). In model 7, family environment could impact health literacy through self-efficacy ( β = 0.475, p < 0.001). In model 8, school and family environments could impact health literacy through self-efficacy ( β = 0.393, p < 0.001). The specific data are shown in . shows that the mediating effect size of self-efficacy between school environment and health literacy was 0.078 (95% CI = [0.119, 0.195]). shows that the mediating effect size of self-efficacy between family environment and health literacy was 0.134 (95% CI = [0.048, 0.236]). A mediating model path diagram was shown in based on the mediating role of self-efficacy between school and family environments and health literacy.
This study revealed that socioeconomic and family-related factors such as low living costs and annual household income, living in rural areas, and poor parents’ education level and occupation type were positively associated with low health literacy. After controlling these characteristics, school and family environments can also significantly predict health literacy. In addition, self-efficacy can mediate the effects of school and family environments on healthy literacy. Socioeconomic status is a well-established social determinant of health . The association of socioeconomic-related characteristics with health literacy was consistent with previous studies. A cross-sectional study in Chinese residents found that education level, occupation and income were identified as factors with the strongest contribution to inadequate or problematic health literacy . A multicenter study among subjects aged ≥15 years in European countries showed that low health literacy is associated with a lower level of education and low socioeconomic status . A cross-sectional study conducted among 903 residents in Kingdom of Saudi Arabia found that participants with adequate health literacy had master’s and PhD degree and an income >30 thousand riyals . Three cross-sectional studies conducted among college students in China showed that place of origin, annual family income and parental educational levels were positively associated with health literacy . The authors concluded that, whether in general population or college students, factors (such as residential area and parents’ occupation type), which were closely related to socioeconomic status, were important factors affecting health literacy. EST theory suggests that behavior is influenced by the interactions between individuals and environments. A qualitative study on the influencing factors of residents’ health literacy based on social ecology model confirmed that health literacy was affected by factors such as family member relationships, whether institutions promote health education propaganda, and community exercise facilities and environments . Two cross-sectional studies performed with adolescents in Brazil found that higher oral health literacy was associated with higher family cohesion scores and lower family adaptability scores . The outcomes were basically in accordance with those of this study with a different health literacy instrument among college students. In addition, soft family environmental factors, especially cohesion, conflict and control, might play an important role in the occurrence of depressive symptoms , which was verified to be associated with health literacy . As stated by FST, a well-balanced family that has harmonious connections among its members might result in increased health literacy. SCT posits that individuals must have self-efficacy and the behavioral capability to perform the specific act. An indicator of health capability is the level to which individuals can acquire, analyze and understand fundamental health information and services in order to make well-informed health choices . A nationally representative household survey including 4,286 adults in Israel found that individuals with a high level of e-health literacy were more likely to access a greater variety of health information . A similar phenomenon was observed when the research participants were college students. Based on a study including 376 participants, it was found that students enrolled in health-related programs who had prior experience in healthcare exhibited higher levels of health literacy . A study conducted with 485 students from several academic fields found that students who relied on family and friends or specialist magazines for obtaining health information exhibited better levels of health literacy . The current study indicated that the scores of SEQ, which encompassed health education courses, medical services, psychological therapy, and other factors, could positively predict the level of health literacy. The rationale behind this may be the assistance they receive from healthcare practitioners and the evaluation of their capacity to access health-related information and engage in communication with healthcare specialists. The mediating effect analysis showed that self-efficacy can play a positive and mediating role in the effect of school and family environments on health literacy. The outcome was consistent with the results of Lei et al., which confirmed that health literacy can regulate health management behavior through self-efficacy among patients with hypertension . Another study on blood pressure control behavior in patients with hypertension also showed that self-efficacy played a mediating role between health literacy and blood pressure control . A study conducted in Germany surveyed 2,000 participants aged 15 years and older indicated a partial mediation effect of self-efficacy on the association between sociodemographic aspects and health literacy . Self-efficacy theory believes that situational conditions, which can provide different information, are one of the main factors affecting self-efficacy . As the final stage before college students enter society, universities play an important role in shaping their health literacy. Health education courses, medical, psychological counseling and other services in universities can lay a foundation for students to develop health literacy. In addition, a close relationship between family members may stimulate students’ self-confidence and enhance their expectations and beliefs about completing tasks. Therefore, conditions in family and school environments can enhance students’ self-efficacy and confidence, and finally improve their health status. In this study, we found that factors related to socioeconomic status such as living costs, residential area, annual household income, and parents’ education level and occupation type had significant impacts on their health literacy. Moreover, the impact of the family environment on college students’ health literacy is greater than that of the school environment. Further analysis also found that the mediating effect of self-efficacy on the relationship between family environment and health literacy is that of the school environment. The reason for this could be that family environment, being the largest microsystem, exerted a more significant influence on students’ health literacy compared to school environment. Our study provided empirical evidence on the correlations among sociodemographic, educational and familial factors, and health literacy. Additionally, we examined the degree to which self-efficacy acted as a significant mediator or moderator in the relationship. Nevertheless, a limited amount of research that examines the role of self-efficacy and environments in determining health literacy still exists. Further quantitative and qualitative research is needed to investigate the complex relationship among an individual self-efficacy, surrounding environments and their level of health literacy. These findings suggested that the government should take measures to raise residents’ income and improve their living conditions. In addition, universities and parents can take several measures, such as offering health education courses, creating a positive family atmosphere and providing learners with understanding and support, to help students build self-confidence, improve self-efficacy and increase their health literacy level.
This study had some limitations. First, sample bias may exist in this work because the sample was limited to a university in Wuhan. Second, the SEQ used in this research was designed from a physical perspective, ignoring the soft environment aspect, which further limited the application of the result. However, some groups were not equivalent, for example, the disciplines (humanities and social sciences) and the year of study (≥ third), which could impact the generalizability of the findings. This could potentially explain why these characteristics do not exhibit a correlation with health literacy. At last, this is a cross-sectional study that cannot establish causal relationships or allow for generalizations to all college students or institutions.
This study confirmed that improving school and family environments could directly or indirectly increase college students’ health literacy through promoting their self-efficacy. Factors related to socioeconomic status had a significant impact on their health literacy. Moreover, other factors that affect students’ health literacy and relationships among self-efficacy, surrounding environments and health literacy may need to be explored in the future.
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Trabeculectomy versus stepwise treatment for breaking the attack of acute primary angle closure in patients with long attack duration: study design and protocol for a multicentre randomised controlled trial (LAAAC) | e332c311-86ce-4353-9ff5-63d73813b01a | 11804192 | Surgical Procedures, Operative[mh] | Previous studies showed Chinese patients with acute primary angle closure (APAC) had longer attack duration and lower success rate in breaking the attack with solely medication or laser therapy. Since timely abortion of the attack of APAC is crucial for preserving vision function, we hypothesise that direct surgery, compared with stepwise treatment, may largely benefit long-attacking patients with APAC in China. The multicentre, randomised controlled design with long follow-up enables this study to provide high-level evidence which may help optimise the current APAC treatment pattern, improve the visual outcome and quality of life of Chinese patients with APAC. Acute primary angle closure (APAC) is a common ophthalmic emergency for Chinese, where intraocular pressure (IOP) rises dramatically (in some cases even over 50 mm Hg). If left untreated, it inevitably leads to glaucomatous optic neuropathy and visual impairment, or even blindness. Studies have shown that the prevalence of APAC in Chinese descendants significantly outruns any other ethnic groups. In mainland China alone, the reported rate of blindness after APAC lies shockingly between 12.54% and 60.77%. Therefore, this blinding emergency causes great concern in China. Our previous work had proposed a critical time window of 4.6 hours for APAC treatment, within which the blindness rate can be controlled under 1%. Attack duration, being the most modifiable risk factor for APAC-related blindness, is crucial. Thus, effective and prompt treatment is of paramount significance to avert poor visual outcomes as it frees the optic nerve from lethal pressure-induced compression. According to guidelines published by developed countries, a stepwise protocol (medication laser surgery) is recommended for managing APAC. On first visit, patients receive medication as initial treatment. Then the treatment will be escalated to laser if IOP remains uncontrolled. Paracentesis may also be implemented. Surgeries are then warranted once the first two steps fail as initial treatment. When developing the Chinese glaucoma guideline, our scholars took this suggestion into consideration and many glaucoma specialists abide by this protocol during clinical practices. However, there has not yet been any robust evidence-based study conducted to back this protocol, neither is there an interventional evaluation of effect. Notably, patients with APAC in China and developed countries differ in disease characteristics. To start with, APAC in mainland China experiences remarkably longer attacks. Results from our previous retrospective study found a mean time from symptom to presentation (TST) of 6.49±9.81 days, which is consistent with other studies regarding the Chinese population, but evidently longer than what was reported in Western countries (mostly within 24 hours, range 2–72 hours). Longer attack duration has been proven to be a risk factor for failed medical or laser therapies. Besides, poorly controlled IOP after initial treatment might play a crucial part in the development of chronic glaucoma, long-term visual impairment and blindness. From our preparatory work (yet to be published), an attack duration of 72 hours distinctively separated satisfying success rate from poor in breaking the attack. Tan et al as well found that a mean presentation within 3 days of symptoms was associated with good visual outcome. Unlike Caucasian subjects with APAC, the effectiveness of medication with or without laser treatment is rather unsatisfying for the Chinese. In light of published research, medication and laser (including laser peripheral iridotomy (LPI) and argon laser peripheral iridoplasty (ALPI)) has an overall success rate of over 70%. While for the Chinese (Asians), the success rate lies between 50% and 60%, with nearly half requiring further surgical intervention. In Aung et al ’s study, approximately 50% of IOP failed to be controlled by medication and laser, with 29.73% (36/111) undergoing trabeculectomy within 1 year. They further argued that early surgical intervention might largely benefit patients with APAC in protecting vision and averting blindness. Though some reported IOP-controlled rate of over 70% in Chinese patients with APAC, heterogeneity exists between this small study sample and the majority of mainland Chinese. In terms of results from our retrospective pilot study, the failure rate of medication in Chinese patients with APAC impended 80%, while the failure rate for ALPI reached beyond 50%. Stepwise protocol for patients with long APAC duration is time-consuming as waiting medication and laser treatment to take effect before planning surgery can take several days. To seize the treatment window and remedy for vision, a more aggressive approach might be the optimal option, which would allow for a safer precondition for ultimate lens extraction. Moreover, despite some achieving temporary remission, the outcome of laser therapy was not that satisfying, and some also believed that a degree of residual angle closure exists even after treatment. Another concerning aspect is that with inadequate initial treatment leading to treatment failure, along with possible poor post-treatment compliance (proven in studies in Hong Kong patients with APAC) resulting in self-terminated follow-up, neglect of IOP increase would be inevitable. Consequently, postponed treatment elevation would exacerbate visual impairment. In China, trabeculectomy is a generally recognised and clinically tested mainstay (which is also advised by the Chinese Glaucoma Guideline), even when the eye is still ‘hot’. According to published studies regarding angle-closure glaucoma in Asians, though trabeculectomy for APAC eye with mean presenting IOP ranging from 30 to 50 mm Hg has more postoperative complications, it is still safe and effective. Complications after trabeculectomy mostly involve shallow anterior chamber (AC), hypotony, choroidal detachment, hyphema, malignant glaucoma and additional surgery. The reported rate of early complications for medically unresponsive angle closure, namely the ‘hot’ eyes, ranged between 23.9% and 33.3%, with shallow AC being the leading cause. Whereas severe complications such as choroidal detachment did not exceed 5% and malignant glaucoma rarely occurred. A substantial amount of researchers are in favour of direct trabeculectomy for APAC as most complications are manageable and can recover with time while benefiting long-term vision preservation. Additionally, the effectiveness of trabeculectomy can be enhanced by cataract surgery combined with goniosynechialysis later when IOP is well-controlled by filtering surgery and phacoemulsification is safe enough. By contrast, some proposed primary lens extraction or primary transscleral cyclophotocoagulation for managing APAC, though lens extraction was mostly studied on medically responsive APAC with controlled IOP, whereas the IOP-lowering efficacy and long-term outcomes of cyclophotocoagulation alone were not satisfying. As far as we know, primary trabeculectomy is rather accessible and practical in clinical settings for the Chinese patients with medically unresponsive APAC. Herein, we propose that for Chinese patients with APAC duration ≥72 hours direct trabeculectomy could be the initial treatment, which possess the potential of improved efficacy in breaking the attack, minimised duration of high IOP and better long-term visual outcomes. To this end, we present a pragmatic, multicentre, randomised controlled trial, comparing direct surgical intervention with stepwise protocol in terms of effectiveness in controlling IOP, short-term and long-term visual function, as well as safety. We aim to provide data and high-level evidence on this matter, possibly develop a new treatment protocol that better fits our patients, and in the meantime bring a better quality of life (QoL). Trial design The Long-Attacking Acute Angle Closure (LAAAC) study is a pragmatic, multicentre, randomised controlled trial, comparing the effectiveness and safety of direct trabeculectomy and stepwise protocol in treating Chinese patients with APAC with symptom duration ≥72 hours. Led by Beijing Tongren Hospital, investigations will be conducted at nine clinical centres in China: Shijiazhuang People’s Hospital, Handan Eye Hospital, the Fourth People’s Hospital of Shenyang, the Fourth Affiliated Hospital of China Medical University, Fushun Eye Hospital, Anyang Eye Hospital, Hebei Eye Hospital and Shanxi Aier Eye Hospital. Primary outcome will be assessed at 1 year after initial treatment. Participants and eligibility Inclusion criteria Conformation of the diagnosis of APAC : (a) presence of at least two of the following symptoms: ocular or periocular pain, nausea and/or vomiting, an antecedent history of intermittent blurring of vision with halos; (b) presenting IOP of ≥30 mm Hg (measured by non-contact tonometry (NCT), measuring range 0–60 mm Hg); (c) presence of at least one of three of the following signs: conjunctival injection, corneal epithelial oedema and mid-dilated unreactive pupil; and (d) presence of shallow AC with slit-lamp exam (defined as less than 1/4 corneal thickness) in both eyes and a fellow unaffected eye with the presence of 180° or more of iridotrabecular contact with or without peripheral anterior synechia on gonioscopy. When the cornea is oedematous owing to the attack, features of APAC in the fellow eye should be examined first under gonioscopy. Gonioscopy for the affected eye should be done as soon as the cornea restore transparency to the confirmed closed angle. TST≥72 hours. Patients with written informed consent. Exclusion criteria Secondary angle closure, such as neovascular glaucoma, uveitic glaucoma and glaucoma secondary to trauma or surgeries. Pre-existing blindness (defined according to the WHO criteria as BCVA below 3/60 in the better eye) or history of any diseases that cause severe visual impairment prior to the attack. History of APAC or symptoms indicating a previous attack. Any history of previous intraocular surgery, laser procedure, paracentesis, usage of antiglaucoma medication or topiramate. TST<72 hours. Patients with severe or malignant diseases with life expectancy less than the follow-up period. Patients who plan to move out of the area, thus leading to attrition. Patients participating in other clinical trials. Patients with contraindications of antiglaucoma therapies. Recruitment procedure Potential eligible patients will be identified at the first medical visit for APAC at either the emergency department or glaucoma outpatient clinics. Recruitment will be performed by ophthalmologists. After being assessed for eligibility and informed consent is given, basic medical treatment will start immediately. Those who declined participation will be asked for a reason for refusal. In the meantime, they will be randomised for subsequent treatment accordingly . If the first APAC consultation were at the emergency departments, patients will be referred to glaucoma clinic at the latest in time (defined as the baseline visit) at the same centre for further evaluation and start preparation for surgery if applicable. On the other hand, if the first APAC consultation were at the glaucoma clinics, all baseline information would be acquired during this visit. Interventions On first medical consultation, medical treatment will be initiated immediately regardless of randomisation. Medical therapies include IOP-lowering eye drops, as well as oral acetazolamide and intravenous mannitol if the presenting IOP exceeds 40 mm Hg. Patients will be monitored for 2–6 hours (based on the clinicians’ judgement) to determine the effectiveness of treatment. IOP>21 mm Hg indicates an escalation of treatment. All interventions will be performed following a Standard Operation Procedure (SOP) among centres. For those who remain unresponsive to medical or laser treatment while awaiting surgery, the performance of paracentesis can be done based on clinicians’ judgement. Stepped treatment After initial medical treatment, patients with uncontrolled IOP in the stepped treatment group will go on to receive laser therapies (LPI or ALPI depending on the mechanism of angle closure and corneal status). When laser therapy is unavailable, AC paracentesis can be performed instead to temporarily reduce IOP, and then the patients will go on to receive laser treatment as soon as there is access. IOP will be measured at 1 hour, 2 hours and 24 hours after the completion of the procedure. IOP over 21 mm Hg after observation for 24 hours warrants surgery (trabeculectomy). Besides, achieving short-term abortion of the attack yet elevation of IOP>21 mm Hg seen during follow-up visits (with maximum medication) is also an indication for trabeculectomy. Trabeculectomy Trabeculectomy will be performed by experienced glaucoma specialists. Preoperatively, levofloxacin eye drops will be administrated. Mitomycin C will be applied under the scleral flap and subconjunctival space for no more than 5 min (judgement based on the patients’ age and appearance of Tenon’s capsule) during the procedure to augment surgical results as a major reason for the failure of trabeculectomy was found to be bleb fibrosis. Releasable sutures are used for better postoperative management. Tobramycin dexamethasone in the form of both drops and ointments will be routinely prescribed for 4 weeks as postoperative management. Subconjunctival or periocular injection of dexamethasone would be administered if a patient presents with postoperative hypotony or signs indicating choroidal detachment. For those exhibiting signs highly suggestive of malignant glaucoma, additional cycloplegic drops can be added according to clinicians’ judgement. Subsequent management During postoperative monitoring, IOP≥21 mm Hg on two consecutive visits warrants additional intervention. This starts with filtration bleb manipulation, including suture lysis, bleb massage, needling or injections of antimetabolites. If bleb management fails to control IOP adequately, antiglaucoma eye drops and oral carbonic anhydrase inhibitors can be added. Cataract surgery For all patients with IOP lower than 21 mm Hg after initial treatment, the combination of phacoemulsification, intraocular lens implantation and goniosynechialysis will be performed at least 1 month after the initial treatment of medication, laser or trabeculectomy. Ethics and dissemination All participating centres received approval from their respective institutional review board and ethics committee. The study was registered with the Chinese Clinical Trial Registry (ChiCTR2200057289), complied with the Health Insurance Portability and Accountability Act regulations and adhered to the tenets of the Declaration of Helsinki. Follow-up visits All eyes will be examined on the first medical visit at each centre, on the day of initial treatment and 1 day, 1 week, 1 month, 3 months, 6 months and 12 months following the initial treatment. The information required at each follow-up visit is demonstrated in . Study endpoints For those completing a 1-year follow-up, the study reaches its endpoint at 1 year after the initial treatment. Participants automatically withdraw from the study when: Missing three consecutive follow-ups or more. With poor treatment compliance. Death or suffering from newly diagnosed systemic diseases impeding regular follow-up. Study endpoint is defined as the last follow-up before withdrawal. However, if any patient violates treatment protocol or lacks essential information which hinders the judgement of efficacy and safety of the trial, they will be excluded from final data analysis. Outcome measurements and data collection Outcomes and adverse events will be recorded on a trial-specific case report form. Demographic information, along with medical history of each recruited participant, will be documented at baseline. Extensive ophthalmological examinations of BCVA, IOP and slit-lamp examination will be performed as well if the patients are first presented at the emergency department. Subsequent examination will be completed after referral at glaucoma clinics including gonioscopy, ultrasound biomicroscopy, anterior segment photography, fundus photography, Humphrey visual field (VF), a visual quality questionnaire, optical coherence tomography, specular microscopy and IOL master. Of note, we attach great significance to recording a precise TST (hours), time from presentation to the subsidence of the attack and (if the cornea is oedematous due to the attack) time from symptoms to cornea restoring transparency will also be recorded. During follow-up, we intend to document any related adverse events, poorly controlled IOP needing treatment upgrade and cataract surgeries. Primary outcomes The primary outcome of this study is BCVA measured using the Early Treatment Diabetic Retinopathy Study (ETDRS) logMAR E visual acuity (VA) charts 1 year post initial treatment. Notably, resolved attack or controlled IOP do not completely equal good visual outcome, considering that patients’ attack duration in different groups may considerably vary due to waiting treatment elevation. Also, since VA most directly reflects one’s visual function and concerns patients with glaucoma the most, it would be the most appropriate option for the primary outcome of this study. ETDRS VA is designed to inspect visual impairment in clinical trials. If no letter could be read on the chart, vision of finger count, hand movement, light perception and no light perception will be recorded accordingly. VA examiners are all verified ophthalmologists, have undergone unified training and masked from treatment assignment. Secondary outcomes Secondary outcomes consist of complete success rate in breaking the attack, IOP, mean deviation on Humphrey VF testing and vision-related QoL collected from the National Eye Institute Visual Function Questionnaire (25 items) (NEI-VFQ25) 1 year post initial treatment. Complete success in breaking the attack is defined as IOP<21 mm Hg without the aid of IOP-lowering medication. If IOP can only be controlled with the assistance of medication, the success should be defined as partial rather than complete. An NCT will be employed for the measurement of IOP, taking several facts into consideration since many patients with APAC are present in an emergency context and that not all centres allow access to Goldmann tonometry. Three measurements will be taken for each eye by a masked examiner, and the mean values will be used. VF testing will be performed on Humphrey 24-2 automated perimetry. Reliable VF results are defined as fixation loss<20%, with both positive and negative false <33%. To further evaluate visual outcomes in patients with APAC, we adopted the NEI-VFQ25 questionnaire, which is a vision-specific health profile measure. Since the original questionnaire was designed for English speakers, our study adopted the Chinese version, which had been tested for good reliability and validity. Ancillary measurements The data presented in will also be recorded. Box 1 Ancillary information and examinations required Time from syndrome to presentation, time from presentation to the subsidence of the attack and time from symptoms to cornea restoring transparency. Number and types of medication needed after laser or surgery. Further lens extraction surgery or other antiglaucoma surgeries during the follow-up period. Gonioscopy. Ultrasound biomicroscopy. Fundus photography. Specular microscopy. Optical coherence tomography. Other examinations required for the preparation of surgeries. Any ancillary examinations that demand direct contact with the cornea will only be performed when the IOP is maximally controlled and when corneal oedema has resolved so that safety is guaranteed. Safety and adverse events We intend to report all serious adverse events, along with complications and unexpected events from each step of intervention. illustrates the expected adverse events and complications related to glaucoma care in this trial. If any, all will be extracted, kept detailed records and reported to the Ethics Committee and the Trial Steering Committee in regular progress reports. Sample size and statistical methods Sample size has been calculated based on the primary outcome (BCVA) using SAS OnDemand for Academics (SAS Institute, Cary, North Carolina, USA). The power was set at 80% and the significance level was 5%, considering an attrition rate of 10%. In order to detect a minimum difference of 5 ETDRS letters (logMAR=0.1, SD approximately 0.2) 1 year after initial treatment, a total of 142 patients (71 per group) is required. Analysis of outcomes will be performed at the end of the trial on an intention-to-treat basis. After testing for data distribution of normality, demographic information, primary and secondary outcomes will be presented and compared between treatment groups accordingly. Moreover, mixed models will be applied for the investigation of outcomes changes over follow-up time. A cost-effective estimation will also be performed to determine the health economics results of two treatment arms. A p value of <0.05 will be considered statistically significant. Randomisation and blinding A remote-automated computer central randomisation service provided by Depa EDC will be used for randomised sequence generation, which guarantees full allocation concealment. Minimisation will be used for treatment allocation to achieve optimal balance between groups. In this regard, two intervention groups will be balanced simultaneously over five stratification factors: (1) centre, (2) age (<60 years, ≥60 years), (3) IOP at presentation (30–45 mm Hg, ≥45 mm Hg including IOP values that exceed NCT examination threshold), (4) education level (middle school and high school or lower, higher than high school) and (5) TST (72–168 hours, ≥168 hours). The service can be easily accessed through a WeChat-based mini program. Patients will be assigned randomly (at a 1:1 ratio) following routine medication. Randomisation unit of this trial is the participant rather than the eye. In light of the nature of surgical intervention, neither the clinical staff nor the participants can be blinded with respect to the intervention. However, outcomes, including BCVA, IOP, VF, NEI-VFQ25 and other ancillary examinations, will be done separately by examiners masked to randomisation. Quality control Researchers participating in this clinical research must be qualified, with professional background and ability to conduct clinical research. All examinations, diagnosis and interventions will be carried out strictly abiding by the SOP. Before the study launches, all centres are visited and convened for training. The steering committee oversees the whole research and formulates study strategies. The safety and data monitoring committee will monitor safety and other data at six regular intervals during the recruitment phase of the trial. Interim analysis will be conducted 6 months after the initiation of the trial. The Long-Attacking Acute Angle Closure (LAAAC) study is a pragmatic, multicentre, randomised controlled trial, comparing the effectiveness and safety of direct trabeculectomy and stepwise protocol in treating Chinese patients with APAC with symptom duration ≥72 hours. Led by Beijing Tongren Hospital, investigations will be conducted at nine clinical centres in China: Shijiazhuang People’s Hospital, Handan Eye Hospital, the Fourth People’s Hospital of Shenyang, the Fourth Affiliated Hospital of China Medical University, Fushun Eye Hospital, Anyang Eye Hospital, Hebei Eye Hospital and Shanxi Aier Eye Hospital. Primary outcome will be assessed at 1 year after initial treatment. Inclusion criteria Conformation of the diagnosis of APAC : (a) presence of at least two of the following symptoms: ocular or periocular pain, nausea and/or vomiting, an antecedent history of intermittent blurring of vision with halos; (b) presenting IOP of ≥30 mm Hg (measured by non-contact tonometry (NCT), measuring range 0–60 mm Hg); (c) presence of at least one of three of the following signs: conjunctival injection, corneal epithelial oedema and mid-dilated unreactive pupil; and (d) presence of shallow AC with slit-lamp exam (defined as less than 1/4 corneal thickness) in both eyes and a fellow unaffected eye with the presence of 180° or more of iridotrabecular contact with or without peripheral anterior synechia on gonioscopy. When the cornea is oedematous owing to the attack, features of APAC in the fellow eye should be examined first under gonioscopy. Gonioscopy for the affected eye should be done as soon as the cornea restore transparency to the confirmed closed angle. TST≥72 hours. Patients with written informed consent. Exclusion criteria Secondary angle closure, such as neovascular glaucoma, uveitic glaucoma and glaucoma secondary to trauma or surgeries. Pre-existing blindness (defined according to the WHO criteria as BCVA below 3/60 in the better eye) or history of any diseases that cause severe visual impairment prior to the attack. History of APAC or symptoms indicating a previous attack. Any history of previous intraocular surgery, laser procedure, paracentesis, usage of antiglaucoma medication or topiramate. TST<72 hours. Patients with severe or malignant diseases with life expectancy less than the follow-up period. Patients who plan to move out of the area, thus leading to attrition. Patients participating in other clinical trials. Patients with contraindications of antiglaucoma therapies. Conformation of the diagnosis of APAC : (a) presence of at least two of the following symptoms: ocular or periocular pain, nausea and/or vomiting, an antecedent history of intermittent blurring of vision with halos; (b) presenting IOP of ≥30 mm Hg (measured by non-contact tonometry (NCT), measuring range 0–60 mm Hg); (c) presence of at least one of three of the following signs: conjunctival injection, corneal epithelial oedema and mid-dilated unreactive pupil; and (d) presence of shallow AC with slit-lamp exam (defined as less than 1/4 corneal thickness) in both eyes and a fellow unaffected eye with the presence of 180° or more of iridotrabecular contact with or without peripheral anterior synechia on gonioscopy. When the cornea is oedematous owing to the attack, features of APAC in the fellow eye should be examined first under gonioscopy. Gonioscopy for the affected eye should be done as soon as the cornea restore transparency to the confirmed closed angle. TST≥72 hours. Patients with written informed consent. Secondary angle closure, such as neovascular glaucoma, uveitic glaucoma and glaucoma secondary to trauma or surgeries. Pre-existing blindness (defined according to the WHO criteria as BCVA below 3/60 in the better eye) or history of any diseases that cause severe visual impairment prior to the attack. History of APAC or symptoms indicating a previous attack. Any history of previous intraocular surgery, laser procedure, paracentesis, usage of antiglaucoma medication or topiramate. TST<72 hours. Patients with severe or malignant diseases with life expectancy less than the follow-up period. Patients who plan to move out of the area, thus leading to attrition. Patients participating in other clinical trials. Patients with contraindications of antiglaucoma therapies. Potential eligible patients will be identified at the first medical visit for APAC at either the emergency department or glaucoma outpatient clinics. Recruitment will be performed by ophthalmologists. After being assessed for eligibility and informed consent is given, basic medical treatment will start immediately. Those who declined participation will be asked for a reason for refusal. In the meantime, they will be randomised for subsequent treatment accordingly . If the first APAC consultation were at the emergency departments, patients will be referred to glaucoma clinic at the latest in time (defined as the baseline visit) at the same centre for further evaluation and start preparation for surgery if applicable. On the other hand, if the first APAC consultation were at the glaucoma clinics, all baseline information would be acquired during this visit. Interventions On first medical consultation, medical treatment will be initiated immediately regardless of randomisation. Medical therapies include IOP-lowering eye drops, as well as oral acetazolamide and intravenous mannitol if the presenting IOP exceeds 40 mm Hg. Patients will be monitored for 2–6 hours (based on the clinicians’ judgement) to determine the effectiveness of treatment. IOP>21 mm Hg indicates an escalation of treatment. All interventions will be performed following a Standard Operation Procedure (SOP) among centres. For those who remain unresponsive to medical or laser treatment while awaiting surgery, the performance of paracentesis can be done based on clinicians’ judgement. Stepped treatment After initial medical treatment, patients with uncontrolled IOP in the stepped treatment group will go on to receive laser therapies (LPI or ALPI depending on the mechanism of angle closure and corneal status). When laser therapy is unavailable, AC paracentesis can be performed instead to temporarily reduce IOP, and then the patients will go on to receive laser treatment as soon as there is access. IOP will be measured at 1 hour, 2 hours and 24 hours after the completion of the procedure. IOP over 21 mm Hg after observation for 24 hours warrants surgery (trabeculectomy). Besides, achieving short-term abortion of the attack yet elevation of IOP>21 mm Hg seen during follow-up visits (with maximum medication) is also an indication for trabeculectomy. Trabeculectomy Trabeculectomy will be performed by experienced glaucoma specialists. Preoperatively, levofloxacin eye drops will be administrated. Mitomycin C will be applied under the scleral flap and subconjunctival space for no more than 5 min (judgement based on the patients’ age and appearance of Tenon’s capsule) during the procedure to augment surgical results as a major reason for the failure of trabeculectomy was found to be bleb fibrosis. Releasable sutures are used for better postoperative management. Tobramycin dexamethasone in the form of both drops and ointments will be routinely prescribed for 4 weeks as postoperative management. Subconjunctival or periocular injection of dexamethasone would be administered if a patient presents with postoperative hypotony or signs indicating choroidal detachment. For those exhibiting signs highly suggestive of malignant glaucoma, additional cycloplegic drops can be added according to clinicians’ judgement. Subsequent management During postoperative monitoring, IOP≥21 mm Hg on two consecutive visits warrants additional intervention. This starts with filtration bleb manipulation, including suture lysis, bleb massage, needling or injections of antimetabolites. If bleb management fails to control IOP adequately, antiglaucoma eye drops and oral carbonic anhydrase inhibitors can be added. Cataract surgery For all patients with IOP lower than 21 mm Hg after initial treatment, the combination of phacoemulsification, intraocular lens implantation and goniosynechialysis will be performed at least 1 month after the initial treatment of medication, laser or trabeculectomy. On first medical consultation, medical treatment will be initiated immediately regardless of randomisation. Medical therapies include IOP-lowering eye drops, as well as oral acetazolamide and intravenous mannitol if the presenting IOP exceeds 40 mm Hg. Patients will be monitored for 2–6 hours (based on the clinicians’ judgement) to determine the effectiveness of treatment. IOP>21 mm Hg indicates an escalation of treatment. All interventions will be performed following a Standard Operation Procedure (SOP) among centres. For those who remain unresponsive to medical or laser treatment while awaiting surgery, the performance of paracentesis can be done based on clinicians’ judgement. Stepped treatment After initial medical treatment, patients with uncontrolled IOP in the stepped treatment group will go on to receive laser therapies (LPI or ALPI depending on the mechanism of angle closure and corneal status). When laser therapy is unavailable, AC paracentesis can be performed instead to temporarily reduce IOP, and then the patients will go on to receive laser treatment as soon as there is access. IOP will be measured at 1 hour, 2 hours and 24 hours after the completion of the procedure. IOP over 21 mm Hg after observation for 24 hours warrants surgery (trabeculectomy). Besides, achieving short-term abortion of the attack yet elevation of IOP>21 mm Hg seen during follow-up visits (with maximum medication) is also an indication for trabeculectomy. Trabeculectomy Trabeculectomy will be performed by experienced glaucoma specialists. Preoperatively, levofloxacin eye drops will be administrated. Mitomycin C will be applied under the scleral flap and subconjunctival space for no more than 5 min (judgement based on the patients’ age and appearance of Tenon’s capsule) during the procedure to augment surgical results as a major reason for the failure of trabeculectomy was found to be bleb fibrosis. Releasable sutures are used for better postoperative management. Tobramycin dexamethasone in the form of both drops and ointments will be routinely prescribed for 4 weeks as postoperative management. Subconjunctival or periocular injection of dexamethasone would be administered if a patient presents with postoperative hypotony or signs indicating choroidal detachment. For those exhibiting signs highly suggestive of malignant glaucoma, additional cycloplegic drops can be added according to clinicians’ judgement. Subsequent management During postoperative monitoring, IOP≥21 mm Hg on two consecutive visits warrants additional intervention. This starts with filtration bleb manipulation, including suture lysis, bleb massage, needling or injections of antimetabolites. If bleb management fails to control IOP adequately, antiglaucoma eye drops and oral carbonic anhydrase inhibitors can be added. Cataract surgery For all patients with IOP lower than 21 mm Hg after initial treatment, the combination of phacoemulsification, intraocular lens implantation and goniosynechialysis will be performed at least 1 month after the initial treatment of medication, laser or trabeculectomy. After initial medical treatment, patients with uncontrolled IOP in the stepped treatment group will go on to receive laser therapies (LPI or ALPI depending on the mechanism of angle closure and corneal status). When laser therapy is unavailable, AC paracentesis can be performed instead to temporarily reduce IOP, and then the patients will go on to receive laser treatment as soon as there is access. IOP will be measured at 1 hour, 2 hours and 24 hours after the completion of the procedure. IOP over 21 mm Hg after observation for 24 hours warrants surgery (trabeculectomy). Besides, achieving short-term abortion of the attack yet elevation of IOP>21 mm Hg seen during follow-up visits (with maximum medication) is also an indication for trabeculectomy. Trabeculectomy will be performed by experienced glaucoma specialists. Preoperatively, levofloxacin eye drops will be administrated. Mitomycin C will be applied under the scleral flap and subconjunctival space for no more than 5 min (judgement based on the patients’ age and appearance of Tenon’s capsule) during the procedure to augment surgical results as a major reason for the failure of trabeculectomy was found to be bleb fibrosis. Releasable sutures are used for better postoperative management. Tobramycin dexamethasone in the form of both drops and ointments will be routinely prescribed for 4 weeks as postoperative management. Subconjunctival or periocular injection of dexamethasone would be administered if a patient presents with postoperative hypotony or signs indicating choroidal detachment. For those exhibiting signs highly suggestive of malignant glaucoma, additional cycloplegic drops can be added according to clinicians’ judgement. During postoperative monitoring, IOP≥21 mm Hg on two consecutive visits warrants additional intervention. This starts with filtration bleb manipulation, including suture lysis, bleb massage, needling or injections of antimetabolites. If bleb management fails to control IOP adequately, antiglaucoma eye drops and oral carbonic anhydrase inhibitors can be added. For all patients with IOP lower than 21 mm Hg after initial treatment, the combination of phacoemulsification, intraocular lens implantation and goniosynechialysis will be performed at least 1 month after the initial treatment of medication, laser or trabeculectomy. All participating centres received approval from their respective institutional review board and ethics committee. The study was registered with the Chinese Clinical Trial Registry (ChiCTR2200057289), complied with the Health Insurance Portability and Accountability Act regulations and adhered to the tenets of the Declaration of Helsinki. All eyes will be examined on the first medical visit at each centre, on the day of initial treatment and 1 day, 1 week, 1 month, 3 months, 6 months and 12 months following the initial treatment. The information required at each follow-up visit is demonstrated in . For those completing a 1-year follow-up, the study reaches its endpoint at 1 year after the initial treatment. Participants automatically withdraw from the study when: Missing three consecutive follow-ups or more. With poor treatment compliance. Death or suffering from newly diagnosed systemic diseases impeding regular follow-up. Study endpoint is defined as the last follow-up before withdrawal. However, if any patient violates treatment protocol or lacks essential information which hinders the judgement of efficacy and safety of the trial, they will be excluded from final data analysis. Outcomes and adverse events will be recorded on a trial-specific case report form. Demographic information, along with medical history of each recruited participant, will be documented at baseline. Extensive ophthalmological examinations of BCVA, IOP and slit-lamp examination will be performed as well if the patients are first presented at the emergency department. Subsequent examination will be completed after referral at glaucoma clinics including gonioscopy, ultrasound biomicroscopy, anterior segment photography, fundus photography, Humphrey visual field (VF), a visual quality questionnaire, optical coherence tomography, specular microscopy and IOL master. Of note, we attach great significance to recording a precise TST (hours), time from presentation to the subsidence of the attack and (if the cornea is oedematous due to the attack) time from symptoms to cornea restoring transparency will also be recorded. During follow-up, we intend to document any related adverse events, poorly controlled IOP needing treatment upgrade and cataract surgeries. Primary outcomes The primary outcome of this study is BCVA measured using the Early Treatment Diabetic Retinopathy Study (ETDRS) logMAR E visual acuity (VA) charts 1 year post initial treatment. Notably, resolved attack or controlled IOP do not completely equal good visual outcome, considering that patients’ attack duration in different groups may considerably vary due to waiting treatment elevation. Also, since VA most directly reflects one’s visual function and concerns patients with glaucoma the most, it would be the most appropriate option for the primary outcome of this study. ETDRS VA is designed to inspect visual impairment in clinical trials. If no letter could be read on the chart, vision of finger count, hand movement, light perception and no light perception will be recorded accordingly. VA examiners are all verified ophthalmologists, have undergone unified training and masked from treatment assignment. Secondary outcomes Secondary outcomes consist of complete success rate in breaking the attack, IOP, mean deviation on Humphrey VF testing and vision-related QoL collected from the National Eye Institute Visual Function Questionnaire (25 items) (NEI-VFQ25) 1 year post initial treatment. Complete success in breaking the attack is defined as IOP<21 mm Hg without the aid of IOP-lowering medication. If IOP can only be controlled with the assistance of medication, the success should be defined as partial rather than complete. An NCT will be employed for the measurement of IOP, taking several facts into consideration since many patients with APAC are present in an emergency context and that not all centres allow access to Goldmann tonometry. Three measurements will be taken for each eye by a masked examiner, and the mean values will be used. VF testing will be performed on Humphrey 24-2 automated perimetry. Reliable VF results are defined as fixation loss<20%, with both positive and negative false <33%. To further evaluate visual outcomes in patients with APAC, we adopted the NEI-VFQ25 questionnaire, which is a vision-specific health profile measure. Since the original questionnaire was designed for English speakers, our study adopted the Chinese version, which had been tested for good reliability and validity. Ancillary measurements The data presented in will also be recorded. Box 1 Ancillary information and examinations required Time from syndrome to presentation, time from presentation to the subsidence of the attack and time from symptoms to cornea restoring transparency. Number and types of medication needed after laser or surgery. Further lens extraction surgery or other antiglaucoma surgeries during the follow-up period. Gonioscopy. Ultrasound biomicroscopy. Fundus photography. Specular microscopy. Optical coherence tomography. Other examinations required for the preparation of surgeries. Any ancillary examinations that demand direct contact with the cornea will only be performed when the IOP is maximally controlled and when corneal oedema has resolved so that safety is guaranteed. Safety and adverse events We intend to report all serious adverse events, along with complications and unexpected events from each step of intervention. illustrates the expected adverse events and complications related to glaucoma care in this trial. If any, all will be extracted, kept detailed records and reported to the Ethics Committee and the Trial Steering Committee in regular progress reports. The primary outcome of this study is BCVA measured using the Early Treatment Diabetic Retinopathy Study (ETDRS) logMAR E visual acuity (VA) charts 1 year post initial treatment. Notably, resolved attack or controlled IOP do not completely equal good visual outcome, considering that patients’ attack duration in different groups may considerably vary due to waiting treatment elevation. Also, since VA most directly reflects one’s visual function and concerns patients with glaucoma the most, it would be the most appropriate option for the primary outcome of this study. ETDRS VA is designed to inspect visual impairment in clinical trials. If no letter could be read on the chart, vision of finger count, hand movement, light perception and no light perception will be recorded accordingly. VA examiners are all verified ophthalmologists, have undergone unified training and masked from treatment assignment. Secondary outcomes consist of complete success rate in breaking the attack, IOP, mean deviation on Humphrey VF testing and vision-related QoL collected from the National Eye Institute Visual Function Questionnaire (25 items) (NEI-VFQ25) 1 year post initial treatment. Complete success in breaking the attack is defined as IOP<21 mm Hg without the aid of IOP-lowering medication. If IOP can only be controlled with the assistance of medication, the success should be defined as partial rather than complete. An NCT will be employed for the measurement of IOP, taking several facts into consideration since many patients with APAC are present in an emergency context and that not all centres allow access to Goldmann tonometry. Three measurements will be taken for each eye by a masked examiner, and the mean values will be used. VF testing will be performed on Humphrey 24-2 automated perimetry. Reliable VF results are defined as fixation loss<20%, with both positive and negative false <33%. To further evaluate visual outcomes in patients with APAC, we adopted the NEI-VFQ25 questionnaire, which is a vision-specific health profile measure. Since the original questionnaire was designed for English speakers, our study adopted the Chinese version, which had been tested for good reliability and validity. The data presented in will also be recorded. Box 1 Ancillary information and examinations required Time from syndrome to presentation, time from presentation to the subsidence of the attack and time from symptoms to cornea restoring transparency. Number and types of medication needed after laser or surgery. Further lens extraction surgery or other antiglaucoma surgeries during the follow-up period. Gonioscopy. Ultrasound biomicroscopy. Fundus photography. Specular microscopy. Optical coherence tomography. Other examinations required for the preparation of surgeries. Any ancillary examinations that demand direct contact with the cornea will only be performed when the IOP is maximally controlled and when corneal oedema has resolved so that safety is guaranteed. We intend to report all serious adverse events, along with complications and unexpected events from each step of intervention. illustrates the expected adverse events and complications related to glaucoma care in this trial. If any, all will be extracted, kept detailed records and reported to the Ethics Committee and the Trial Steering Committee in regular progress reports. Sample size has been calculated based on the primary outcome (BCVA) using SAS OnDemand for Academics (SAS Institute, Cary, North Carolina, USA). The power was set at 80% and the significance level was 5%, considering an attrition rate of 10%. In order to detect a minimum difference of 5 ETDRS letters (logMAR=0.1, SD approximately 0.2) 1 year after initial treatment, a total of 142 patients (71 per group) is required. Analysis of outcomes will be performed at the end of the trial on an intention-to-treat basis. After testing for data distribution of normality, demographic information, primary and secondary outcomes will be presented and compared between treatment groups accordingly. Moreover, mixed models will be applied for the investigation of outcomes changes over follow-up time. A cost-effective estimation will also be performed to determine the health economics results of two treatment arms. A p value of <0.05 will be considered statistically significant. A remote-automated computer central randomisation service provided by Depa EDC will be used for randomised sequence generation, which guarantees full allocation concealment. Minimisation will be used for treatment allocation to achieve optimal balance between groups. In this regard, two intervention groups will be balanced simultaneously over five stratification factors: (1) centre, (2) age (<60 years, ≥60 years), (3) IOP at presentation (30–45 mm Hg, ≥45 mm Hg including IOP values that exceed NCT examination threshold), (4) education level (middle school and high school or lower, higher than high school) and (5) TST (72–168 hours, ≥168 hours). The service can be easily accessed through a WeChat-based mini program. Patients will be assigned randomly (at a 1:1 ratio) following routine medication. Randomisation unit of this trial is the participant rather than the eye. In light of the nature of surgical intervention, neither the clinical staff nor the participants can be blinded with respect to the intervention. However, outcomes, including BCVA, IOP, VF, NEI-VFQ25 and other ancillary examinations, will be done separately by examiners masked to randomisation. Researchers participating in this clinical research must be qualified, with professional background and ability to conduct clinical research. All examinations, diagnosis and interventions will be carried out strictly abiding by the SOP. Before the study launches, all centres are visited and convened for training. The steering committee oversees the whole research and formulates study strategies. The safety and data monitoring committee will monitor safety and other data at six regular intervals during the recruitment phase of the trial. Interim analysis will be conducted 6 months after the initiation of the trial. 10.1136/bmjophth-2024-001934 online supplemental file 1 |
Disrespect and abuse during childbirth and associated factors among women: a cross-sectional study | 9eaeb2f9-606f-4f47-a23d-693518f7804f | 11871645 | Surgical Procedures, Operative[mh] | Pregnancy and childbirth represent a pivotal phase in a woman’s life, carrying significant physical, psychological, emotional, social, and cultural implications that render individuals vulnerable to potential harm . Empathy, support, confidentiality, and respect are essential requirements for women during the childbirth process. Nevertheless, numerous women, particularly those residing in low-income countries, encounter various explicit and concealed forms of disrespect and abuse from midwives when seeking childbirth services . These negative experiences can directly and indirectly impact the quality of care and the well-being of both the mother and the infant . The primary objective of implementing Respectful Maternity Care (RMC) is to enhance the interpersonal interactions between expectant mothers and healthcare providers throughout the stages of pregnancy, delivery, and postpartum, fostering a supportive and empowering environment while eradicating any disrespect towards the mother. The ultimate aim is to ensure the woman’s satisfaction during the childbirth process. RMC encompasses the ideals of respecting autonomy, dignity, emotions, privacy, choice, and freedom from abuse and coercion, while considering individual preferences during childbirth . Providing respectful care is a fundamental strategy for improving healthcare quality and outcomes, and it is recognized as a universal right . The disrespect of pregnant women not only impacts the quality of healthcare services provided but also indirectly contributes to maternal mortality . Disrespect and disrespect experienced during childbirth can result in a negative experience of pregnancy and childbirth . Moreover, negative experiences have long-lasting effects on mothers’ attitudes toward their preferred mode of delivery, often contributing to an increased rate of cesarean Section . Previous studies have consistently demonstrated a significant association between experiencing disrespectful behavior during the prenatal period and an increased preference for cesarean Sect. . This issue also has implications for breastfeeding intention and may influence newborn care practices . Due to concerns about experiencing disrespect and abuse, mothers may decline essential newborn care services, such as growth monitoring and vaccination . Every woman has the right to receive high-quality, respectful, non-violent, and non-discriminatory care throughout pregnancy and childbirth. Experiencing disrespect or mistreatment during childbirth constitutes a violation of internationally recognized human rights . Such instances can occur within the relationship between women and healthcare providers or within the healthcare system itself, involving acts such as physical abuse, discrimination, privacy violations, unfair treatment, disrespectful care, and failure to honor patient requests . The World Health Organization recognizes that positive birth outcomes depend on effective interactions, including respecting women’s autonomy, providing adequate information, and offering emotional support throughout pregnancy and childbirth. In response to growing evidence of disrespect during these critical periods, the organization has called for the prevention and elimination of mistreatment during childbirth through the implementation of the Respectful Maternity Care (RMC) initiative . Several studies have investigated the extent and nature of disrespect and abuse during childbirth, highlighting its significant global prevalence. Shakibazadeh et al. conducted a study in Tehran, reporting a relatively high prevalence of disrespectful behavior toward mothers. The findings indicated that nearly all mothers had experienced at least one form of mistreatment, with ‘restriction of movement and companionship’ being the most prevalent (99.7%), while ‘stigmatization and discrimination’ had the lowest prevalence (5.4%) . Similarly, a study by Hajizadeh et al. in 2020 conducted in the city of Tabriz reported a prevalence of disrespect and abuse at 74.7% . In another study by Hosseini Tabar et al. in the city of Paveh, the prevalence of disrespect and abuse was found to be 100%, indicating that all participants had encountered at least one form of disrespect . In response to these issues, the Charter of Mothers’ Rights was established in 2003 to advocate for women’s rights during labor, with an emphasis on respecting women in Iran’s national childbirth guidelines . Nevertheless, studies indicate a persistently high prevalence of disrespect and mistreatment. Research findings suggest that multiple factors contribute to the prevalence of disrespect and abuse during childbirth. These factors include insufficient supervision of prenatal care, limited awareness among women about their rights in prenatal care, the spouse’s employment status, low income, unfavorable economic conditions, parity (number of previous births), type of delivery, and HIV positivity . Furthermore, inadequate resources, understaffing, job burnout, low motivation, and insufficient training are additional factors that contribute to the prevalence of disrespectful behaviors and abuse during childbirth . Additionally, it is worth noting that healthcare providers themselves may experience disrespect from higher institutions, which can further contribute to the perpetuation of disrespectful behaviors and abuse towards mothers . Despite the existing evidence highlighting the increasing prevalence of disrespect within maternity service centers, limited research has been conducted to explore the underlying factors contributing to the disrespect of mothers. Identifying the determinants of disrespect and abuse during pregnancy and childbirth can provide valuable insights for policymakers and planners in developing and implementing targeted interventions to improve maternal care and reduce instances of disrespect. Therefore, the present study aims to assess the prevalence of disrespect and abuse during childbirth and its associated factors among women attending comprehensive health centers in Tehran in 2021.
Study setting and design The present study is a cross-sectional study conducted in Tehran in 2021. The study sample comprised Iranian women residing in Tehran who had given birth vaginally within the previous five days, and sought care at comprehensive health centers. Childbirth management in Iran depends on the type of hospital. In public hospitals affiliated with medical universities, deliveries are primarily conducted by obstetrics residents and midwifery students. In public hospitals not affiliated with medical universities, as well as in private hospitals, deliveries are managed by midwives and obstetricians. However, although the labor and delivery process may be managed by midwives, all actions taken by the midwife during this process are carried out under the supervision of a physician. Tehran, the capital of Iran, has a population of 14 million according to the latest census. Comprehensive health centers are prominent healthcare establishments offering an extensive array of resources and services, encompassing prenatal and postnatal care, as well as neonatal screenings. all services rendered within these centers are provided free of charge. Study population To enhance access to the study population and reduce recall bias, sampling was conducted among women who visited comprehensive health centers for newborn screening. Women who reported experiencing vaginal delivery within the past five days were included in the study. However, women who were critically ill, illiterate, or had preterm deliveries were excluded. Study sample and sampling procedure In the present study, the sample size required to estimate the prevalence of disrespect and abuse towards mothers during childbirth was determined to be at least 300 individuals. This estimation was based on a confidence level of 95% and a level of precision set at 0.05, as calculated using the following formula. [12pt]{minimal}
$$\:n=_{1-\!/\:\!.}^{2}\:pq}{{d}^{2}}=^{2}\:0.75\:0.25}{{0.05}^{2}}\:300$$ To ensure a representative sample across Tehran, a two-stage cluster sampling method was employed. First, Tehran was divided into five geographic regions (central, north, south, east, and west). Within each region, referral data from the official websites of Tehran, Iran and Shahid Beheshti Universities of Medical Sciences were used to identify comprehensive health centers with the highest patient volumes. Finally, ten health centers were selected for sampling. Participants were selected using systematic random sampling technique. The information was gathered from every other postnatal woman until the anticipated sample was achieved. Outcome measures and measurements The variables in this study were measured as follow: Demographic and midwifery characteristics The researchers developed a two-part questionnaire to collect data. The first section focused on mother’s personal characteristics, including age, educational level, ethnicity, occupation, and Economic status. The second section was dedicated to pregnancy-related information, such as the number of previous births, history of abortion, infant death, pregnancy intention (planned or unplanned), location of prenatal care, the person responsible for maternal care during labor, presence of a companion during labor, birth agent, timing of delivery, utilization of labor induction methods, perineal status, type of delivery room, administration of epidural or spinal anesthesia, use of analgesic medications or Entonox, and the use of forceps or vacuum during delivery. Disrespect and abuse questionnaire Data relating to perceived disrespectful maternity care were collected using a Disrespect and Abuse Questionnaire, which contains 23 items in seven dimensions: (1) protection of pregnant women against physical harm (6 items); (2) observing women’s rights with regard to being informed about her condition/ informed consent/ choice of childbirth position (8 items); (3) observing women’s privacy and confidentiality (1 item); (4) observing women’s dignity and respect (2 items); (5) receiving equitable care without discrimination (2 items); (6) maintaining care and not neglecting pregnant women (3 items); (7) delayed discharge or detention of pregnant women (1 item). A positive answer to each of the items in one dimension was defined as abuse for that dimension. If a mother was identified as having faced D&A in at least one of the seven dimensions, she was considered “disrespected and abused”. This questionnaire was completed 6 to 18 h after birth and designed by Asefa et al. and approved by the Maternal and Child Health Integrated Program (USAID, 2011) . This questionnaire was translated into Persian by Hajizadeh et al. In the face validity assessment, all items received a minimum impact score of 1.5. In the content validity assessment, all items achieved the minimum acceptable values for CVR (> 0.69) and CVI (> 0.79). Exploratory factor analysis revealed that the Disrespect and Abuse Questionnaire consists of 23 items and five factors, including abandoning the mother, improper care, mother’s immobility, lack of communication with the mother, and deprivation of the mother. The construct validity of the scale was confirmed through confirmatory factor analysis, with a X2/df ratio of less than 5 and a root mean square error of approximation (RMSEA) of less than 0.08 . Data analysis Data analysis was conducted utilizing SPSS V.26 software. Descriptive statistics, such as frequency and percentage, mean, and standard deviation, were employed to depict the individual characteristics and experiences of disrespect and abuse variables. The Chi-square test was utilized to compare disrespect and abuse based on individual and obstetric characteristics. To explore the factors influencing the prevalence of disrespect and abuse, variables that exhibited statistical significance were incorporated into a multiple logistic regression model.
The present study is a cross-sectional study conducted in Tehran in 2021. The study sample comprised Iranian women residing in Tehran who had given birth vaginally within the previous five days, and sought care at comprehensive health centers. Childbirth management in Iran depends on the type of hospital. In public hospitals affiliated with medical universities, deliveries are primarily conducted by obstetrics residents and midwifery students. In public hospitals not affiliated with medical universities, as well as in private hospitals, deliveries are managed by midwives and obstetricians. However, although the labor and delivery process may be managed by midwives, all actions taken by the midwife during this process are carried out under the supervision of a physician. Tehran, the capital of Iran, has a population of 14 million according to the latest census. Comprehensive health centers are prominent healthcare establishments offering an extensive array of resources and services, encompassing prenatal and postnatal care, as well as neonatal screenings. all services rendered within these centers are provided free of charge.
To enhance access to the study population and reduce recall bias, sampling was conducted among women who visited comprehensive health centers for newborn screening. Women who reported experiencing vaginal delivery within the past five days were included in the study. However, women who were critically ill, illiterate, or had preterm deliveries were excluded.
In the present study, the sample size required to estimate the prevalence of disrespect and abuse towards mothers during childbirth was determined to be at least 300 individuals. This estimation was based on a confidence level of 95% and a level of precision set at 0.05, as calculated using the following formula. [12pt]{minimal}
$$\:n=_{1-\!/\:\!.}^{2}\:pq}{{d}^{2}}=^{2}\:0.75\:0.25}{{0.05}^{2}}\:300$$ To ensure a representative sample across Tehran, a two-stage cluster sampling method was employed. First, Tehran was divided into five geographic regions (central, north, south, east, and west). Within each region, referral data from the official websites of Tehran, Iran and Shahid Beheshti Universities of Medical Sciences were used to identify comprehensive health centers with the highest patient volumes. Finally, ten health centers were selected for sampling. Participants were selected using systematic random sampling technique. The information was gathered from every other postnatal woman until the anticipated sample was achieved.
The variables in this study were measured as follow:
The researchers developed a two-part questionnaire to collect data. The first section focused on mother’s personal characteristics, including age, educational level, ethnicity, occupation, and Economic status. The second section was dedicated to pregnancy-related information, such as the number of previous births, history of abortion, infant death, pregnancy intention (planned or unplanned), location of prenatal care, the person responsible for maternal care during labor, presence of a companion during labor, birth agent, timing of delivery, utilization of labor induction methods, perineal status, type of delivery room, administration of epidural or spinal anesthesia, use of analgesic medications or Entonox, and the use of forceps or vacuum during delivery.
Data relating to perceived disrespectful maternity care were collected using a Disrespect and Abuse Questionnaire, which contains 23 items in seven dimensions: (1) protection of pregnant women against physical harm (6 items); (2) observing women’s rights with regard to being informed about her condition/ informed consent/ choice of childbirth position (8 items); (3) observing women’s privacy and confidentiality (1 item); (4) observing women’s dignity and respect (2 items); (5) receiving equitable care without discrimination (2 items); (6) maintaining care and not neglecting pregnant women (3 items); (7) delayed discharge or detention of pregnant women (1 item). A positive answer to each of the items in one dimension was defined as abuse for that dimension. If a mother was identified as having faced D&A in at least one of the seven dimensions, she was considered “disrespected and abused”. This questionnaire was completed 6 to 18 h after birth and designed by Asefa et al. and approved by the Maternal and Child Health Integrated Program (USAID, 2011) . This questionnaire was translated into Persian by Hajizadeh et al. In the face validity assessment, all items received a minimum impact score of 1.5. In the content validity assessment, all items achieved the minimum acceptable values for CVR (> 0.69) and CVI (> 0.79). Exploratory factor analysis revealed that the Disrespect and Abuse Questionnaire consists of 23 items and five factors, including abandoning the mother, improper care, mother’s immobility, lack of communication with the mother, and deprivation of the mother. The construct validity of the scale was confirmed through confirmatory factor analysis, with a X2/df ratio of less than 5 and a root mean square error of approximation (RMSEA) of less than 0.08 .
Data analysis was conducted utilizing SPSS V.26 software. Descriptive statistics, such as frequency and percentage, mean, and standard deviation, were employed to depict the individual characteristics and experiences of disrespect and abuse variables. The Chi-square test was utilized to compare disrespect and abuse based on individual and obstetric characteristics. To explore the factors influencing the prevalence of disrespect and abuse, variables that exhibited statistical significance were incorporated into a multiple logistic regression model.
51% ( n = 153) of the participants in the study fell within the age range of 20 to 29 years. A significant proportion of the participants (70%, n = 210) possessed a high school and Diploma level of education. Turk women constituted 25.3% ( n = 76) of the sample, while Fars women accounted for 31.7% ( n = 95). The remaining 43% consisted of women from diverse ethnic backgrounds, including Kurd, Lor, Baluch, Gilak, Arab, and others. The majority of women (80.3%, n = 241) were housewives. In terms of socioeconomic status, approximately half of the participants reported a relatively unfavorable financial situation (51.3%, n = 154), while the other half mentioned a relatively favorable situation (48.7%, n = 146). Out of the participating women in the study, 111 (37%) experienced their first childbirth, 114 (38%) had their second childbirth, and 75 (25%) had given birth three or more times. The majority of women had no prior history of abortion ( n = 209; 69.7%) or infant death ( n = 284; 94.7%). Regarding pregnancy status, approximately half of the women reported an unwanted pregnancy in the recent past ( n = 151; 50.3%). The demographic and obstetric information of the women is summarized in Tables and . In relation to prevalence of disrespect and abuse, a total of 205 women (68.3%) encountered at least one of the seven dimensions associated with Disrespect and Abuse. The dimension that exhibited the highest prevalence was rights protection ( n = 189; 63.0%), whereas the dimension of detained or confined against one’s will had the lowest prevalence ( n = 28; 9.3%) (Table ). A statistically significant relationship was observed between the variables of age ( p = 0.004), education level ( p = 0.021), ethnicity ( p = 0.001), economic status ( p = 0.018), pregnancy status ( p = 0.001), location of prenatal care ( p = 0.002), person responsible for maternal care during labor ( p = 0.001), presence of a companion during labor ( p = 0.001), birth agent ( p = 0.001), perineal status ( p = 0.001), delivery room ( p = 0.009), receiving analgesia ( p = 0.019), and instrumental delivery ( p = 0.01), with the prevalence of Disrespect and Abuse. The statistical analysis presented in Tables and revealed that variables such as occupation, number of births and abortions, history of infant death, time of delivery, use of labor induction methods, and receipt of pharmaceutical analgesia did not demonstrate a significant relationship with the prevalence of Disrespect and Abuse. Additional information can be found in Tables and which display the associations between women’s personal characteristics, midwifery factors, and the Dimensions of disrespect and abuse. To further investigate the relationship between each independent variable (including individual characteristics and midwifery) and the dependent variable (disrespect and abuse), only those variables that exhibited statistical significance ( p < 0.05) were included in the multiple logistic regression model. A forward stepwise logistic regression model was used to investigate factors affecting the experience of disrespect and abuse during childbirth. The findings from this analysis revealed that several demographic and midwifery characteristics of the participants have a significant impact on the prevalence of disrespect and abuse during childbirth. These influential factors include age, education level, economic status, ethnicity, pregnancy status, location of prenatal care, person responsible for maternal care during labor, presence of a companion during delivery, birth agent, instrumental delivery, perineal status, delivery room, and receipt of analgesia. In order to mitigate potential confounding variables, a regression analysis was employed to incorporate all the identified factors that exhibited a significant impact on disrespect and abuse. This comprehensive approach enabled the removal of confounding factors, ensuring a more accurate evaluation of the relationship between the identified factors and the prevalence of disrespect and abuse. When developing the logistic regression model analyzing the relationship within mothers’ profiles and disrespect/abuse committed towards them during the childbirth process, the following problems were noted: disrespect and abuse during childbirth met the conditions for logistic regression, as it has a value of range 0 to 1, or binary form. It was coded as [“1 for presence of disrespect and abuse, 0 for absence”]. The findings from this multivariate model demonstrated that ethnicity, instrumental delivery, presence of a companion during labor, caregiver during labor, and desired pregnancy, exhibited predictive significance in relation to the prevalence of disrespect and abuse during childbirth. The prevalence of disrespect and abuse was significantly lower among Turk women compared to Fars women, with a 67% reduction in odds (OR: 0.339; CI: 0.141 to 0.815). Women who experienced unwanted pregnancy were 2.3 times more likely to encounter disrespect and abuse during labor (OR: 2.317; CI: 1.147 to 4.680). The prevalence of disrespect was 88% lower among women whose caregiver during labor was a physician, as opposed to a midwife (OR: 0.129; CI: 0.043 to 0.388). In cases where the mother was unaware of the responsible caregiver, the likelihood of experiencing disrespect increased by 7.1 times (OR: 7.190; CI: 0.866 to 59.711). Mothers without a companion during labor had a 74% lower likelihood of disrespect compared to those who were accompanied (OR: 0.263; CI: 0.100 to 0.694), and the prevalence of disrespect was 92% lower among women who did not undergo instrumental delivery (OR: 0.088; CI: 0.015 to 0.527) (Table ).
The present study was conducted to assess the prevalence of disrespect and abuse during childbirth among Iranian women, as well as the associated factors. Disrespect and mistreatment during childbirth not only affect the immediate experience of labor but also have profound psychological ramifications that can impact a mother’s well-being and her parenting in the long term. Negative experiences, such as neglect, abuse, or feeling disrespected during labor, can lead to mental health challenges like heightened anxiety, stress, and even post-traumatic stress disorder (PTSD) . These psychological problems may impair a mother’s ability to nurture her child, resulting in significant changes in her parenting style . Additionally, mothers subjected to such treatment often develop feelings of inadequacy, mistrust towards the healthcare system, and strained relationships with their children. On the other hand, studies suggest that respectful and supportive care during childbirth not only facilitates a smoother birthing process but also positively influences a mother’s overall well-being and approach to caregiving . Incorporating the principles of respectful parenting into discussions about childbirth care can provide deeper insights into how mistreatment impacts maternal caregiving and child development. A focus on respectful care during childbirth may therefore foster healthier maternal-child relationships and improve long-term outcomes for families . Findings revealed that 68.3% of the women had experienced at least one of the seven dimensions of disrespect and abuse. These results align with Sharma et al.‘s research, which demonstrated that all women experienced some form of disrespect during labor, delivery, or the postpartum period. This finding serves as a cautionary indication, emphasizing the significance of providing respectful care during childbirth . Similar outcomes were observed in other studies . However, the prevalence of reported disrespect and abuse in Pathak and Goli’s study was considerably lower than in our investigation . This inconsistency may be attributed to differences in the study period, participants’ understanding of service provision, economic status, socio-cultural disparities, service quality, and the ability of participants to report instances of disrespect and abuse. One possible explanation for the high prevalence of disrespectful practices during childbirth in Iran is the dominant focus on medical interventions rather than prioritizing women’s needs. For example, the routine use of episiotomy and the administration of oxytocin during labor without obtaining informed consent or involving women in the decision-making process are widely accepted practices in Iran . In contrast, European countries take a different approach, where fewer than half of women undergo oxytocin administration or episiotomy during childbirth . Moreover, the routine use of these interventions without obtaining the woman’s consent is considered a form of obstetric violence in these countries . The current study identified lack of support for women’s rights and physical abuse as the most prevalent forms of disrespect during childbirth. This finding aligns with Wassihun and Banks’ research, which also indicated that physical abuse and non-consensual care were the primary categories of disrespect and abuse . Specifically, approximately 68% of women did not receive informed consent before undergoing any procedure during labor, which is inconsistent with the recommendation that healthcare providers should inform women about the necessity and risks of interventions, allowing them to provide informed consent. It is crucial for women to receive comprehensive information about their care and actively participate in the decision-making process regarding their treatment . In this regard, Asefa et al.‘s study reported similar findings, indicating that more than half of the women did not provide informed consent before undergoing procedures during childbirth, which is consistent with the present study . Comparable findings have also been reported in other studies . Midwifery care has distinct characteristics that require a higher level of satisfaction compared to other forms of medical care. Ethical practices stipulate that women’s informed consent should be obtained before performing any procedure . Gynecologists and midwives acknowledge humanizing birth as a form of care that upholds honor, respect, and the right to choose and participate in the decision-making process. These findings strongly emphasize the need for further action to meet mothers’ expectations within the labor sector . In this study, approximately half of the participants reported experiencing physical abuse during childbirth. This finding is consistent with Bulto’s research, which revealed that over two-thirds (67%) of participants had encountered physical abuse, including instances of scolding, yelling, and even slappin . However, Nawab et al.‘s study yielded contrasting results, with fewer than 6% of participants reporting physical disrespect and abuse . Research has indicated that certain midwives perceive physical abuse as a response to mothers who fail to cooperate during labor, believing that both the mother and fetus are at risk . hese midwives attribute their disrespect during childbirth to the reluctance of pregnant women to comply with their instructions, which they justify as an attempt to prevent adverse outcomes . Furthermore, some studies have noted that mothers believed midwives engaged in physical violence during childbirth in their pursuit of a safe delivery. While some mothers considered these behaviors to be in their best interest, others regarded them as inhumane . Roughly 40% of mothers reported experiencing a lack of care or attention during childbirth. This finding aligns with previous research that has identified neglect as one of the most prevalent forms of disrespect and abuse during labor . However, it is important to note that Wassihun et al.‘s study reported a lower incidence of inattention and neglect, less than 2% , which contrasts with the results of the aforementioned studies. The high prevalence of neglect is concerning because the presence of a healthcare provider during labor is considered crucial by women who have undergone childbirth . Teaching hospitals, as large referral centers, may experience a higher influx of women compared to private hospitals. This can impact staffing capacity and contribute to mothers feeling neglected. Moreover, service providers may prioritize women with labor complications over those with uncomplicated pregnancies, which can further amplify feelings of neglect among women without complications . Due to privacy concerns, 22% of mothers reported experiencing privacy violations during childbirth. Similar findings have been reported in other studies . However, the results of other studies did not align with the findings of the present study . The challenge of maintaining privacy in healthcare facilities may arise due to the presence of a large number of professionals, staff, and students who may inadvertently violate women’s privacy, particularly in teaching hospitals. Some studies have indicated that mothers attribute privacy violations to the lack of privacy-enhancing measures, such as bed curtains and open spaces within the wards. This suggests that mothers perceive insufficient privacy in healthcare centers as a result of system deficiencies rather than the actions of midwives . However, midwives’ personal biases can also impact the provision of privacy. Some midwives believe that all women are in labor for the same reason and therefore do not see the need to prioritize privacy . These factors may have implications for the continuity of care that women receive from healthcare providers . In our study, 22% of women reported experiencing discriminatory care during childbirth. In contrast to our findings, Sharma et al.‘s research revealed that none of the individuals included in their study experienced discrimination based on any specific characteristic . Similar to our study, lower rates of discrimination were also observed in other studies . Discrimination against women for any reason constitutes a violation of their fundamental health rights. Therefore, it is imperative to provide appropriate training and implement ongoing monitoring of obstetric and gynecological healthcare providers to ensure non-discriminatory maternal care . In the current study, 9% of mothers indicated that they were kept in the hospital against their wishes to be discharged. Similar findings have been reported by other researchers . However, several other studies have reported a high incidence of hospital detention . The disparity in study results could be attributed to variations in research settings, economic conditions, and the availability of services and care during pregnancy and childbirth. In many South African countries, delivery services and maternity care are provided free of charge due to the high rates of maternal and perinatal mortality . The present study aimed to investigate the factors contributing to the prevalence of disrespectful behaviors. The results obtained after incorporating the variables into the logistic regression model showed that the prevalence of disrespect was 67% lower among Turk women compared to Persian women. In contrast to the findings of the current study, Mirzania et al. found that non-Persian-speaking women often faced harsh reactions from service providers due to their limited understanding and unfamiliarity with the Persian language, resulting in a lack of necessary cooperation . Similarly, Jardim et al. reported higher rates of violence during childbirth among women from ethnic minorities . The divergent outcomes may be influenced by various factors, such as the type of hospital and healthcare personnel involved in service provision, the level of women’s expectations regarding care, and socio-cultural considerations. The prevalence of disrespect was significantly lower—by 88%—among women who received care from specialized physicians during the various stages of labor. One contributing factor to the rise in disrespect among midwives toward physicians is the heavier workload, increased fatigue, and lower salaries experienced by midwives in hospital settings, which can lead to frustration during childbirth . Another factor contributing to the higher prevalence of violence during childbirth among midwives compared to specialist physicians is the lack of recognition of midwives’ professional identity by physicians. The conflict between physicians and midwives in Iran may stem from various factors, including unclear roles, inadequate management, hierarchical issues, and insufficient skills and knowledge . A systematic review on midwives’ perspectives in sub-Saharan Africa identified the low status of midwives within the healthcare system hierarchy as a contributing factor to disrespect and abuse during childbirth . Another factor contributing to the increased prevalence of disrespect and ause by midwives compared to physicians is the limitation in their decision-making power. In many hospitals in Iran, midwives have a less prominent role in the childbirth process than specialist physicians and are often required to follow physicians’ orders, even in cases where these directives may conflict with the immediate needs of the mothers. This limitation in decision-making authority leads to a sense of lack of control over the childbirth process and related care, which may ultimately reduce the quality of interactions between the midwife and the pregnant woman . In the current study, when mothers were unaware of who was responsible for providing their care, the likelihood of experiencing disrespect was 1.7 times higher. Similar findings have been reported in other studies . The introduction of healthcare providers can help mothers establish better communication and reduce anxiety during childbirth . Women with unintended pregnancies had a 2.3 times higher likelihood of experiencing disrespect during labor. Consistent with the findings of the present study, Ferede et al. also reported a higher incidence of disrespect among women with unwanted pregnancies . The Bante study found that planned pregnancies tripled the odds of receiving Respectful Maternity Care (RMC) . In Kawish et al.‘s systematic review, unwanted pregnancy was identified as one of the factors contributing to the experience of disrespect during labor . In planned pregnancies, mothers tend to receive greater emotional and social support from family members, leading to improved childbirth outcomes, increased utilization of maternal health services, and enhanced preparedness and awareness for childbirth. Lack of awareness plays a significant role in non-cooperation during childbirth and the escalation of violence during the birthing process . Contrary to the findings of the present study, Wassihun et al. and Bulto et al. reported that women with unwanted pregnancies experienced less disrespect compared to women who desired their pregnancies . The prevalence of disrespect was 20% lower in women who did not undergo instrumental delivery compared to those who had a vacuum-assisted delivery, which aligns with findings from other studies . This difference may be attributed to the nature of instrumental delivery, which can cause discomfort for the mother and require increased cooperation, potentially leading to tension and disrespectful behaviors from healthcare providers. Moreover, the experience of greater maternal pain and increased maternal and fetal complications associated with instrumental delivery could contribute to these results. Furthermore, in cases of complicated pregnancies necessitating instrument-assisted deliveries, mothers may perceive such complications as indicative of lower-quality services, leading to a negative perception of service providers and a sense of inadequacy. Experiences of disrespect was 74% lower in mothers who did not have a companion during labor, contrasting with previous studies that emphasized the positive role of companions in reducing disrespect during childbirth . Seth et al. found that the presence of a companion at birth was linked to a reduction in physical and verbal abuse, as well as the promotion of confidential care . In Werdofa et al.‘s study, women reported the absence of a companion during labor and delivery as an experience of violence and abuse. These women expressed that the lack of a companion prolongs their hospital stay, as they are deprived of the support for expressing their feelings. However, while the presence of a companion generally contributes to a positive childbirth experience, some women express concerns about privacy infringement. Additionally, the presence of companions can lead to excessive and unnecessary crowding in the labor room, potentially impacting the patient care process . This study employed a quantitative approach to assess disrespect and abuse during childbirth, utilizing indicators specifically designed for ongoing quality improvement initiatives. However, this study has several limitations. The cross-sectional design of the study limits the ability to establish causal relationships or assess long-term outcomes. Additionally, it is important to acknowledge that quantitative methods have inherent limitations in capturing the nuanced aspects of disrespect and abuse. The completion of the questionnaire by mothers shortly after delivery in this study helped reduce recall bias during data collection. However, this approach does not allow for an exploration of how the passage of time and specific circumstances may influence women’s perceptions of disrespect and abuse, which could be crucial for understanding their subsequent decision-making. Another limitation of the present study is the lack of analysis of disrespect and abuse based on hospital type, which may limit the generalizability of the findings. Furthermore, this research focused solely on women’s experiences and did not examine midwives themselves, including their training, psychological support, or the systemic pressures that may contribute to such behaviors. This omission may hinder a more comprehensive understanding of the factors underlying disrespect and abuse in maternity care. Therefore, future research should consider exploring these factors, alongside a larger sample size, a longer postpartum interval, and both private and public healthcare sectors to enhance the generalizability of the results.
The high prevalence of disrespect and abuse experienced by mothers during labor and delivery is a concerning issue that can have significant implications for maternal health and well-being. Several factors were identified as influential in these behaviors, including pregnancy status, ethnicity, the person responsible for maternal care during labor, the presence of a labor companion, and the occurrence of instrumental delivery. Addressing disrespect and abuse in delivery centers is essential to promote a positive and respectful childbirth experience for mothers. It involves creating a mother-friendly care environment where women feel safe, supported, and empowered during labor and delivery. Regular supervision and monitoring of healthcare providers can help ensure adherence to respectful care practices. Additionally, promoting awareness and education among healthcare providers about the importance of respectful care and cultural sensitivity is crucial. Sensitizing healthcare professionals to the unique needs and experiences of diverse populations can contribute to reducing disparities in the provision of care.
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Tumor-associated macrophages in canine visceral hemangiosarcoma | 1b2a7969-41ff-4cde-a88f-f588e2f00f00 | 10687809 | Anatomy[mh] | Study Population and Sample Collection Tissue samples of all necropsy cases performed between January 2018 and December 2021 of privately owned, treatment-naïve dogs with naturally occurring visceral HSA, with and without disseminated disease, were collected retrospectively from the pathology archives of the Department of Preclinical Sciences and Pathology of the Norwegian University of Life Sciences. Corresponding clinical metadata (breed, age, sex, medication, and comorbidities) were obtained from the medical records. The owners’ written consent was required for the dogs to be included in the study. Dogs were excluded if they had undergone surgical treatment for HSA, had a history of occult atopic or allergic disease, had previous or concurrent neoplastic disease, were currently on immunomodulating medication (glucocorticoids or cytotoxic drugs), or had systemic inflammatory disorders (infectious or immune-mediated). Necropsy There were 17 dogs included in the study . All cases had undergone a standard necropsy performed by a veterinary pathologist. Detailed pathology records for each dog were available and included complete macroscopic and histologic descriptions of all major organ systems and any abnormal findings. Standard organ samples and samples from all tumors were collected and fixed in 10% buffered formalin, paraffin-embedded, and stored as formalin-fixed paraffin-embedded (FFPE) blocks at room temperature. There were 27 tumor samples in total, of which 13 were regarded as primary tumors and 14 as metastases in the pathology records. Seven cases (41.2%) had HSA located in 1 organ, while 10 (58.8%) had tumors in 2 or more locations. Since it was not always possible to establish which tumor represented the primary tumor, tumors were grouped into splenic, cardiac, and other locations. Staging and Histological Parameters Hematoxylin and eosin-stained sections were reviewed by a board-certified veterinary pathologist to confirm the histopathological diagnosis and to assess the histological parameters of the tumors. Histological parameters were scored according to the grading system used by Ogilvie et al. Briefly, the overall differentiation was scored from 1 to 3, with 3 being the least differentiated, nuclear pleomorphism from 0 to 3, with 3 being marked nuclear pleomorphism, the amount of necrosis from 0 to 3 (assessed histologically after trimming of macroscopically visible necrotic areas before paraffin-embedding), with 3 being more than 50% necrosis, and the number of mitosis from 0 to 3, with 3 being above 30 mitoses per 10 high-power fields (HPFs) (400x, equivalent to a total area of 2.37 mm 2 ). A total score was calculated, with 0–5 being graded as 1, 6–9 as Grade 2, and 10–12 as Grade 3. The necropsy reports were used retrospectively to assign a clinical stage for each case from I to III, according to the previously established staging scheme .
Tissue samples of all necropsy cases performed between January 2018 and December 2021 of privately owned, treatment-naïve dogs with naturally occurring visceral HSA, with and without disseminated disease, were collected retrospectively from the pathology archives of the Department of Preclinical Sciences and Pathology of the Norwegian University of Life Sciences. Corresponding clinical metadata (breed, age, sex, medication, and comorbidities) were obtained from the medical records. The owners’ written consent was required for the dogs to be included in the study. Dogs were excluded if they had undergone surgical treatment for HSA, had a history of occult atopic or allergic disease, had previous or concurrent neoplastic disease, were currently on immunomodulating medication (glucocorticoids or cytotoxic drugs), or had systemic inflammatory disorders (infectious or immune-mediated).
There were 17 dogs included in the study . All cases had undergone a standard necropsy performed by a veterinary pathologist. Detailed pathology records for each dog were available and included complete macroscopic and histologic descriptions of all major organ systems and any abnormal findings. Standard organ samples and samples from all tumors were collected and fixed in 10% buffered formalin, paraffin-embedded, and stored as formalin-fixed paraffin-embedded (FFPE) blocks at room temperature. There were 27 tumor samples in total, of which 13 were regarded as primary tumors and 14 as metastases in the pathology records. Seven cases (41.2%) had HSA located in 1 organ, while 10 (58.8%) had tumors in 2 or more locations. Since it was not always possible to establish which tumor represented the primary tumor, tumors were grouped into splenic, cardiac, and other locations.
Hematoxylin and eosin-stained sections were reviewed by a board-certified veterinary pathologist to confirm the histopathological diagnosis and to assess the histological parameters of the tumors. Histological parameters were scored according to the grading system used by Ogilvie et al. Briefly, the overall differentiation was scored from 1 to 3, with 3 being the least differentiated, nuclear pleomorphism from 0 to 3, with 3 being marked nuclear pleomorphism, the amount of necrosis from 0 to 3 (assessed histologically after trimming of macroscopically visible necrotic areas before paraffin-embedding), with 3 being more than 50% necrosis, and the number of mitosis from 0 to 3, with 3 being above 30 mitoses per 10 high-power fields (HPFs) (400x, equivalent to a total area of 2.37 mm 2 ). A total score was calculated, with 0–5 being graded as 1, 6–9 as Grade 2, and 10–12 as Grade 3. The necropsy reports were used retrospectively to assign a clinical stage for each case from I to III, according to the previously established staging scheme .
For the present study, new tissue sections were made from the FFPE blocks and immunohistochemically (IHC) labeled. Sequential sections were made from each block for labeling with different antibodies to reduce variability between sections. Tissues were sectioned at 4 µm thickness using a microtome and mounted on poly-lysin-coated SuperFrost TM Plus slides (Thermo Fisher Scientific, Oslo, Norway). All washing steps were performed using 2 changes of phosphate-buffered saline (PBS), each for 5 minutes, and all incubations were performed in a moisture chamber on a rotating table at room temperature. Tissue sections were deparaffinized in xylene and rehydrated through an ethanol gradient using a standard protocol on an automated slide stainer. Heat-induced epitope retrieval was performed in a pressure cooker at 110 o C for 10 minutes using Diva Decloaked (Biocare Medical, Histolab, Gothenburg, Sweden). Tissues were encircled using a water-repelling liquid blocker pen (PAP Pen, Dako, Glostrup, Denmark). Endogenous peroxidase activity was inhibited using Peroxidazed 1 (Biocare Medical, Histolab, Gothenburg, Sweden) for 10 minutes. The sections were blocked using Background Sniper (Biocare Medical, Histolab, Gothenburg, Sweden) for 10 minutes. The sections were then incubated with primary antibodies diluted in Da Vinci Diluent Green (Biocare Medical, Histolab, Gothenburg, Sweden) for 30 minutes. CD204 (mouse antihuman, clone SRA-E5, Abnova) was applied at a 1:100 dilution, and CD206 (rabbit antihuman, polyclonal, Abcam) at 1:800 dilution. Both antibodies have been validated in dogs for immunohistochemistry on FFPE tissues. , , Sections were incubated with Mach 1 mouse probe (Biocare Medical, Histolab, Gothenburg, Sweden) for CD204 for 15 minutes and Mach 1 universal probe (Biocare Medical, Histolab, Gothenburg, Sweden) for CD206 for 30 minutes. Sections were then incubated with 3-amino-9-ethylcarbazole (AEC) substrate chromogen (Biocare Medical, Histolab, Gothenburg, Sweden) for 10 minutes, then counterstained with Meyer’s hematoxylin for 10 seconds. Tissue sections were mounted using a water-soluble mounting medium (Aquatex ® , Merck, Darmstadt, Germany) and left to dry overnight. Each tissue sample was stained with both CD204 and CD206 antibodies. Negative controls were processed by omitting primary antibodies. Isotype controls were labeled as part of the protocol optimization to ensure no unspecific labeling of the tissues. A sample from each tissue (spleen, heart, lung, peritoneum, liver, kidney, and muscle) was labeled using the same protocol while replacing the primary antibody with a species-specific isotype control at the same dilution (mouse IgG and rabbit IgG isotype controls, Invitrogen, Thermo Fisher Scientific, Oslo, Norway). Splenic tissue sections from a necropsied control dog without pathological findings were used as positive controls.
All slides were manually scanned using a Zeiss AX10 microscope, equipped with a Zeiss axiocam 506 color camera, coupled with Zen pro 2012 (blue edition) image acquiring software (Carl Zeiss Microscopy GmbH, Jena, Germany). Sections that showed weak or no immunolabeling were excluded from the analysis. Areas with folded or detached tissues were also excluded. When present, areas of necrosis and hemorrhage were avoided. Within tumor tissues ( n = 27), CD204- and CD206-positive cells were counted both in the areas with the highest densities of positive cells, known as hot spots, and outside the hot spots. Hot spots were identified subjectively by the investigators. Cells were counted in 5 randomly selected HPFs within the hot spots and similarly in the tumor tissues outside of the hot spots and in normal tissue surrounding the tumor and reported as a mean number per HPF. The area of a HPF was 0.078 mm 2 . If there was no normal tissue surrounding the tumor, positive cells were counted only within the tumor. The ratio of CD206-positive macrophages was estimated using the ratio of CD206-/CD204-positive cells.
All statistical analyses were performed using JMP pro 16.1.0 (SAS Institute Inc., Cary, NC). The number of CD204- and CD206-positive cells, as well as the ratio of CD206/CD204-positive cells were log-transformed, before being fitted into a mixed model, using the model building function in JMP. To compare these numbers between the different counted regions, tumor location and counted region were set as fixed factors, while case number and tissue sample number were set as random factors. When assessing differences between tumor locations, tumor location was set as a fixed factor, case number and tissue sample number were set as random factors and counted regions within the tumors were assessed individually. When assessing the effect of histological parameter scores on the counting data, the mitotic score, mitotic count, necrosis score, nuclear pleomorphism score, and differentiation score were set as fixed factors, whereas case number and tissue sample number were set as random factors and the counted regions within the tumors were assessed individually. When assessing the effect of clinical stage, stage was set as a fixed factor, case number and tissue sample number were set as random factors, and counted regions within the tumors were assessed individually. All models fulfilled the conditions for using mixed models (independence, homogeneity of variance, linearity, and normality of residuals) when assessed using graphical methods (residual by predicted plots and normal quantile plots of residuals). Tukey’s tests were used to compare differences between different fixed factors when there were more than 2 groups. P -values < .05 were considered statistically significant for the statistical testing.
Clinical Characteristics and Tumor Samples The mean age was 9.7 years (range: 6–13). There were 10 (58.8%) male and 7 (41.2%) female dogs. Seven (41.2%) dogs were classified as having Stage II and 10 (58.8%) dogs as having Stage III disease. Twelve (70.6%) of the tumors were graded as Grade 1 and 5 (29.4%) as Grade 2. None of the tumors were classified as grade 3. In 8 of the tumor samples, there was not enough surrounding normal tissue to quantify positive cells outside the tumor tissue. One tumor sample in each of 2 dogs (1 from a splenic tumor and 1 from a liver metastasis) had extensive necrosis, making it impossible to quantify immunolabeled cells. These tumors were excluded from the analysis, while the remaining tumors from these dogs were included. Quantification of CD204- and CD206-Positive Cells in Tumor Tissues and Normal Surrounding Tissues Tissue-resident macrophages and tumor-infiltrating macrophages strongly immunolabeled for CD204 . There were areas with high densities of CD204-positive cells, forming hot spots within the tumors , but not in the surrounding tissue. Mean log(CD204) was significantly higher both within tumor hot spots and in the tumor outside of hot spots than in the normal surrounding tissues ( P = .0002 and P = .009, respectively) . Mean log(CD204) was not significantly different between tumor hot spots and in the tumor outside of hot spots ( P = .43). Similarly, tumor-infiltrating macrophages strongly immunolabeled for CD206 . Tissue-resident macrophages in the surrounding normal tissue had more variable immunolabeling, depending on the organ . Interstitial and alveolar macrophages in the lungs were mostly CD206 positive, as were interstitial macrophages in the renal cortex. On the other hand, only a very small proportion of macrophages within the splenic red pulp were CD206 positive. Similarly, Kupffer cells in the liver were CD206-negative, with few interstitial macrophages or intravascular monocytes showing positive labeling in the liver. Interstitial macrophages in the heart were also mostly CD206-negative. Mean log(CD206) was significantly higher both within tumor hot spots and in the tumor outside of hot spots than in the normal surrounding tissues ( P < .0001 and P = .002, respectively) . In addition, mean log(CD206) was significantly higher within tumor hot spot than in the tumor outside of hot spots ( P = .001). The mean log(CD206/CD204) was also significantly higher both within tumor hot spots and in the tumor outside of hot spots than in the normal surrounding tissues ( P = .0002 and P = .007, respectively) . Mean log(CD206/CD204) was not significantly different between tumor hot spots and in the tumor outside of hot spots ( P = .45). Comparison of CD204- and CD206-Positive Cells Between Tumor Locations The mean log(CD204) was not significantly different between tumor locations (spleen, heart, or other sites), either in tumor hot spots ( P = .59) or in tumor tissues outside hot spots ( P = .70) . Similarly, the mean log(CD206) in hot spots or in the tumor tissues outside of hot spots were not significantly different between different tumor locations ( P = .46 and P = .46, respectively). The mean log(CD206/CD204) was not significantly different between different tumor locations in either hot spots ( P = .94) or the tumor tissues outside of hot spots ( P = .14) . Comparison of CD204- and CD206-Positive Cells Between Tumors of Different Stages and Histological Parameter Scores The mean log(CD204) was significantly higher in tumor hot spots among tumors with a pleomorphism score of 0 than those with a score of 1 or 2 ( P = .01 and P = .02, respectively). The mean log(CD204) in tumor tissues outside of hot spots was, however, not significantly different between tumors with different pleomorphism scores ( P = .39). Mean log(CD204) in tumor hot spots and in the tumor tissues outside of hot spots did not vary significantly between different mitotic scores ( P = .06 and P = .45), necrosis scores ( P = .10 and P = .70), differentiation scores ( P = .06 and P = .34), or show any correlation with mitotic count ( P = .10 and P = .64). The mean log(CD206) in tumor hot spots and in the tumor tissues outside of hot spots did not vary significantly between mitotic scores ( P = .85 and P = .25), necrosis score ( P = .81 and P = .34), nuclear pleomorphism ( P = .28 and P = .19), or differentiation scores ( P = .45 and P = .70), or correlate with mitotic count ( P = .37 and P = .19). Similarly, the mean log(CD206/CD204) in tumor hot spots and in the tumor tissues outside of hot spots did not vary significantly between mitotic scores ( P = .27 and P = .65), necrosis score ( P = .47 and P = .65), nuclear pleomorphism ( P = .16 and P = .90), differentiation scores ( P = .44 and P = .50), or correlate with mitotic count ( P = .61 and P = .72). Neither mean log(CD204), mean log(CD206), or mean log(CD206/CD204) in tumor hot spots were significantly different between tumors of different clinical stages ( P = .40, P = .77, and P = .70, respectively). Similarly, mean log(CD204), mean log(CD206), or mean log(CD206/CD204) were not significantly different in tumor tissues outside of hot spots for different clinical stages ( P = .26, P = .88, and P = .70, respectively).
The mean age was 9.7 years (range: 6–13). There were 10 (58.8%) male and 7 (41.2%) female dogs. Seven (41.2%) dogs were classified as having Stage II and 10 (58.8%) dogs as having Stage III disease. Twelve (70.6%) of the tumors were graded as Grade 1 and 5 (29.4%) as Grade 2. None of the tumors were classified as grade 3. In 8 of the tumor samples, there was not enough surrounding normal tissue to quantify positive cells outside the tumor tissue. One tumor sample in each of 2 dogs (1 from a splenic tumor and 1 from a liver metastasis) had extensive necrosis, making it impossible to quantify immunolabeled cells. These tumors were excluded from the analysis, while the remaining tumors from these dogs were included.
Tissue-resident macrophages and tumor-infiltrating macrophages strongly immunolabeled for CD204 . There were areas with high densities of CD204-positive cells, forming hot spots within the tumors , but not in the surrounding tissue. Mean log(CD204) was significantly higher both within tumor hot spots and in the tumor outside of hot spots than in the normal surrounding tissues ( P = .0002 and P = .009, respectively) . Mean log(CD204) was not significantly different between tumor hot spots and in the tumor outside of hot spots ( P = .43). Similarly, tumor-infiltrating macrophages strongly immunolabeled for CD206 . Tissue-resident macrophages in the surrounding normal tissue had more variable immunolabeling, depending on the organ . Interstitial and alveolar macrophages in the lungs were mostly CD206 positive, as were interstitial macrophages in the renal cortex. On the other hand, only a very small proportion of macrophages within the splenic red pulp were CD206 positive. Similarly, Kupffer cells in the liver were CD206-negative, with few interstitial macrophages or intravascular monocytes showing positive labeling in the liver. Interstitial macrophages in the heart were also mostly CD206-negative. Mean log(CD206) was significantly higher both within tumor hot spots and in the tumor outside of hot spots than in the normal surrounding tissues ( P < .0001 and P = .002, respectively) . In addition, mean log(CD206) was significantly higher within tumor hot spot than in the tumor outside of hot spots ( P = .001). The mean log(CD206/CD204) was also significantly higher both within tumor hot spots and in the tumor outside of hot spots than in the normal surrounding tissues ( P = .0002 and P = .007, respectively) . Mean log(CD206/CD204) was not significantly different between tumor hot spots and in the tumor outside of hot spots ( P = .45).
The mean log(CD204) was not significantly different between tumor locations (spleen, heart, or other sites), either in tumor hot spots ( P = .59) or in tumor tissues outside hot spots ( P = .70) . Similarly, the mean log(CD206) in hot spots or in the tumor tissues outside of hot spots were not significantly different between different tumor locations ( P = .46 and P = .46, respectively). The mean log(CD206/CD204) was not significantly different between different tumor locations in either hot spots ( P = .94) or the tumor tissues outside of hot spots ( P = .14) .
The mean log(CD204) was significantly higher in tumor hot spots among tumors with a pleomorphism score of 0 than those with a score of 1 or 2 ( P = .01 and P = .02, respectively). The mean log(CD204) in tumor tissues outside of hot spots was, however, not significantly different between tumors with different pleomorphism scores ( P = .39). Mean log(CD204) in tumor hot spots and in the tumor tissues outside of hot spots did not vary significantly between different mitotic scores ( P = .06 and P = .45), necrosis scores ( P = .10 and P = .70), differentiation scores ( P = .06 and P = .34), or show any correlation with mitotic count ( P = .10 and P = .64). The mean log(CD206) in tumor hot spots and in the tumor tissues outside of hot spots did not vary significantly between mitotic scores ( P = .85 and P = .25), necrosis score ( P = .81 and P = .34), nuclear pleomorphism ( P = .28 and P = .19), or differentiation scores ( P = .45 and P = .70), or correlate with mitotic count ( P = .37 and P = .19). Similarly, the mean log(CD206/CD204) in tumor hot spots and in the tumor tissues outside of hot spots did not vary significantly between mitotic scores ( P = .27 and P = .65), necrosis score ( P = .47 and P = .65), nuclear pleomorphism ( P = .16 and P = .90), differentiation scores ( P = .44 and P = .50), or correlate with mitotic count ( P = .61 and P = .72). Neither mean log(CD204), mean log(CD206), or mean log(CD206/CD204) in tumor hot spots were significantly different between tumors of different clinical stages ( P = .40, P = .77, and P = .70, respectively). Similarly, mean log(CD204), mean log(CD206), or mean log(CD206/CD204) were not significantly different in tumor tissues outside of hot spots for different clinical stages ( P = .26, P = .88, and P = .70, respectively).
We found that the mean ratio of CD206-/CD204-positive cells was significantly higher within tumor hot spots and in the tumor tissue outside of hot spots than in the surrounding normal tissues. Hence, there were more M2-skewed macrophages within the tumor tissue than outside, indicating an active protumoral polarization within the tumor. Our results are in line with those reported in human and murine tumors, suggesting TAM-targeting therapies may represent a viable treatment option for canine HSA. , In this study, there were more CD204- and CD206-positive cells within tumor hot spots and in the tumor tissue outside of hot spots than in normal surrounding tissues (spleen, heart, liver, kidneys, and lungs). This finding supports the notion that there is either active recruitment of circulating monocytes that differentiate into macrophages and/or that local tissue-resident macrophages expand in response to the growth of HSA. In murine models of lung cancer, pancreatic cancer, and glioblastoma, TAMs originate from both expanding tissue-resident macrophages and circulating monocytes. , , Regan et al showed that canine HSA cells secreted high levels of the chemokine CCL2, stimulating monocyte migration and recruitment to metastases. However, they did not explore the relative contribution of the recruited monocytes to the total TAM number, nor can this be determined based on our results. We found no significant differences in the numbers of CD204-positive or CD206-positive cells per HPF (0.078 mm 2 ) between tumor locations, nor in the ratio of CD206-/CD204-positive cells. The number of CD204-positive cells per HPF was higher in the splenic tumors than in the tumors in the heart and other organs, but not significantly so. Knowing whether the splenic or cardiac lesion represents the primary tumor in dogs with HSA can be challenging, especially in a retrospective study design. Therefore, we divided the lesions into cardiac, splenic, and other sites. The fact that the number of CD204-positive cells per HPF was higher in the splenic lesions, which is the most commonly reported primary tumor site, could correspond to higher numbers of TAMs within the primary tumor compared with metastases. , Given the small number of dogs, the study was probably underpowered to show any such difference. Similar results were reported in a study comparing the phenotypical differences between paired primary and metastatic tumor samples in humans with clear cell renal cell carcinoma. The number of CD204-positive cells seemed to be higher within primary tumors than in metastases. They also observed higher ratios of CD163 (an M2-associated marker in humans)-positive macrophages in the primary tumors compared with metastases. Interestingly, the number of ionized calcium-binding adaptor molecule 1 (IBA1)-positive cells, another macrophage marker, showed the opposite pattern. On the other hand, Withers et al showed that the number of CD204-positive macrophages in pulmonary metastases was significantly higher than in the primary tumors of dogs with osteosarcoma. Whether these differences in numbers of TAMs between primary and metastatic lesions are due to biological differences between tumor types, or investigated organs, remains unexplored. Since our aim was to study macrophage phenotypes in treatment-naïve dogs, in the different tumor sites, we could not relate our findings to clinical outcomes. Most chemotherapies, including those used for treating dogs with HSA, can impact the number of TAMs and their phenotype. , , Chemotherapies will generally shift TAMs toward a proinflammatory, antitumoral M1 phenotype, which would probably have changed the results of our study. Furthermore, the acute stress associated with surgery can affect macrophage phenotypes and TAM infiltrates. , , Surgery has been shown to increase the numbers of TAMs within the primary tumor and metastases and skew TAMs and macrophages in the metastatic target organ toward a protumoral M2 phenotype. , , When comparing clinical stage and histological parameters with TAM numbers and phenotype, nuclear pleomorphism was the only score that showed a significant correlation. Higher pleomorphism scores were associated with lower numbers of CD204-positive cells. However, there were only 2 dogs with tumors having a pleomorphism score of 0, which puts the validity of this finding in question. Several studies have shown that higher TAMs numbers correlate with poor clinical outcomes in several human cancers. Similar findings are emerging within the veterinary field, with higher TAMs numbers correlating with either poor STs or high histological grades. , , , Whether our findings reflect the fact that there is little association between TAM numbers and histological parameters in dogs with HSA or whether our study was underpowered remains unclear. Since most HSAs have similar biological behavior, with widespread metastasis and poor survival, there might not be any differences in TAM numbers and phenotype between tumors. , , Targeting TAMs in cancer therapy has gained a lot of interest in the last decade. , Several strategies have been suggested and include either (1) depletion of TAMs, (2) inhibition of recruitment of blood monocytes, or (3) reprogramming of TAMs. Results from trials based on inhibition of recruitment and depletion of TAMs have been disappointing, so the focus seems to be changing toward reprogramming. The drug MDP was one of the earliest immunotherapies used in dogs. , MDP had a significant clinical benefit in dogs with osteosarcoma and HSA compared with those treated with surgery alone. MDP binds to the intracellular NOD2 receptor of macrophages, dendritic cells, monocytes, and Paneth cells. , NOD2 binding results in activation of the nuclear factor kappa B (NF-κB) pathway and downstream production of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and IL-12. These cytokines are usually associated with M1-skewed macrophages and improved tumoricidal abilities. The clinical benefit of MDP gives us hope that more selective TAM-targeting immunotherapies might improve the survival of dogs with HSA in the future. The expression of CD206 in normal tissue-resident macrophages in various organs has not been previously studied in dogs. Tissue-resident macrophages in the normal tissue surrounding the tumor in the liver, spleen, and heart were largely CD206-negative. Most alveolar and interstitial macrophages in the lungs were CD206-positive. Whether this is also the case in healthy dogs without cancer or was an effect of premetastatic niche formation remains unexplored. In humans, most alveolar macrophages of healthy individuals seem to be CD206 positive, which corresponds well with our findings. We also observed that tissue-resident macrophages in the kidney cortex were CD206 positive, but only 1 dog had renal metastasis. Our study has several possible limitations. First, we did not include tissue samples from noncancer patients as a control group, although we included the healthy surrounding tissues from the same dogs as an internal control. The number and phenotype of macrophages in the healthy surrounding tissues may have been influenced by the tumor and could be different under physiological conditions in healthy dogs. Second, the study was retrospective, and the representativity of the included cases could have been improved. There is also a risk that relevant clinical and histopathological data were missed during data collection. Third, the number of included dogs was small, and there is a risk that differences in macrophage numbers and phenotype in HSA lesions between different locations were missed. Finally, performing CD204 and CD206 immunohistochemistry on separate slides instead of using methods like multiplex IHC/immunofluorescence introduces some errors when estimating double-positive cells. Although staining sequential sections should result in limited variation, counting the exact same areas is impossible. In conclusion, we have shown that the number of CD206-expressing M2-polarized macrophages was significantly higher within tumor tissues than in the normal surrounding tissues. This is in line with findings in human medicine and supports the notion that TAMs are immunosuppressive and protumoral. We found no significant differences in the number or phenotype of macrophages related to the location of tumors. There was no association between clinical staging or histologic parameters and the numbers or phenotype of TAMs, except for higher nuclear pleomorphism scores being associated with lower TAM numbers. Given the translational value of dogs with cancer, dogs with HSA could serve as a good model to evaluate new therapies aimed at reprogramming TAMs.
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Evaluation of self-perceived halitosis and effective conditions in periodontology clinic applicants: A cross-sectional study | 142eff96-bd1b-43d5-97b9-8e4a1b7f8f94 | 11651456 | Dentistry[mh] | Halitosis, halitosis, or oral malodor, is the expression describing the unpleasant odor emanating from the oral cavity. Halitosis can affect people’s social relationships and lives, and it can also be an indicator of some diseases. Its etiology includes non-oral causes such as smoking, alcohol and some foods, and oral or systemic causes. Generally, poor oral hygiene and intraoral conditions such as periodontitis are considered the most important cause (85%) for bad breath. Acute necrotizing ulcerative gingivitis is one of the most significant reasons of foul breath, despite the fact that most forms of gingivitis and periodontitis can also contribute to it. Oral malodor can vary in intensity as a result of adult periodontitis, which is characterized by a gradual loss of periodontal attachment brought on by plaque. Strong oral malodors can result from aggressive periodontitis, which is characterized by a rapid loss of periodontal bone and related tooth movement. Lack of oral hygiene brought on by xerostomia (dry mouth) also has the potential to induce or exacerbate malodor, and some research indicates that wearing dentures can do the same, probably because of increased deposits on the tongue surface. The prevalence of halitosis may be underestimated since some people are unaware of their poor respiratory conditions. According to a 2006 study by Al-Ansari et al on a sample of 1500 people, 25% of the population had halitosis. In addition, Liu et al studied 2000 people in China, and they discovered that 25% of them had halitosis. Halitosis can have an impact on a person’s life, and it can also have psychological repercussions that result in social anxiety disorders that hinder social interaction. Productivity can be affected by halitosis, and the majority of sufferers express personal discomfort. The aim of this study is to determine the prevalence of self-reported halitosis among patients at the periodontology clinic and to link oral hygiene practices to self-reported halitosis.
The Necmettin Erbakan University Faculty of Dentistry Ethics Committee for Non-Pharmaceutical and Medical Device Clinical Research approved this cross-sectional study (Decision number: 2022/240). After signing the informed consent form, participants completed a survey that included demographic information and questions about how they perceived halitosis. 2.1. Population and sampling Five hundred nine participants who chose to take part in the study and applied to our clinic for a variety of reasons were included in it. They did not have any medical, mental, or psychiatric conditions that would have made it impossible for them to respond to the survey questions. According to the power analysis results of the G Power program (G * Power 3.1 software; Heinrich Heine University, Düsseldorf, Germany); for logistic regression analysis, it was determined that a minimum of 509 samples were required in two-tailed analysis (two-tailed), 1.5 odds ratio, 0.05 margin of error (α), 0.20 effect value (pr), 0.95 power (1−β) level. 2.2. Questionnaire items The 10-question survey was divided into 2 parts: the first asked about sociodemographic information such as age, gender, marital status, education level, and smoking status (current, former, or nonsmoker); the second asked about the presence or absence of medical conditions that were thought to be linked to bad breath, such as diabetes mellitus, kidney disease, gastrointestinal tract disorders, respiratory disease, chronic sinusitis, xerostomia, and drug use. Individuals’ oral hygiene habits were assessed using questions on how frequently they used a toothbrush, dental floss, miswak (a chewing stick), and mouthwash. In the final section of the survey, participants were asked whether they believed they had foul breath, how they learned they did (by self-report, dental diagnosis, or word of mouth from a relative or friend), and whether they had previously sought therapy for the issue. The demographic and periodontal clinical data (plaque index, gingival index, bleeding on probing) from the study participants’ patient forms were evaluated. 2.3. Statistical analysis Using the Windows version of IBM SPSS Statistics, version 23.0, statistical analyses for the study were conducted. For categorical data, the frequency and percentage are provided as well as the mean and standard deviation. The correlations between brushing practices, demographic factors, general health information, diagnosis and plaque, gingival index levels, and the presence of bleeding on probing in relation to halitosis were examined using the Pearson chi-square test and Fisher Exact test. The analysis of binary logistic regression was utilized to pinpoint key causes of bad breath. A value of P ≤ .05 was considered significant.
Five hundred nine participants who chose to take part in the study and applied to our clinic for a variety of reasons were included in it. They did not have any medical, mental, or psychiatric conditions that would have made it impossible for them to respond to the survey questions. According to the power analysis results of the G Power program (G * Power 3.1 software; Heinrich Heine University, Düsseldorf, Germany); for logistic regression analysis, it was determined that a minimum of 509 samples were required in two-tailed analysis (two-tailed), 1.5 odds ratio, 0.05 margin of error (α), 0.20 effect value (pr), 0.95 power (1−β) level.
The 10-question survey was divided into 2 parts: the first asked about sociodemographic information such as age, gender, marital status, education level, and smoking status (current, former, or nonsmoker); the second asked about the presence or absence of medical conditions that were thought to be linked to bad breath, such as diabetes mellitus, kidney disease, gastrointestinal tract disorders, respiratory disease, chronic sinusitis, xerostomia, and drug use. Individuals’ oral hygiene habits were assessed using questions on how frequently they used a toothbrush, dental floss, miswak (a chewing stick), and mouthwash. In the final section of the survey, participants were asked whether they believed they had foul breath, how they learned they did (by self-report, dental diagnosis, or word of mouth from a relative or friend), and whether they had previously sought therapy for the issue. The demographic and periodontal clinical data (plaque index, gingival index, bleeding on probing) from the study participants’ patient forms were evaluated.
Using the Windows version of IBM SPSS Statistics, version 23.0, statistical analyses for the study were conducted. For categorical data, the frequency and percentage are provided as well as the mean and standard deviation. The correlations between brushing practices, demographic factors, general health information, diagnosis and plaque, gingival index levels, and the presence of bleeding on probing in relation to halitosis were examined using the Pearson chi-square test and Fisher Exact test. The analysis of binary logistic regression was utilized to pinpoint key causes of bad breath. A value of P ≤ .05 was considered significant.
Male participants in the study make up 34.6% of the total, while female participants make up 65.4%. Table contains demographic information as well as the questions and their responses. The patients’ self-reported halitosis prevalence is 34.2%. While 83.3% of the individuals who believed they had foul breath were aware of their own perceptions, 14.9% and 0.6% of them claimed to have received a diagnosis from a dentist, and 1.1% claimed to have received 1 from their relatives or friends. Only 70 of the 174 individuals with foul breath admitted to having sought therapy for the condition in the past (Table ). The correlation between teeth brushing practices and halitosis is seen in Table . When compared to people who brushed, miswaked, or flossed at least once a day, individuals who brushed their teeth less frequently or never did so reported a higher prevalence of self-perceived bad breath. Patients who reported using mouthwash daily had self-reported bad breath levels that were comparable to those of nonusers (Table ). The medical diseases linked to halitosis are listed in Table . In comparison to patients without these medical history factors, patients with diabetes mellitus, chronic sinusitis, kidney disease, xerostomia, and any medication reported a higher prevalence of halitosis, whereas people with gastrointestinal system disorders and respiratory system diseases reported similar values. According to Table , there was no discernible difference in the prevalence of self-reported halitosis between men and women. The difference between smokers and nonsmokers was also not statistically significant ( P > .05). A considerably higher prevalence of self-perceived halitosis was observed by those with lower levels of education and by those who were not married. The prevalence of self-reported halitosis and their periodontal health were shown to be significantly correlated (Table ) ( P < .05). Individuals with poor periodontal health reported a higher prevalence of halitosis, compared to those with gingival health. As the participants’ periodontal health deteriorates, the participants’ judgments of halitosis rise.
In order to assess the prevalence of self-reported oral malodor in patients enrolled in the department of periodontology at Necmettin Erbakan University and identify the risk factors for this disease, this study was carried out. A third of people said they had unpleasant breath. Additionally, those who brushed their teeth less frequently or not at all, did not floss, had dry mouth, or had systemic disease had considerably higher self-reported halitosis. However, in this study, there was no correlation between gender, oral hygiene practices, or smoking and self-reported halitosis. The prevalence of oral malodor in this patient sample is 34.2%. Jassem M. El-Ansari et al in a study evaluating the self-reported prevalence and factors of halitosis in 1551 Kuwaiti patients, the prevalence of halitosis was 23.3%. Halitosis has a self-reported prevalence ranging from 22% to 40% in various populations. Certain population groups were studied in some research, which also revealed various prevalence percentages ranging from 7% to 50%. In a 2016 research of dental students, the prevalence of self-reported halitosis was found to be 21.8%. Another study on patients undergoing orthodontic treatment discovered a frequency of 41.2%. Studies that employ differing prevalence estimates imply that different criteria were applied, which may have had an impact on these prevalence rates. Also, population characteristics can affect the results. Although some studies have demonstrated that self-reported assessments can be highly erroneous, it was discovered that the self-estimated and clinical assessments of halitosis correlated well. Self-evaluation of halitosis has many limitations because it is a subjective assessment. The fact that it considers any offensive odor that has a detrimental effect on quality of life means that it is still clinically significant. Numerous research have linked oral hygiene practices including brushing and flossing with self-reported bad breath. According to this study, which is in line with earlier research, cleaning teeth less frequently results in more instances of subjective bad breath. The Nalçaci et al study found that using the toothbrush less frequently than once a day was the factor most closely linked to self-perceived bad breath. According to Kayombo et al, a low prevalence of halitosis was connected with regular brushing and tongue cleansing. Adult halitosis may be brought on by a number of conditions, including the use of drugs, drug use, hyposalivation, and sociodemographic and behavioral variables. In this study, like in the study by Moreno et al, participants who take regular medication have a greater rate of halitosis evaluation. Additionally, although it is not statistically significant, patients with systemic diseases have a higher frequency of halitosis. It is well known that some medications alter the onset of foul breath by decreasing salivation. According to the findings of Saniya Setia et al’s study with dentistry students, women practiced superior oral hygiene and much less halitosis than men. According to Milanesi et al, women are 2.57 times more likely than men to report having unpleasant breath. Men are more likely than women to self-report having halitosis, according to some studies. However, there was no difference between the genders in the frequency of halitosis in several studies. Although there was no statistically significant difference between the assessments of halitosis in the male and female study participants, they are also very close to one another. In terms of awareness and oral health, education level is a crucial factor. Additionally, greater oral hygiene may be linked to improved oral health knowledge and habits, which would lead to a decrease in the occurrence of oral health issues. Higher rates of self-perceived oral malodor were reported by subjects with less education than by those with a college degree. A greater education level was linked to less self-reported halitosis, according to another study. Similar to the literature, the prevalence of reporting halitosis in this study was lower among people with higher education levels. Self-reported foul breath was significantly linked to smoking, according to Kayambo et al. Teshome et al in 2021, it was revealed, in line with earlier studies, that oral halitosis is more common in smokers, with smokers having a higher risk of developing halitosis than nonsmokers. It was discovered that the sample taking part in the study had a low smoking rate and that there was no statistically significant correlation between smoking and self-perceived halitosis. It was discovered that assessments of halitosis in smokers and nonsmokers were rather similar. According to Silva et al, people with periodontitis had a 90% higher incidence of self-reported halitosis than people in good health. In this study, gingival health was associated with lower levels of halitosis, whereas periodontal disease was associated with higher levels of halitosis. The association between halitosis and bleeding, gingival index scores, and plaque index scores in recorded periodontal diagnosis and probing were examined in this study. Halitosis and the severity of periodontal disease were revealed to be significantly correlated by the evaluation. Our research was carried out in a single hospital and may not necessarily represent the broader community in the area. Our study’s most significant flaw is that it only took into account the patients’ perceptions of their own self-reported levels of halitosis. In addition sinusitis problems, respiratory problems, diabetes and dry mouth can cause bad breath. Sometimes these are noticeable to people, while sometimes individuals may not notice them. In our study, the necessary equipment could be used to compare the bad breath that individuals know with the real bad breath. This is another limitation of our study. However, unlike other studies, it was conducted with patients who came to the periodontology clinic. There are limited number of studies on this subject in our country. Actually, it is important to come to a clinic where conditions that may cause bad breath will be treated, but it is interesting that many people come to the clinic because bad breath is not the reason. The fact that a human perceptible malodor truly indicates the presence of this illness makes this approach remain crucial for therapeutic usage. Furthermore, no special tools are required to diagnose self-reported foul breath. Our study’s findings demonstrated that whether someone has halitosis or not, they are most likely to notice it in themselves and their relatives. The dentists’ reported rates are the lowest. The fact that most people forego dental care and visits to the dentist could be the cause of this. Dentists should especially detect the presence of bad breath and plan treatments to eliminate the causative factor, and increase the awareness of the patients. Sinusitis problems, respiratory problems, diabetes and dry mouth can cause bad breath. Sometimes these are noticeable to individuals, while sometimes individuals may not notice them. In our study, the necessary equipment could have been used to compare the bad breath individuals know with the real bad breath. 4.1. Clinical relevance 4.1.1. Scientific relevance of the study Halitosis is a complex problem that adversely affects the quality of life of those who suffer from it. Although there are many studies on this subject, the studies generally carried out with dentistry students. There are very few studies evaluating the prevalence of halitosis, which come to the periodontology clinic for any reason. 4.1.2. Principal findings Our study reveals the situations in which self-perceived halitosis is relevant. Periodontal diseases, regular drug use, inadequate oral hygiene, low education level, advanced age, marital status, increase in plaque index-gingival index scores, and presence of bleeding on probing are factors associated with halitosis. 4.1.3. Practical implications Halitosis is an important health problem that affects people’s social relationships and lives. In planning the treatment of the disease, the underlying causes should be learned first.
4.1.1. Scientific relevance of the study Halitosis is a complex problem that adversely affects the quality of life of those who suffer from it. Although there are many studies on this subject, the studies generally carried out with dentistry students. There are very few studies evaluating the prevalence of halitosis, which come to the periodontology clinic for any reason. 4.1.2. Principal findings Our study reveals the situations in which self-perceived halitosis is relevant. Periodontal diseases, regular drug use, inadequate oral hygiene, low education level, advanced age, marital status, increase in plaque index-gingival index scores, and presence of bleeding on probing are factors associated with halitosis. 4.1.3. Practical implications Halitosis is an important health problem that affects people’s social relationships and lives. In planning the treatment of the disease, the underlying causes should be learned first.
Halitosis is a complex problem that adversely affects the quality of life of those who suffer from it. Although there are many studies on this subject, the studies generally carried out with dentistry students. There are very few studies evaluating the prevalence of halitosis, which come to the periodontology clinic for any reason.
Our study reveals the situations in which self-perceived halitosis is relevant. Periodontal diseases, regular drug use, inadequate oral hygiene, low education level, advanced age, marital status, increase in plaque index-gingival index scores, and presence of bleeding on probing are factors associated with halitosis.
Halitosis is an important health problem that affects people’s social relationships and lives. In planning the treatment of the disease, the underlying causes should be learned first.
The authors thank HK for his assistance with statistical calculations and for providing Statistical Consulting.
Conceptualization: Ozkan Sen Dilek. Data curation: Ozkan Sen Dilek, Durmaz Esra Nur. Formal analysis: Ozkan Sen Dilek. Methodology: Ozkan Sen Dilek. Writing – original draft: Ozkan Sen Dilek, Durmaz Esra Nur, Saraç Fatma. Writing – review & editing: Ozkan Sen Dilek.
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Vitamin D3/VDR alleviates double-stranded RNA virus -induced biliary epithelial cell damage by inhibiting autophagy | 789fdce8-3ac3-4dcd-9968-325c87d474c1 | 11780797 | Surgical Procedures, Operative[mh] | Biliary atresia (BA), which is characterized by progressive fibro-inflammation of the extrahepatic and intrahepatic bile ducts, may lead to cirrhosis and liver failure . Kasai portoenterostomy (KPE) can restore bile drainage and remains the first choice of treatment for BA at present. Hormones, antibiotics, hepatoprotective agents, fat-soluble vitamins, and probiotics are often used to treat cholangitis and complications after KPE . However, approximately 60% of patients develop liver failure after KPE and consequently require life-saving liver transplantation . Currently, the etiology and pathogenesis of BA have not been elucidated, and effective strategies have not been developed to prevent progressive liver fibrosis postoperatively . Therefore, the mechanisms involved in the occurrence and development of BA must be revealed. The early inhibition of BA progression can improve the prognosis of patients with BA. In patients with BA, 1,25(OH)2D3 (1,25-VD3) deficiency is common due to the vitamin D activation disorder, and 1,25-VD3 deficiency is inversely correlated with liver fibrosis . Vitamin D receptor (VDR) knockout mice spontaneously develop hepatic fibrosis . In the liver, VDR is mainly expressed in hepatic non-parenchymal cells such as hepatic stellate cells, sinus endothelial cells, Kupffer cells, and bile duct epithelial cells . VDR plays an important role in maintaining the innate immunity of biliary epithelial cells (BECs) . In mice, VDR deficiency in BECs disrupts cell junctions . The apoptosis of intrahepatic bile duct epithelial cells (IBDECs) and extrahepatic bile duct epithelial cells is significantly upregulated in BA and is considered the factor initiating progressive liver fibrosis in BA patients . The roles of VDR in the occurrence and progression of BA and the underlying molecular mechanisms have not been previously reported. In this study, we examined the roles of 1,25-VD3 and VDR in the pathogenesis of BA along with the underlying mechanisms.
The significant reduction of VDR expression in IBDECs may be a poor prognostic marker for BA As shown in Fig. , VDR protein expression was localized to the cytoplasm of IBDECs with tan granular and diffusely distributed patterns (Fig. A). The mean immunohistochemical positive score of VDR in the IBDECs of children with choledochal cysts was approximately 3. Of the 38 pediatric patients with BA, 23 patients with a mean immunohistochemical VDR score ≥ 3 were classified into the “no-reduced VDR expression” (VDR-N) group. Meanwhile, 15 patients with a mean immunohistochemical VDR score of < 3 were classified into the VDR-R group (Fig. B). Patients in the VDR-R group were associated with an increased rate of cholangitis ( p = 0.037; Table ) and decreased native liver survival time after KPE ( p = 0.032 , Fig. C). The clinicopathological characteristics of the patients were retrieved from their clinical records and are shown in Table . Poly(I: C) promoted HIBEC damage and apoptosis by inducing autophagy Poly(I: C) induced autophagy in HIBEC in a concentration-dependent manner. The level of autophagy induced by poly(I: C) at a concentration of 5 µg/mL was comparable to that induced by higher concentrations (Fig. ); thus, poly(I: C) at a concentration of 5 µg/mL was used in subsequent experiments. The induction of autophagy in HIBEC by poly(I: C) was time dependent (Fig. A). Chloroquine (an inhibitor of autophagy) mitigated the poly(I: C)-induced downregulation of P62/SQSTM1, an autophagy-related protein (Fig. B). Consistently, co-treatment with chloroquine and poly(I: C) significantly decreased the formation of RFP-LC3 puncta, indicating that chloroquine mitigated poly(I: C)-induced autophagy ( p < 0.05; Fig. C, D), further demonstrating that chloroquine mitigated the poly(I: C)-induced autophagy of HIBECs. Notably, poly(I: C) promoted apoptosis and inhibited the activity of HIBECs, whereas chloroquine suppressed the poly(I: C)-induced upregulation of apoptosis ( p < 0.01, Fig. E, F) and inhibition of cell activity ( p < 0.01, p < 0.01, Fig. G). Chloroquine also suppressed the poly(I: C)-induced secretion of inflammatory cytokines, such as INF-β, TNF-α, and IL-6 ( p < 0.01, p < 0.05, p < 0.01, respectively, Fig. H, I, J). These findings suggest that poly(I: C) induced HIBEC damage and apoptosis by promoting autophagy. The autophagy inhibitor mitigated poly(I: C)-induced HIBEC injury and apoptosis by suppressing autophagy. 1,25-VD3 mitigated dsRNA-induced BEC damage and apoptosis by inhibiting autophagy Poly(I: C) significantly downregulated VDR expression in HIBECs. As the treatment concentration of 1,25-VD3 increased, the decreased expression of VDR in HIBECs caused by poly(I: C) was gradually restored, demonstrating that 1,25-VD3 suppressed the inhibitory effect of poly(I: C) on VDR expression in a dose-dependent manner (Fig. A). Compared with the control group, poly(I: C) significantly increased the expression ratio of LC3B II to LC3BI in HIBECs while significantly decreasing the expression of P62. As the treatment concentration of 1,25-VD3 increased, the above phenomena were reversed, suggesting that 1,25-VD3 can inhibit the degree of autophagy induction in HIBECs by poly(I: C) in a concentration-dependent manner (Fig. B). 1,25-VD3 suppressed the autophagic flux in poly(I: C)-induced HIBECs ( p < 0.05, Fig. C, D). The effects of poly(I: C) on the apoptosis ( p < 0.05, Fig. E, F), cell viability (Fig. G), and the secretion of inflammatory cytokines were obviously reversed by 1,25-VD3 in a dose-dependent manner (IFN-β, p < 0.05, TNF-α, p < 0.05 and IL-6, p < 0.05, respectively, Fig. H, I, J). VDR activator mitigated dsRNA-induced BEC damage and apoptosis by inhibiting autophagy. Because the protective effect of 1,25-VD3 at 50 nM against poly(I: C)-induced HIBEC damage was like that of higher concentrations, 50 nM was used in subsequent experiments. VDR knockdown suppressed the protective effect of 1,25-VD3 against poly(I: C)-induced HIBEC damage 1,25-VD3 upregulated VDR expression in poly(I: C)-treated HIBECs. To further confirm the role of VDR in poly(I: C)-treated HIBECs, HIBECs were transfected with si-VDR, pcDNA3.1-VDR, or the corresponding negative controls. Transfection with si-VDR and pcDNA3.1-VDR significantly downregulated and upregulated VDR expression, respectively ( p < 0.01, p < 0.01, respectively, Fig. A). Next, HIBECs were transfected with si-VDR and subsequently treated with poly(I: C) and 1,25-VD3. VDR knockdown significantly mitigated the protective effects of 1,25-VD3 on the poly(I: C)-induced upregulation of apoptosis ( p < 0.05, Fig. B, C), downregulation of cell activity ( p < 0.05, Fig. D), and secretion of inflammatory factors in HIBECs ( IFN-β , p < 0.05 TNF-α , p < 0.05 and IL-6, p < 0.05, respectively, Fig. E). Western blotting analysis indicated that VDR knockdown suppressed the inhibitory effect of 1,25-VD3 on poly(I: C)-induced autophagy, as evidenced by analyzing the numbers of red puncta in different treatment groups (Fig. F, G). This was further confirmed by the downregulation of P62/SQSTM1 (Fig. H, I). These findings indicate that VDR mediated the protective effects of 1,25-VD3 on poly(I: C)-induced HIBEC damage and apoptosis. 1,25-VD3 exerted a protective effect on poly(I: C)-induced HIBEC damage through the VDR/SRC and PLA2/PKC/ERK pathways The molecular mechanism underlying the effects of VDR on poly(I: C)-induced autophagy in HIBECs was examined. CAV1 and SRC are thought to be involved in VDR signaling . The interaction between VDR and SRC was examined through Co-IP assay. VDR protein was enriched in the plasma membranes immunoprecipitated with anti-c-Src antibodies (IP-c-Src) (Fig. A, left panel). Meanwhile, SRC protein was enriched in the sample immunoprecipitated with anti-VDR (IP-VDR) antibodies (Fig. A, right panel). HIBECs were treated with 1,25-VD3 and subjected to immunoprecipitation assay. As shown in Fig. B, SRC and p-Y419-SRC coimmunoprecipitated with VDR in the plasma membrane fractions isolated from 1,25-VD3-treated HIBECs. Subsequently, the expressions of Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM1, also called TRIF), TLR3, and VDR proteins were examined. VDR knockdown significantly suppressed the effects of 1,25-VD3 on the expressions of VDR, TRIF and TLR3 (Fig. C, D, E). The interaction between 1,25-VD3 and VDR results in the activation of PLA2 and PKC downstream. Thus, the activities of PLA2 and PKC in HIBECs transfected with VDR siRNA and subsequently treated with poly(I: C) and 1,25-VD3 were examined. VDR knockdown partly mitigated the 1,25-VD3-induced upregulation of PLA2 and PKC activity ( p < 0.05, p < 0.05, respectively, Fig. F and G). Additionally, 1,25-VD3 significantly suppressed the poly(I: C)-induced downregulation of SRC and ERK1/2 phosphorylation. In contrast, transfection with si-VDR mitigated the effects of 1,25-VD3 on SRC and ERK1/2 phosphorylation ( p < 0.05, p < 0.05, respectively, Fig. H, I, J). VDR activators may provide an effective approach for the treatment of BA The rate of jaundice was significantly lower in the RRV + 1,25-VD3 group than in the RRV group at day 12 after viral inoculation (100% in RRV group vs. 50% in the RRV + 1,25-VD3 group; Fig. A and B, left). None of the mice in the WT + RRV group survived more than 14 d, while 40% of the mice in the 1,25-VD3 + RRV group survived for more than 18 d (Fig. B, right). In the 1,25-VD3 + RRV group, no blockages were observed in the extra-hepatic bile ducts (Fig. C), and no obvious infiltration of inflammatory cells was found around the intrahepatic bile ducts (Fig. D). The TEM images showed that no obvious autophagosomes were observed in the EBDECs of the control group. In the RRV group, a small amount of autophagic vesicles appeared in the EBDECs at day 6, and more autophagic vesicles were observed at day 12. The mitochondria were extremely swollen, and the crista process was disorganized, broken, or missing in the EBDECs. Compared with the RRV group, the amount of autophagic vesicles at day 12 was significantly reduced in the RRV + 1,25-VD3 group (Fig. E). The TEM images revealed that RRV induced the autophagosomal encapsulation of mitochondria in the EBDECs, while 1,25-VD3 significantly inhibited the autophagy induced by RRV in EBDECs. We conducted a retrospective analysis of patients with BA who received oral calcitriol after KPE. The incidence of cholangitis after surgery in the calcitriol-treated group was significantly lower than that in the control group (Table ). Additionally, the native liver survival time was higher in the calcitriol-treated group ( p = 0.035, Fig. ). The clinicopathological characteristics of the patients with BA are shown in Table . Thus, we can conclude that VDR activators exert a protective effect against dsRNA virus-induced BEC damage by inhibiting autophagy. These findings indicate that VDR activator is a potentially effective therapeutic approach for BA.
As shown in Fig. , VDR protein expression was localized to the cytoplasm of IBDECs with tan granular and diffusely distributed patterns (Fig. A). The mean immunohistochemical positive score of VDR in the IBDECs of children with choledochal cysts was approximately 3. Of the 38 pediatric patients with BA, 23 patients with a mean immunohistochemical VDR score ≥ 3 were classified into the “no-reduced VDR expression” (VDR-N) group. Meanwhile, 15 patients with a mean immunohistochemical VDR score of < 3 were classified into the VDR-R group (Fig. B). Patients in the VDR-R group were associated with an increased rate of cholangitis ( p = 0.037; Table ) and decreased native liver survival time after KPE ( p = 0.032 , Fig. C). The clinicopathological characteristics of the patients were retrieved from their clinical records and are shown in Table .
Poly(I: C) induced autophagy in HIBEC in a concentration-dependent manner. The level of autophagy induced by poly(I: C) at a concentration of 5 µg/mL was comparable to that induced by higher concentrations (Fig. ); thus, poly(I: C) at a concentration of 5 µg/mL was used in subsequent experiments. The induction of autophagy in HIBEC by poly(I: C) was time dependent (Fig. A). Chloroquine (an inhibitor of autophagy) mitigated the poly(I: C)-induced downregulation of P62/SQSTM1, an autophagy-related protein (Fig. B). Consistently, co-treatment with chloroquine and poly(I: C) significantly decreased the formation of RFP-LC3 puncta, indicating that chloroquine mitigated poly(I: C)-induced autophagy ( p < 0.05; Fig. C, D), further demonstrating that chloroquine mitigated the poly(I: C)-induced autophagy of HIBECs. Notably, poly(I: C) promoted apoptosis and inhibited the activity of HIBECs, whereas chloroquine suppressed the poly(I: C)-induced upregulation of apoptosis ( p < 0.01, Fig. E, F) and inhibition of cell activity ( p < 0.01, p < 0.01, Fig. G). Chloroquine also suppressed the poly(I: C)-induced secretion of inflammatory cytokines, such as INF-β, TNF-α, and IL-6 ( p < 0.01, p < 0.05, p < 0.01, respectively, Fig. H, I, J). These findings suggest that poly(I: C) induced HIBEC damage and apoptosis by promoting autophagy. The autophagy inhibitor mitigated poly(I: C)-induced HIBEC injury and apoptosis by suppressing autophagy.
Poly(I: C) significantly downregulated VDR expression in HIBECs. As the treatment concentration of 1,25-VD3 increased, the decreased expression of VDR in HIBECs caused by poly(I: C) was gradually restored, demonstrating that 1,25-VD3 suppressed the inhibitory effect of poly(I: C) on VDR expression in a dose-dependent manner (Fig. A). Compared with the control group, poly(I: C) significantly increased the expression ratio of LC3B II to LC3BI in HIBECs while significantly decreasing the expression of P62. As the treatment concentration of 1,25-VD3 increased, the above phenomena were reversed, suggesting that 1,25-VD3 can inhibit the degree of autophagy induction in HIBECs by poly(I: C) in a concentration-dependent manner (Fig. B). 1,25-VD3 suppressed the autophagic flux in poly(I: C)-induced HIBECs ( p < 0.05, Fig. C, D). The effects of poly(I: C) on the apoptosis ( p < 0.05, Fig. E, F), cell viability (Fig. G), and the secretion of inflammatory cytokines were obviously reversed by 1,25-VD3 in a dose-dependent manner (IFN-β, p < 0.05, TNF-α, p < 0.05 and IL-6, p < 0.05, respectively, Fig. H, I, J). VDR activator mitigated dsRNA-induced BEC damage and apoptosis by inhibiting autophagy. Because the protective effect of 1,25-VD3 at 50 nM against poly(I: C)-induced HIBEC damage was like that of higher concentrations, 50 nM was used in subsequent experiments.
1,25-VD3 upregulated VDR expression in poly(I: C)-treated HIBECs. To further confirm the role of VDR in poly(I: C)-treated HIBECs, HIBECs were transfected with si-VDR, pcDNA3.1-VDR, or the corresponding negative controls. Transfection with si-VDR and pcDNA3.1-VDR significantly downregulated and upregulated VDR expression, respectively ( p < 0.01, p < 0.01, respectively, Fig. A). Next, HIBECs were transfected with si-VDR and subsequently treated with poly(I: C) and 1,25-VD3. VDR knockdown significantly mitigated the protective effects of 1,25-VD3 on the poly(I: C)-induced upregulation of apoptosis ( p < 0.05, Fig. B, C), downregulation of cell activity ( p < 0.05, Fig. D), and secretion of inflammatory factors in HIBECs ( IFN-β , p < 0.05 TNF-α , p < 0.05 and IL-6, p < 0.05, respectively, Fig. E). Western blotting analysis indicated that VDR knockdown suppressed the inhibitory effect of 1,25-VD3 on poly(I: C)-induced autophagy, as evidenced by analyzing the numbers of red puncta in different treatment groups (Fig. F, G). This was further confirmed by the downregulation of P62/SQSTM1 (Fig. H, I). These findings indicate that VDR mediated the protective effects of 1,25-VD3 on poly(I: C)-induced HIBEC damage and apoptosis.
The molecular mechanism underlying the effects of VDR on poly(I: C)-induced autophagy in HIBECs was examined. CAV1 and SRC are thought to be involved in VDR signaling . The interaction between VDR and SRC was examined through Co-IP assay. VDR protein was enriched in the plasma membranes immunoprecipitated with anti-c-Src antibodies (IP-c-Src) (Fig. A, left panel). Meanwhile, SRC protein was enriched in the sample immunoprecipitated with anti-VDR (IP-VDR) antibodies (Fig. A, right panel). HIBECs were treated with 1,25-VD3 and subjected to immunoprecipitation assay. As shown in Fig. B, SRC and p-Y419-SRC coimmunoprecipitated with VDR in the plasma membrane fractions isolated from 1,25-VD3-treated HIBECs. Subsequently, the expressions of Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM1, also called TRIF), TLR3, and VDR proteins were examined. VDR knockdown significantly suppressed the effects of 1,25-VD3 on the expressions of VDR, TRIF and TLR3 (Fig. C, D, E). The interaction between 1,25-VD3 and VDR results in the activation of PLA2 and PKC downstream. Thus, the activities of PLA2 and PKC in HIBECs transfected with VDR siRNA and subsequently treated with poly(I: C) and 1,25-VD3 were examined. VDR knockdown partly mitigated the 1,25-VD3-induced upregulation of PLA2 and PKC activity ( p < 0.05, p < 0.05, respectively, Fig. F and G). Additionally, 1,25-VD3 significantly suppressed the poly(I: C)-induced downregulation of SRC and ERK1/2 phosphorylation. In contrast, transfection with si-VDR mitigated the effects of 1,25-VD3 on SRC and ERK1/2 phosphorylation ( p < 0.05, p < 0.05, respectively, Fig. H, I, J).
The rate of jaundice was significantly lower in the RRV + 1,25-VD3 group than in the RRV group at day 12 after viral inoculation (100% in RRV group vs. 50% in the RRV + 1,25-VD3 group; Fig. A and B, left). None of the mice in the WT + RRV group survived more than 14 d, while 40% of the mice in the 1,25-VD3 + RRV group survived for more than 18 d (Fig. B, right). In the 1,25-VD3 + RRV group, no blockages were observed in the extra-hepatic bile ducts (Fig. C), and no obvious infiltration of inflammatory cells was found around the intrahepatic bile ducts (Fig. D). The TEM images showed that no obvious autophagosomes were observed in the EBDECs of the control group. In the RRV group, a small amount of autophagic vesicles appeared in the EBDECs at day 6, and more autophagic vesicles were observed at day 12. The mitochondria were extremely swollen, and the crista process was disorganized, broken, or missing in the EBDECs. Compared with the RRV group, the amount of autophagic vesicles at day 12 was significantly reduced in the RRV + 1,25-VD3 group (Fig. E). The TEM images revealed that RRV induced the autophagosomal encapsulation of mitochondria in the EBDECs, while 1,25-VD3 significantly inhibited the autophagy induced by RRV in EBDECs. We conducted a retrospective analysis of patients with BA who received oral calcitriol after KPE. The incidence of cholangitis after surgery in the calcitriol-treated group was significantly lower than that in the control group (Table ). Additionally, the native liver survival time was higher in the calcitriol-treated group ( p = 0.035, Fig. ). The clinicopathological characteristics of the patients with BA are shown in Table . Thus, we can conclude that VDR activators exert a protective effect against dsRNA virus-induced BEC damage by inhibiting autophagy. These findings indicate that VDR activator is a potentially effective therapeutic approach for BA.
The etiology and pathogenesis of BA have not been elucidated. Previous studies have demonstrated that inflammatory and immune responses are involved in the pathogenesis of BA, with viral infection and genetic variation being the etiological factors . In BA, the apoptosis and epithelial–mesenchymal transition of BECs are associated with dsRNA virus-induced impaired innate immune response . The direct virus-induced damage of BECs and secondary autoimmune reaction-induced damage to BECs lead to the occlusion of bile duct, resulting in the development of BA and liver fibrosis . Poly(I: C) is an authentic mimic of viral dsRNA that elicits immune responses in HIBECs . In this study, poly(I: C) significantly promoted autophagy and inflammatory response in HIBECs, decreased HIBEC viability, and enhanced HIBEC apoptosis. Chloroquine, an inhibitor of autophagy, significantly suppressed poly(I: C)-induced autophagy, inflammation, and apoptosis in HIBECs. These results reveal that the pathological mechanism underlying poly(I: C)-induced apoptosis in HIBECs involves autophagy, and the inhibition of autophagy alleviates dsRNA virus-induced damage and apoptosis in HIBECs. Autophagy plays an important role in maintaining the physiological functions of cells . The dysregulation of autophagy can lead to ubiquitination inhibition, reactive oxygen species accumulation, mitochondrial dysfunction, increased genomic instability, and disrupted cellular homeostasis, thereby promoting the onset and progression of various diseases . Dysregulated autophagy can also help induce BEC senescence . The markers of senescence (p53 and senescence-related β-galactosidase) are expressed in IBDECs of children with BA . TEM revealed autophagic bodies in the IBDECs of pediatric patients with BA (Fig. ). In patients with BA, the expression of P62 protein was significantly elevated in hepatocytes and IBDECs compared to children with choledochal cysts (Fig. ). This suggests that autophagy dysregulation in BECs is a potential mechanism underlying BA pathogenesis. Autophagy has differential roles in different liver diseases. For example, autophagy induction exerts therapeutic effects in alcoholic hepatitis and nonalcoholic cirrhosis. Meanwhile, autophagy inhibition exerts therapeutic effects in liver fibrosis . Thus, autophagy regulation may a potentially effective therapeutic strategy for BA. Vitamin D activates autophagy through genomic and non-genomic signaling pathways, modulates various physiological functions of different organs, and regulates bone health and calcium metabolism . Additionally, vitamin D is a natural agonist of VDR that induces autophagy as a protective mechanism to inhibit oxidative stress and apoptosis and, consequently, regulates cell proliferation, differentiation, and immune response . For the first time, this study demonstrated that poly(I: C) promotes apoptosis and downregulates VDR expression in HIBECs. 1,25-VD3 exerted a protective effect against poly(I: C)-induced autophagy in HIBECs. Additionally, compared with the VDR-N group, pediatric patients with BA in the VDR-R group showed an increased frequency of cholangitis and a decreased native liver survival time after KPE. These results suggest that VDR downregulation in IBDECs may be a biomarker of poor prognosis in pediatric patients with BA. Hepatic VDR expression is markedly downregulated in patients with primary biliary cholangitis . VDR plays an important role in maintaining the innate immunity of the bile duct epithelium . A deficiency in VDR results in the loss of cell adhesion in BECs and disrupts bile duct integrity . We speculated that dsRNA viruses induce BEC damage and apoptosis, significantly downregulate VDR expression, and promote bile duct damage, thereby contributing to poor prognosis in pediatric patients with BA who exhibit downregulation VDR expression in IBDECs. In this study, 1,25-VD3 alleviated poly(I: C)-induced HIBEC damage by inhibiting autophagy in vitro, indicating that VDR activators are potential therapeutic agents for virus-induced BA. An RRV-induced BA murine model was used to verify the potential therapeutic effect of VDR activator on BA. 1,25-VD3 alleviated RRV-induced extrahepatic bile duct stenosis and intrahepatic inflammation in vivo. TEM revealed that 1,25-VD3 exerted a protective effect in extrahepatic BDECs by inhibiting RRV-induced autophagy. 1,25-VD3-induced autophagy is a protective mechanism that inhibits oxidative stress and apoptosis and regulates cell proliferation, differentiation, and immune response . Vitamin D supplementation has been shown to have potential therapeutic effects in lupus animal models by modulating autophagy . In intervertebral disc degeneration, VDR activation alleviates oxidative damage and suppresses nucleus pulposus apoptosis by promoting mitochondrial autophagy . Sun et al. reported that pediatric patients with BA show impaired vitamin D activation, and vitamin D deficiency is common and inversely correlated with liver fibrosis in BA patients. Calcitriol, an active metabolite of vitamin D and an agonist of VDR, has been used in the treatment of rickets in infants , demonstrating the basic safety of calcitriol in the treatment of pediatric diseases. After KPE, BA patients treated with calcitriol showed a lower frequency of postoperative cholangitis, slower progression of liver fibrosis, and longer native liver survival time compared with untreated BA patients. These findings suggest that VDR activation is a potentially effective method for the treatment of BA. In RRV-infected BECs, the expressions of pattern recognition receptors (e.g., Toll-like receptors) are upregulated, and the intracellular signaling pathways are activated, resulting in cell damage . Poly(I: C), an extensively used immune stimulant, activates TLR3 ligand, induces an innate immune response, and upregulates TLR3 in BECs . The lack of innate immune tolerance of poly(I: C) by TLR3 in BECs may contribute to the development of BA . TLR3 mediates the host response against RNA virus through TRIF . The TLR3 signaling pathway is dysregulated in TRIF-deficient or TRIF mutant mice , and TRIF overexpression can regulate the autophagy ubiquitination pathway . This suggests that TRIF regulates TLR3 and autophagy ubiquitination. In this study, 1,25-VD3 mitigated the significant poly(I: C)-induced upregulation of TLR3 and TRIF3 in HIBECs. Meanwhile, VDR knockdown suppressed the beneficial effect of 1,25-VD3, suggesting that 1,25-VD3 activates VDR to regulate the autophagy ubiquitination pathway. 1,25-VD3 binds to VDR, which activates VDR-associated SRC to promote the release of the PDIA3/PLAA complex from CAV1 and subsequently PLA, thereby activating ERK1/2 . This study also demonstrated that VDR knockdown suppresses the 1,25-VD3-induced upregulation of PLA2 and PKC activities and ERK1/2 phosphorylation. In summary, the 1,25-VD3/VDR/SRC axis mitigates poly(I: C)-induced HIBEC damage and apoptosis through the PLA2/PKC/ERK pathway. This study has several limitations that must be addressed when considering clinical applications. First, this study only preliminarily demonstrated that the inhibition of autophagy can alleviate dsRNA virus-induced BEC damage and intrahepatic inflammation. The biliary atresia (BA) animal model used in this study cannot account for BA caused by cytomegalovirus infection or environmental toxins (such as biliatresone), which means the findings may not fully address damage caused by these other risk factors. Therefore, further studies are needed to determine the proportion of patients with BA who could benefit from clinical treatment with VDR activators. Second, the molecular mechanisms by which 1,25-VD3 alleviates cholangiocyte injury induced by RRV in animals and poly(I: C) in cells still need further verification to determine if they are consistent. Third, autophagy plays different roles in various liver diseases . Promoting autophagy has potential therapeutic effects in alcoholic liver hepatitis and non-alcoholic liver cirrhosis, while inhibiting autophagy shows potential therapeutic effects in liver fibrosis and dsRNA virus-induced BEC damage. Our results did not reveal a significant correlation between the expression levels of VDR and P62 in IBDECs. Autophagy is a dynamic process, and its regulation may vary with the progression of the disease . Thus, further clarification is needed to (1) determine which stage of autophagy is specifically affected by dsRNA virus-induced injury and bile duct epithelial cell apoptosis and (2) reveal the changes in autophagy levels at different stages of BA progression. These factors are crucial for treating BA through autophagy modulation. Therefore, much work is still needed to understand how to precisely modulate autophagy for the early-stage treatment of BA. Fourth, existing studies have shown that Vitamin D3/VDR can regulate inflammation and gut microbiota imbalance , both of which may be contributing factors to the progression of BA. Whether Vitamin D3/VDR exerts its effects through mechanisms other than the regulation of autophagy is worthy of further investigation. Fifth, chloroquine and hydroxychloroquine are well-known inhibitors of autophagy that are used to treat malaria (including neonates) and coronavirus disease in children . However, the effectiveness of autophagy inhibitors in the early treatment of BA must be elucidated. Sixth, the number of pediatric patients BA included in this study was limited. Further large-scale randomized controlled trials are needed to validate the effects, optimal dosage, and duration of calcitriol administration for the treatment of BA. Finally, the prognosis of BA can be improved if the pathological process of BA can be blocked before extrahepatic bile duct obstruction. The effectiveness of VDR agonists in the early treatment of BA will be evaluated in future studies. In conclusion, this study demonstrated that VDR downregulation in IBDECs is a marker of poor prognosis in BA patients. VDR agonists/activators are potential therapeutic agents for virus-induced BA. Furthermore, the 1,25-VD3/VDR/SRC axis alleviated poly(I: C)-induced HIBEC damage and apoptosis. The findings of this study provided novel insights for developing therapeutic interventions for BA.
Human tissue specimens In this study, 38 BA and 10 choledochal cyst liver tissue samples were collected from patients undergoing surgery at the Second Affiliated Hospital and Children’s Hospital of Xi’an Jiaotong University between January 2015 and December 2019. For clinical observation, 30 patients with BA who underwent KPE between January 2021 and June 2023 were recruited. Among them, 16 patients with BA were assigned to the control group, and 14 were assigned to the treatment group. The children in the control group received anti-inflammatory therapy (intravenous cefoperazone sodium and sulbactam sodium), hormone therapy [intravenous methylprednisolone (4 mg/kg/d for 2 weeks) and oral prednisolone (2 mg/kg/d for 2 weeks)], and oral vitamin D after KPE surgery. The patients in the treatment group received Rocaltrol (calcitriol) after KPE (0.25 µg, qod) instead of vitamin D until the hormone therapy was stopped. The remaining post-surgery adjuvant therapies in the treatment group were identical to those in the control group. The study was approved by the Ethics Committee of Xi’an Jiaotong University and informed consent (2020 − 660) was obtained from all patients and voluntary participation prior to the study. Cell culture and treatment Human intrahepatic BECs (HIBECs) were purchased from Jennio Biotechnology (Guangzhou, China) and cultured in Dulbecco’s modified Eagle medium/Ham’s F12 medium (1:1) supplemented with 10% (v/v) fetal bovine serum, 5 ng/mL epidermal growth factor, 0.4 µg/mL succinyl hydrocortisone, 2 nM triiodothyronine, 5 µg/mL insulin, 10 µg/mL recombinant human hepatocyte growth factor, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in an atmosphere containing 5% (v/v) CO 2 . HIBECs passaged 4–8 times were used in this study. After reaching 75% confluency, the HIBECs were treated with 2.5–10 µg/mL poly(I: C) (Invitrogen, San Diego, CA, USA) for up to 48 h or with 5 µg/mL poly(I: C) for 24 h. Subsequently, the cells were treated with various concentrations of 1,25-VD3 for 24 h. Cell transfection The VDR overexpression vector (pcDNA3.1-VDR) and the control vector (pcDNA 3.1) were purchased from GenScript Biotech Corp. (Nanjing, China). Small interfering RNA (siRNA) against VDR (si-VDR) and its negative control (scramble) were purchased from ThermoFisher Scientific (Waltham, MA, USA). Plasmids and oligonucleotides were transfected into HIBECs using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. At 48 h after transfection, the cells were harvested for further analysis. Cell viability Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St Louis, USA) colorimetric assay. Briefly, cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well and subjected to different treatment regimens. After treatment for the indicated duration, the cells were incubated with MTT solution (20 µL, 5 mg/mL; Sigma) at 37 °C for 4 h. The medium was then removed, and the sample was incubated with 0.1 mL of dimethyl sulfoxide (Sigma) to dissolve the formazan product. Subsequently, the absorbance of the reaction mixture at 450 nm was measured using a microplate reader (BioTek, Winooski, USA). Cell apoptosis analysis Cell apoptosis was examined using an annexin V/fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (Becton Dickinson, USA) following the manufacturer’s instructions. At 48 h after transfection, the HIBECs were suspended in annexin-binding buffer and incubated with annexin V-FITC/PI solution for 15 min in the dark at room temperature. The apoptosis rate was determined using flow cytometry (CYTOMICS FC 500, Beckman Coulter, USA). Enzyme-linked immunosorbent assay (ELISA) The secretion levels of interleukin (IL)-6, interferon (IFN)-β and tumor necrosis factor (TNF)-α were determined by commercially available ELISA kit (Enzo Life Science, Inc., Farmingdale, NY, USA) according to the manufacturer’s protocol. Briefly, the culture supernatants were added to each well, incubated with the standard biotin solutions, absorbance was read at 450 nm using an ELISA reader (iMark microplate reader, Bio-Rad, Hercules, California, USA). The concentration of each cytokine was calculated by recombinant human polyclonal IFN-β, TNF-α and IL-6 standard curves. Western blotting Harvested cells were lysed for 30 min in radioimmunoprecipitation assay buffer (Sigma-Aldrich, St Louis, MO) supplemented with protease inhibitors. Total protein was extracted from the treated HIBECs using M-PER TM Mammalian Protein Extraction Reagent (ThermoFisher Scientific, Waltham, MA, USA). The extracted protein was quantified using a bicinchoninic acid assay kit (ThermoFisher Scientific, Waltham, MA, USA). The protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was sectioned before antibody hybridization and was individually probed with the following primary antibodies overnight at 4 °C: anti-VDR (ab32042, 1:500), anti-P62/SQSTM1 (ab91526, 1:1000), anti-LC3B (ab48394; 1:400), anti-C-SRC (ab109381; 1:1000), anti-Y419-phospho-SRC (ab185617; 1:1000), anti-TRIF (ab13810; 1:1000), anti-TLR3 (ab137722; 1:1000), anti-ERK1/2 (ab17942; 1:1000), anti-p-ERK1/2 (ab214362; 1:1000), and anti-GAPDH (ab8245, 1:5000) antibodies. Next, each membrane was washed twice with PBS and incubated with goat anti-rabbit IgG HRP (1:2000; Santa Cruz, CA, USA) for 1 h at room temperature. Immunoreactive signals were visualized using enhanced chemiluminescence and quantified using Image J software (NIH, Bethesda, MD, USA). GAPDH was used as the loading control. Detection of PKC activity PKC activity was measured using a PKC kinase activity assay kit (Abcam). Briefly, the lysates of treated HIBECs were transferred to a PKC substrate microtiter plate and incubated with 10 µL of ATP at 30 °C for 2 h to initiate substrate phosphorylation. The sample was then incubated with a specific antibody against the phosphorylated substrate at room temperature for 1 h followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 30 min. Immunoreactive signals were developed using tetramethylbenzidine. The reaction was terminated using the stop solution. The absorbance of the reaction mixture at 450 nm was evaluated using a microplate reader (BioTek, Winooski, USA). The PKC kinase activity was expressed as the optical density at 450 nm (OD 450 ). Detection of PLA2 activity The PLA2 activity was evaluated using a cPLA2 activity assay kit. After washing with PBS, cells were lysed to obtain the cell supernatant. The lysate was transferred to a microplate and incubated with arachidonoyl thio-phosphatidylcholine at room temperature for 1 h (following the manufacturer’s instructions). Subsequently, the sample was incubated with 5,5′-dithiobis (2-nitrobenzoic acid) for 5 min to terminate the reaction. The absorbance of the reaction mixture at 450 nm was measured using a microplate reader (BioTek, Winooski, USA). Co-immunoprecipitation (Co-IP) assay Co-immunoprecipitation was performed using a Thermo Scientific Pierce co-IP kit according to the manufacturer’s instructions. Whole cell lysate (WCL) was incubated overnight at 4 °C with A/G magnetic beads (B23202; Biotool) in Western/IP lysis buffer (Beyotime, Haimen, China). The immunoprecipitates were washed five times with Western/IP lysis buffer prior to IB. Samples were subjected to Western blot analysis using the following antibodies: anti-IgG (Proteintech, #30000-0-AP), anti-VDR (Abcam, #ab109234), anti-C-SRC (Proteintech, #11097-1-AP), anti-Y419-phospho-SRC (R&D, #DYC2685-2). IgG was regarded as a negative control. Examination of red fluorescence protein (RFP)-LC3B puncta To construct mRFP-LC3B-expressing cells, adenoviruses containing mRFP-LC3B coding sequences (Hanbio Biotechnology Co., Ltd, Shanghai, China) were transfected into the cells following the manufacturer’s instructions. At 24 h after transfection, the cells were fixed with 4% formaldehyde for 15 min at room temperature. The number of red puncta was counted under a confocal microscope (Molecular Devices, ImageXpress Micro Confocal System). Six non-overlapping fields were observed, and at least 20 cells were counted in each group. Immunohistochemical analysis Formalin-fixed, paraffin-embedded liver tissues were sectioned to a thickness of 5 μm. The sections were then deparaffinized with xylene, hydrated using a graded alcohol series, and subjected to antigen retrieval and blocking. The sections were then incubated with anti-VDR antibodies (ab3508, 1:200), anti-cytokeratin19 antibody (ab76539, 1:200), anti-SQSTM1/P62 antibody (ab207305, 1:200) at 4 °C overnight followed by incubation with secondary antibodies. Immunoreactive signals were detected using 3,3′-diaminobenzidine and hematoxylin. The expression of VDR and P62 protein were determined using a semiquantitative scoring method as described previously . Establishment of the rhesus rotavirus (RRV)-induced BA mouse model Timed pregnant BALB/c mice were purchased from Guangdong Animal Experimental Center, China. Newborn pups (within 24 h of birth) were intraperitoneally injected with RRV (20 µL of 1.5 × 10 6 plaque-forming units/mL) or the same dose of saline. The neonatal pups were then divided into the following three groups: control group (10, regardless of sex), RRV group (15, regardless of sex), and RRV + 1,25-VD3 (15, regardless of sex, 1,25-VD3 50 µg/kg bodyweight once every other day). The weight, appearance, and survival of the mice were recorded daily. The extrahepatic bile ducts of the mice were examined using cholangiography. The mice were anesthetized with 0.5–2.5% isoflurane, dissected under a microscope on days 6 and 12, and blood and liver samples were collected for analysis. Following blood collection, euthanize the mice by inhalation of 4% isoflurane for 10 min. Autophagosome formation in extrahepatic bile duct epithelial cells (EBDECs) was observed by transmission electron microscopy (TEM). All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Laboratory Animal Center of Xi’an Jiaotong University. Statistical analysis All data are represented as mean ± standard error of the mean from three independent experiments. The correlations between the expression of VDR and clinicopathologic characteristics were evaluated using Chi-square tests. Means between two groups were compared using Student’s t-test, while means between more than two groups were compared using one-way analysis of variance. All statistical analyses were performed in SPSS 22.0. Differences were considered significant at p < 0.05.
In this study, 38 BA and 10 choledochal cyst liver tissue samples were collected from patients undergoing surgery at the Second Affiliated Hospital and Children’s Hospital of Xi’an Jiaotong University between January 2015 and December 2019. For clinical observation, 30 patients with BA who underwent KPE between January 2021 and June 2023 were recruited. Among them, 16 patients with BA were assigned to the control group, and 14 were assigned to the treatment group. The children in the control group received anti-inflammatory therapy (intravenous cefoperazone sodium and sulbactam sodium), hormone therapy [intravenous methylprednisolone (4 mg/kg/d for 2 weeks) and oral prednisolone (2 mg/kg/d for 2 weeks)], and oral vitamin D after KPE surgery. The patients in the treatment group received Rocaltrol (calcitriol) after KPE (0.25 µg, qod) instead of vitamin D until the hormone therapy was stopped. The remaining post-surgery adjuvant therapies in the treatment group were identical to those in the control group. The study was approved by the Ethics Committee of Xi’an Jiaotong University and informed consent (2020 − 660) was obtained from all patients and voluntary participation prior to the study.
Human intrahepatic BECs (HIBECs) were purchased from Jennio Biotechnology (Guangzhou, China) and cultured in Dulbecco’s modified Eagle medium/Ham’s F12 medium (1:1) supplemented with 10% (v/v) fetal bovine serum, 5 ng/mL epidermal growth factor, 0.4 µg/mL succinyl hydrocortisone, 2 nM triiodothyronine, 5 µg/mL insulin, 10 µg/mL recombinant human hepatocyte growth factor, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in an atmosphere containing 5% (v/v) CO 2 . HIBECs passaged 4–8 times were used in this study. After reaching 75% confluency, the HIBECs were treated with 2.5–10 µg/mL poly(I: C) (Invitrogen, San Diego, CA, USA) for up to 48 h or with 5 µg/mL poly(I: C) for 24 h. Subsequently, the cells were treated with various concentrations of 1,25-VD3 for 24 h.
The VDR overexpression vector (pcDNA3.1-VDR) and the control vector (pcDNA 3.1) were purchased from GenScript Biotech Corp. (Nanjing, China). Small interfering RNA (siRNA) against VDR (si-VDR) and its negative control (scramble) were purchased from ThermoFisher Scientific (Waltham, MA, USA). Plasmids and oligonucleotides were transfected into HIBECs using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. At 48 h after transfection, the cells were harvested for further analysis.
Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St Louis, USA) colorimetric assay. Briefly, cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well and subjected to different treatment regimens. After treatment for the indicated duration, the cells were incubated with MTT solution (20 µL, 5 mg/mL; Sigma) at 37 °C for 4 h. The medium was then removed, and the sample was incubated with 0.1 mL of dimethyl sulfoxide (Sigma) to dissolve the formazan product. Subsequently, the absorbance of the reaction mixture at 450 nm was measured using a microplate reader (BioTek, Winooski, USA).
Cell apoptosis was examined using an annexin V/fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (Becton Dickinson, USA) following the manufacturer’s instructions. At 48 h after transfection, the HIBECs were suspended in annexin-binding buffer and incubated with annexin V-FITC/PI solution for 15 min in the dark at room temperature. The apoptosis rate was determined using flow cytometry (CYTOMICS FC 500, Beckman Coulter, USA).
The secretion levels of interleukin (IL)-6, interferon (IFN)-β and tumor necrosis factor (TNF)-α were determined by commercially available ELISA kit (Enzo Life Science, Inc., Farmingdale, NY, USA) according to the manufacturer’s protocol. Briefly, the culture supernatants were added to each well, incubated with the standard biotin solutions, absorbance was read at 450 nm using an ELISA reader (iMark microplate reader, Bio-Rad, Hercules, California, USA). The concentration of each cytokine was calculated by recombinant human polyclonal IFN-β, TNF-α and IL-6 standard curves.
Harvested cells were lysed for 30 min in radioimmunoprecipitation assay buffer (Sigma-Aldrich, St Louis, MO) supplemented with protease inhibitors. Total protein was extracted from the treated HIBECs using M-PER TM Mammalian Protein Extraction Reagent (ThermoFisher Scientific, Waltham, MA, USA). The extracted protein was quantified using a bicinchoninic acid assay kit (ThermoFisher Scientific, Waltham, MA, USA). The protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was sectioned before antibody hybridization and was individually probed with the following primary antibodies overnight at 4 °C: anti-VDR (ab32042, 1:500), anti-P62/SQSTM1 (ab91526, 1:1000), anti-LC3B (ab48394; 1:400), anti-C-SRC (ab109381; 1:1000), anti-Y419-phospho-SRC (ab185617; 1:1000), anti-TRIF (ab13810; 1:1000), anti-TLR3 (ab137722; 1:1000), anti-ERK1/2 (ab17942; 1:1000), anti-p-ERK1/2 (ab214362; 1:1000), and anti-GAPDH (ab8245, 1:5000) antibodies. Next, each membrane was washed twice with PBS and incubated with goat anti-rabbit IgG HRP (1:2000; Santa Cruz, CA, USA) for 1 h at room temperature. Immunoreactive signals were visualized using enhanced chemiluminescence and quantified using Image J software (NIH, Bethesda, MD, USA). GAPDH was used as the loading control.
PKC activity was measured using a PKC kinase activity assay kit (Abcam). Briefly, the lysates of treated HIBECs were transferred to a PKC substrate microtiter plate and incubated with 10 µL of ATP at 30 °C for 2 h to initiate substrate phosphorylation. The sample was then incubated with a specific antibody against the phosphorylated substrate at room temperature for 1 h followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 30 min. Immunoreactive signals were developed using tetramethylbenzidine. The reaction was terminated using the stop solution. The absorbance of the reaction mixture at 450 nm was evaluated using a microplate reader (BioTek, Winooski, USA). The PKC kinase activity was expressed as the optical density at 450 nm (OD 450 ).
The PLA2 activity was evaluated using a cPLA2 activity assay kit. After washing with PBS, cells were lysed to obtain the cell supernatant. The lysate was transferred to a microplate and incubated with arachidonoyl thio-phosphatidylcholine at room temperature for 1 h (following the manufacturer’s instructions). Subsequently, the sample was incubated with 5,5′-dithiobis (2-nitrobenzoic acid) for 5 min to terminate the reaction. The absorbance of the reaction mixture at 450 nm was measured using a microplate reader (BioTek, Winooski, USA).
Co-immunoprecipitation was performed using a Thermo Scientific Pierce co-IP kit according to the manufacturer’s instructions. Whole cell lysate (WCL) was incubated overnight at 4 °C with A/G magnetic beads (B23202; Biotool) in Western/IP lysis buffer (Beyotime, Haimen, China). The immunoprecipitates were washed five times with Western/IP lysis buffer prior to IB. Samples were subjected to Western blot analysis using the following antibodies: anti-IgG (Proteintech, #30000-0-AP), anti-VDR (Abcam, #ab109234), anti-C-SRC (Proteintech, #11097-1-AP), anti-Y419-phospho-SRC (R&D, #DYC2685-2). IgG was regarded as a negative control.
To construct mRFP-LC3B-expressing cells, adenoviruses containing mRFP-LC3B coding sequences (Hanbio Biotechnology Co., Ltd, Shanghai, China) were transfected into the cells following the manufacturer’s instructions. At 24 h after transfection, the cells were fixed with 4% formaldehyde for 15 min at room temperature. The number of red puncta was counted under a confocal microscope (Molecular Devices, ImageXpress Micro Confocal System). Six non-overlapping fields were observed, and at least 20 cells were counted in each group.
Formalin-fixed, paraffin-embedded liver tissues were sectioned to a thickness of 5 μm. The sections were then deparaffinized with xylene, hydrated using a graded alcohol series, and subjected to antigen retrieval and blocking. The sections were then incubated with anti-VDR antibodies (ab3508, 1:200), anti-cytokeratin19 antibody (ab76539, 1:200), anti-SQSTM1/P62 antibody (ab207305, 1:200) at 4 °C overnight followed by incubation with secondary antibodies. Immunoreactive signals were detected using 3,3′-diaminobenzidine and hematoxylin. The expression of VDR and P62 protein were determined using a semiquantitative scoring method as described previously .
] Timed pregnant BALB/c mice were purchased from Guangdong Animal Experimental Center, China. Newborn pups (within 24 h of birth) were intraperitoneally injected with RRV (20 µL of 1.5 × 10 6 plaque-forming units/mL) or the same dose of saline. The neonatal pups were then divided into the following three groups: control group (10, regardless of sex), RRV group (15, regardless of sex), and RRV + 1,25-VD3 (15, regardless of sex, 1,25-VD3 50 µg/kg bodyweight once every other day). The weight, appearance, and survival of the mice were recorded daily. The extrahepatic bile ducts of the mice were examined using cholangiography. The mice were anesthetized with 0.5–2.5% isoflurane, dissected under a microscope on days 6 and 12, and blood and liver samples were collected for analysis. Following blood collection, euthanize the mice by inhalation of 4% isoflurane for 10 min. Autophagosome formation in extrahepatic bile duct epithelial cells (EBDECs) was observed by transmission electron microscopy (TEM). All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Laboratory Animal Center of Xi’an Jiaotong University.
All data are represented as mean ± standard error of the mean from three independent experiments. The correlations between the expression of VDR and clinicopathologic characteristics were evaluated using Chi-square tests. Means between two groups were compared using Student’s t-test, while means between more than two groups were compared using one-way analysis of variance. All statistical analyses were performed in SPSS 22.0. Differences were considered significant at p < 0.05.
Below is the link to the electronic supplementary material. Supplementary Material 1
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Hypoglycaemia Prevention, Awareness of Symptoms, and Treatment (HypoPAST): protocol for a 24-week hybrid type 1 randomised controlled trial of a fully online psycho-educational programme for adults with type 1 diabetes | 4f653c3f-4711-48cf-a741-488b56fc069e | 11520494 | Patient Education as Topic[mh] |
Background and rationale People with type 1 diabetes (T1D) need insulin for survival. However, even with modern insulins and delivery systems, exogenous insulin delivery cannot fully mimic endogenous insulin supply . Hypoglycaemia (low blood glucose) remains a common side effect, caused by relative insulin excess in the absence of sufficient blood glucose. Around 20% of adults with T1D have experienced severe hypoglycaemia (requiring assistance from another person for recovery) in the past 6 months . Hypoglycaemia can be unpleasant, sudden, embarrassing, unpredictable and dangerous (e.g. causing accident or injury) . If undetected and untreated, hypoglycaemia can be life-threatening and associated with adverse outcomes . Repeated exposure to hypoglycaemia can lead to impaired awareness of hypoglycaemia symptoms, increasing a person’s risk of severe episodes, as they have reduced visibility of onset, reducing the window of opportunity for self-treatment . Unsurprisingly, repeated exposure to hypoglycaemia, severe hypoglycaemia, and impaired awareness of hypoglycaemia symptoms can lead to distress and hypoglycaemia-related anxiety, known as ‘fear of hypoglycaemia’ . Fear of hypoglycaemia can compromise diabetes self-management (e.g. maintaining higher glucose levels) and quality of life (e.g. limiting spontaneity, independence and activities in which hypoglycaemia may be a risk or problematic if it occurred) . ‘Worries about low blood glucose’ are consistently among the top five problem areas experienced by adults with T1D, with 75% experiencing it as at least a ‘mild problem’ and 40% as a ‘moderate problem’ . Fear of hypoglycaemia can be triggered by the anticipation, frequency, severity, and sequalae of hypoglycaemia, while asleep or awake, and by impaired awareness of hypoglycaemia . Further, a US study found that 26% of adults with T1D met the diagnostic criteria for post-traumatic stress disorder in relation to their experience of hypoglycaemia . Thus, managing fear of hypoglycaemia requires attention to multiple, modifiable risk factors for hypoglycaemia, in addition to general coping, and anxiety-reduction strategies. The main therapeutic option for reducing fear of hypoglycaemia is the use of diabetes technologies, such as continuous glucose monitoring (CGM) and automated insulin delivery, which can reduce problematic hypoglycaemia . However, they do not necessarily eliminate (fear of) hypoglycaemia , and the psychological and cognitive burden can be increased by continual attachment to devices, visibility of data, and information overload . Further, financial burden remains a considerable barrier to widespread use . Several group-based psycho-educational programmes, involving glucose awareness training, have shown benefits for reducing fear of hypoglycaemia . However, none of these studies have measured fear of hypoglycaemia as a primary outcome and there remains a gap in the literature regarding the effectiveness of psycho-educational programmes in reducing fear of hypoglycaemia among people with high fear at baseline . Furthermore, as such programmes are resource intensive, they are not implemented in routine clinical care in Australia or most other countries. Despite Australia’s National Health and Medical Research Council (NHMRC) T1D guidelines asserting the urgent need for minimal resource interventions to prevent severe hypoglycaemia , no Australian studies have focused on this unmet need. Furthermore, numerous international studies now demonstrate that adults with T1D need and want more clinical, psychological and educational support for hypoglycaemia . Objectives This study aims to examine the effectiveness of a fully online, self-directed, psycho-educational programme for reducing fear of hypoglycaemia among adults with T1D: HypoPAST, which stands for ‘ Hypo glycaemia P revention, A wareness of S ymptoms, and T reatment’. There are four objectives: To assess the effect of HypoPAST on fear of hypoglycaemia among adults with T1D; To assess the effect of HypoPAST on secondary psychological, clinical and behavioural outcomes; To assess the cost-effectiveness of HypoPAST; and To explore the reach, acceptability, usability and sustainability of HypoPAST to adults with T1D, and understand the extent to which learnings from the programme are implementable in the ‘real-world’, using a mixed-methods process evaluation. Trial design This study is a two-arm, parallel-group, hybrid type 1 mixed-method randomised controlled trial (RCT) . Eligible participants will be allocated on a 1:1 basis to intervention (HypoPAST) or control (usual care). The trial is designed to determine the superiority of HypoPAST compared to usual care. Data will be collected at baseline, mid-trial (week 12) and end-trial (week 24): survey data collected at baseline, mid-trial and end-trial; ecological momentary assessment (EMA) data collected twice daily for 2 weeks at baseline and end-trial; interview data collected at end-trial; and website analytics collected during implementation (weeks 0–23).
People with type 1 diabetes (T1D) need insulin for survival. However, even with modern insulins and delivery systems, exogenous insulin delivery cannot fully mimic endogenous insulin supply . Hypoglycaemia (low blood glucose) remains a common side effect, caused by relative insulin excess in the absence of sufficient blood glucose. Around 20% of adults with T1D have experienced severe hypoglycaemia (requiring assistance from another person for recovery) in the past 6 months . Hypoglycaemia can be unpleasant, sudden, embarrassing, unpredictable and dangerous (e.g. causing accident or injury) . If undetected and untreated, hypoglycaemia can be life-threatening and associated with adverse outcomes . Repeated exposure to hypoglycaemia can lead to impaired awareness of hypoglycaemia symptoms, increasing a person’s risk of severe episodes, as they have reduced visibility of onset, reducing the window of opportunity for self-treatment . Unsurprisingly, repeated exposure to hypoglycaemia, severe hypoglycaemia, and impaired awareness of hypoglycaemia symptoms can lead to distress and hypoglycaemia-related anxiety, known as ‘fear of hypoglycaemia’ . Fear of hypoglycaemia can compromise diabetes self-management (e.g. maintaining higher glucose levels) and quality of life (e.g. limiting spontaneity, independence and activities in which hypoglycaemia may be a risk or problematic if it occurred) . ‘Worries about low blood glucose’ are consistently among the top five problem areas experienced by adults with T1D, with 75% experiencing it as at least a ‘mild problem’ and 40% as a ‘moderate problem’ . Fear of hypoglycaemia can be triggered by the anticipation, frequency, severity, and sequalae of hypoglycaemia, while asleep or awake, and by impaired awareness of hypoglycaemia . Further, a US study found that 26% of adults with T1D met the diagnostic criteria for post-traumatic stress disorder in relation to their experience of hypoglycaemia . Thus, managing fear of hypoglycaemia requires attention to multiple, modifiable risk factors for hypoglycaemia, in addition to general coping, and anxiety-reduction strategies. The main therapeutic option for reducing fear of hypoglycaemia is the use of diabetes technologies, such as continuous glucose monitoring (CGM) and automated insulin delivery, which can reduce problematic hypoglycaemia . However, they do not necessarily eliminate (fear of) hypoglycaemia , and the psychological and cognitive burden can be increased by continual attachment to devices, visibility of data, and information overload . Further, financial burden remains a considerable barrier to widespread use . Several group-based psycho-educational programmes, involving glucose awareness training, have shown benefits for reducing fear of hypoglycaemia . However, none of these studies have measured fear of hypoglycaemia as a primary outcome and there remains a gap in the literature regarding the effectiveness of psycho-educational programmes in reducing fear of hypoglycaemia among people with high fear at baseline . Furthermore, as such programmes are resource intensive, they are not implemented in routine clinical care in Australia or most other countries. Despite Australia’s National Health and Medical Research Council (NHMRC) T1D guidelines asserting the urgent need for minimal resource interventions to prevent severe hypoglycaemia , no Australian studies have focused on this unmet need. Furthermore, numerous international studies now demonstrate that adults with T1D need and want more clinical, psychological and educational support for hypoglycaemia .
This study aims to examine the effectiveness of a fully online, self-directed, psycho-educational programme for reducing fear of hypoglycaemia among adults with T1D: HypoPAST, which stands for ‘ Hypo glycaemia P revention, A wareness of S ymptoms, and T reatment’. There are four objectives: To assess the effect of HypoPAST on fear of hypoglycaemia among adults with T1D; To assess the effect of HypoPAST on secondary psychological, clinical and behavioural outcomes; To assess the cost-effectiveness of HypoPAST; and To explore the reach, acceptability, usability and sustainability of HypoPAST to adults with T1D, and understand the extent to which learnings from the programme are implementable in the ‘real-world’, using a mixed-methods process evaluation.
This study is a two-arm, parallel-group, hybrid type 1 mixed-method randomised controlled trial (RCT) . Eligible participants will be allocated on a 1:1 basis to intervention (HypoPAST) or control (usual care). The trial is designed to determine the superiority of HypoPAST compared to usual care. Data will be collected at baseline, mid-trial (week 12) and end-trial (week 24): survey data collected at baseline, mid-trial and end-trial; ecological momentary assessment (EMA) data collected twice daily for 2 weeks at baseline and end-trial; interview data collected at end-trial; and website analytics collected during implementation (weeks 0–23).
Study setting The study will be conducted in Australia, using primarily online methods. Participants will use their own device(s) (i.e. laptop, desktop, smartphone, and/or tablet computer) from a physical location of their choice. Platform O, a Deakin University owned and developed e-research platform, will be used to host the online intervention and automate data collection and email reminder systems. Platform O is a widely used evidence-based research tool which provides an end-to-end e-Research solution and complies with Australian ethics requirements . It includes a dynamic intervention builder, which allows researchers to develop highly interactive contents without programming knowledge, including integration with Qualtrics (Provo, UT), Microsoft PowerPoint and Word, PDFs, Videos (You Tube and Vimeo), third party games and animations and text and images. The platform can be used on any device; however, HypoPAST participants will be encouraged to use a tablet, laptop or desktop computer to access the HypoPAST intervention, due to their larger display sizes. Survey data will be collected via Qualtrics, web analytics will be collected via Google Analytics and AUDCI framework and telephone interviews will be conducted via Microsoft Teams. EMA data will be collected via an ‘app’ using a smartphone or tablet device. The app was developed for a study of hypoglycaemia-related experiences (Hypoglycemia MEasurement, ThResholds and ImpaCtS:HypoMETRICS) among adults with T1D and insulin-treated type 2 diabetes , and adapted for HypoPAST. The app is administered via the uMotif Limited platform (umotif.com), which is used widely for EMA in person-centred data capture studies. Eligibility criteria Eligible participants will be adults (≥ 18 years) living in Australia; with self-reported T1D; with self-reported fear of hypoglycaemia (response to a single item from the Problem Areas in Diabetes indicating that worry about low blood glucose is at least a ‘moderate problem’) scale ; and access to the internet via one of the following combinations: (1) a smartphone and tablet, (2) tablet only or (3) desktop or laptop and tablet or smartphone. Members of the HypoPAST Type 1 Diabetes Lived Experience Steering Group (comprising adults with T1D) will be excluded from participation in the trial. Informed consent Potential participants will be directed to visit the recruitment website (acbrd.org.au/take-part/hypopast-rct), which will include brief descriptions of the HypoPAST study and intervention, the plain language statement and a hyperlink to a Qualtrics survey for participant consent and eligibility screening enabling self-enrolment into the study. Interventions Intervention description HypoPAST is an online psycho-educational training programme informed by several group-based programmes ; behavioural, psychological, educational and clinical insights from a multidisciplinary team; and lived experience insights from a Steering Group of adults with T1D. Participants allocated to the intervention group will receive email instructions about how to access the HypoPAST programme via the website (hypopast.org.au). They will be advised that HypoPAST is intended to enhance, not replace, the individualised medical advice of their diabetes health professional(s); and to continue with their usual diabetes management, clinical care and support during the trial. Participants will be advised that the recommendation is to complete all 7 modules and to do so over 4–8 weeks. This recommendation is made to provide participants with opportunity to digest the content and practice skills. In total, the programme is estimated to take 4 to 8 h (about 30 to 60 min per module), depending on the number of modules undertaken and the time an individual spends on activities and practising skills between modules. As HypoPAST is self-guided, participants will choose how many and which modules they complete, in what order, how many times and the timeframe (e.g. in one or multiple sessions, in 1 day or over multiple days/weeks). There is no limit on how often they access HypoPAST and they will have access to the programme for the full 24 weeks of the trial. Upon entry to the programme, the brief ‘Getting started’ module demonstrates how to make the most of the programme and enables participants, via self-assessment, to prioritise the modules most relevant to their needs. The HypoPAST programme comprises seven modules, covering topics such as timely and appropriate treatment of hypoglycaemia; personal risks for hypoglycaemia; recognising warning signs of hypoglycaemia; drivers of frequent hypoglycaemia; talking about hypoglycaemia with family, friends, health professionals and colleagues; hypoglycaemia while asleep; and managing worries. HypoPAST includes reflection and problem-solving activities enabling participants to discover for themselves how to strengthen their skills and develop preventative habits enabling them to reduce both their fear and their risk of hypoglycaemia (Fig. ). In a separate section of the online platform, ‘My Resources’, participants will have access to information leaflets, activity sheets and videos related to topics covered in the modules. Control description There is currently no specific pathway for managing fear of hypoglycaemia among adults with T1D in Australia. Thus, participants allocated to the control group will be advised to continue with their usual T1D management, clinical care and support, which is assumed to involve strategies for minimising problematic hypoglycaemia. This may include contact with their diabetes specialist or healthcare team and/or the National Diabetes Services Scheme (NDSS) Helpline. At trial end, they will be granted access to the HypoPAST intervention for 24 weeks. Strategies to improve adherence to interventions Intervention group participants will receive three automated email reminders to access the intervention, at 2, 11 and 23 weeks post-randomisation. Intervention uptake will be monitored during the trial. Concomitant care Concomitant care will not be prohibited during the trial. All participants will be advised to continue with their routine diabetes self-management, as discussed with their diabetes health professional. With permission of the participants, we will inform their primary diabetes health professional that they are participating in the study. Participants will be advised, in the plain language study information, to contact their diabetes health professional if they experience severe hypoglycaemia or another diabetes-related issue during the study. Provisions for post-trial care This study will not include administration, manipulation or investigation of the effects, of any pharmacological or therapeutic goods. Therefore, provisions for ancillary or post-trial care are not deemed necessary. However, adverse event monitoring will take place, per Australian Government requirements (see ‘ ’). The study is covered by Deakin University’s clinical trial insurance. Participants will be notified, in the plain language information, that they will not receive payment/reimbursement for medical expenses if they experience a severe hypoglycaemic event or another health-related issue during the study. Outcomes Table details all outcomes, self-reported demographic and clinical characteristics to be collected, the method by which they will be collected, and the timepoint(s) at which they will be collected. Primary outcome The primary outcome is the between-group difference at 24 weeks in fear of hypoglycaemia, assessed using the 18-item Worry subscale of the Hypoglycaemia Fear Survey, version 2 (HFS-II) . We hypothesise a statistically significant difference of at least 9 points will be observed in the Worry score, favouring the intervention. Secondary outcomes Clinical, psychological and behavioural At each timepoint, several clinical, psychological and behavioural secondary outcomes are assessed using validated and study-specific survey measures. These include IAH, hypoglycaemia frequency and severity, attitudes to hypoglycaemia awareness, avoidant behaviours related to fear of hypoglycaemia, hypoglycaemia-specific quality of life, hypoglycaemia related post-traumatic stress, diabetes distress, generalised anxiety symptoms and depressive symptoms. Further details of the methods for assessment are provided in Additional File 1. We hypothesise that clinically significant between-group differences will be observed by 24 weeks in these variables, favouring the intervention. Health economics At each timepoint, participants will complete a resource use questionnaire in the online survey, to collect information about the use of other healthcare resources used and lost productivity . They will also complete a generic health-related quality of life questionnaire, the Assessment of Quality of Life 4 dimension (AQoL-4D), to assess utility values and calculate quality adjusted life years (QALYs) . We hypothesise that HypoPAST will be cost-effective from health sector and societal perspectives compared to the control (usual care), with an incremental cost-effectiveness ratio below the commonly used willingness to pay threshold of $50,000/quality-adjusted life year. Ecological momentary assessments Ecological momentary assessment (EMA) provides opportunities to collect data in ‘real time’ on experiences of hypoglycaemia (e.g. timing, glucose level, symptoms) and their impacts (e.g. on sleep quality, productivity, mood). The EMA comprises two parts, both collected via an app. The questions were previously trialled in the 10-week HypoMETRICS study of hypoglycaemia-related experiences among adults with T1D and insulin-treated type 2 diabetes . For the EMA analysis, we hypothesise that significant between-group differences at end-trial will be observed in effects of person-reported hypoglycaemia on daily functioning scores, number of hypoglycaemic episodes, awareness of symptoms and hypoglycaemia burden, all favouring intervention. Reach, acceptability, usability, fidelity and sustainability A mixed-methods approach will be used to examine the reach of HypoPAST, its acceptability and usability to adults with T1D, their experiences of integrating learnings from HypoPAST into their lives, and their views about sustainability and roll-out of HypoPAST. A mixed-methods approach enables collection of both quantitative data (e.g. intervention uptake, survey responses) and qualitative data (e.g. semi-structured interviews). This process evaluation will draw upon two highly relevant and complementary frameworks: Behaviour Interventions using Technology and Reach, Effectiveness, Adoption, Implementation, and Maintenance . Measures of reach will include study registration rates; proportion of registrants meeting the eligibility criteria; method of referral into the study; demographic and clinical characteristics; and study attrition. Additionally, various website analytics will be collected (Table ) to assess protocol fulfilment with the intervention (i.e. the proportion of participants who accessed the HypoPAST at least once). We will also examine any association(s) between participant outcome and type/duration of content accessed. Intervention group participants will complete study-specific survey items about the intervention’s acceptability and usability (e.g. reasons for accessing/not accessing modules, ease of understanding, trustworthiness, convenience, presentation, likes and dislikes, suggestions for improvement, technical problems) and sustainability (e.g. recommendations to others, suggestions for referral into the programme, potential costs for access). They will also complete the mARM to measure therapeutic alliance with the HypoPAST programme . A semi-structured interview schedule (Additional File 2) will build upon topics from the survey, with the intention to gain in-depth insights into (1) the intervention group participant’s views about the HypoPAST intervention, (2) their experiences implementing principles and strategies from the intervention into their daily lives and (3) their views about sustainability and roll-out of HypoPAST. Participant timeline Self-enrolled, eligible participants will participate in 2 weeks of baseline data collection (survey plus 14-day EMA) before randomisation. Those who do not complete the baseline EMA (defined as missing ≥ 7 the 28 check-ins) will not proceed to randomisation. Those allocated to the intervention will be encouraged to complete the online HypoPAST training in the first 4 to 8 weeks, but will retain access throughout the full 24-week trial. Further data collections will take place at mid-trial (survey) and end-trial (EMA, survey, interviews). From entry to exit, participation will take ≤ 32 weeks, including all data collection. Figure shows the study flow, and Table shows the timeline of activities (e.g. enrolment, randomisation, intervention and data collection). Sample size The RCT sample size and statistical power calculations were conducted using STATA MP (StataCorp) ‘power twomeans’ sample size estimation package. Based on previous research , a standard deviation of 17 was used to estimate a sample size of N = 196 for this study. Given there is no minimal clinically important difference in fear of hypoglycaemia reported in the literature, the anticipated effect size was estimated from previous research . To detect a between-group difference of nine points (standardised effect size of 0.5) in the primary outcome at 6 months, a total of 150 participants (75 per arm) is required. This sample size is a conservative estimate based on 1:1 group allocation for a two-sided test, at the 5% significance level, and a high level of power (90%). Allowing for 30% attrition at 24-week follow-up, the final required sample size is N = 196 participants ( N = 150 × 1.3; n = 98 per study group). For the qualitative sub-study, a purposive sample size of n = 25 was estimated to maximise diversity based on baseline survey responses (e.g. gender, age, education, glucose monitoring method, awareness of hypoglycaemia symptoms, and experience of severe hypoglycaemia). Recruitment The primary recruitment method will be via the NDSS, an initiative of the Australian government administered by Diabetes Australia. Approximately 137,000 Australian adults with T1D are registered with the NDSS. NDSS staff will email the study invitation to a random of sample of adult registrants with T1D who have consented to being contacted about research (30%). Additional NDSS emails will be sent, if needed, to ensure timely achievement of the required sample size. In addition, the study will be advertised via the e-newsletter, social media and website of the Australian Centre for Behavioural Research in Diabetes, and via direct email to adults with T1D who have previously requested to be notified of relevant research opportunities. Participant demographics will be monitored throughout recruitment to examine diversity in representation. If needed, the study may be promoted in a more targeted manner (e.g. to encourage certain demographics to participate, within the channels listed above). For the qualitative sub-study, participants will be recruited from intervention group participants using purposive sampling (see above). Engagement with the intervention will also be considered, as the interview questions require participants to comment on their experiences using and implementing the intervention into their daily lives. Randomisation Participants will be allocated at random (1:1) to the intervention or control (usual care) arm. The allocation sequence will be generated by computer (via Qualtrics/Platform O) using randomly permuted block sizes. As stratification prevents imbalance in treatment groups for important variables thought to influence the outcome, randomisation will be stratified by: Gender: men versus women. We anticipate that gender may influence the expression of emotional well-being (including the primary outcome and several secondary endpoints). If participants have a ‘non-binary’ or ‘another term’ gender identity, they will be allocated at random to either the male or female gender strata, so there is approximately equal representation of these smaller sub-groups in each arm. Glucose monitoring method: finger-prick versus CGM or intermittent-scanned CGM (isCGM, also known as ‘flash’ monitoring). We anticipate that monitoring method may be associated with fear of hypoglycaemia, frequency and severity of hypoglycaemia, IAH and/or the extent to which participants benefit from HypoPAST (given their access or not to real-time glucose data). The allocation sequence will be stored in the Deakin data centre server system with password protection, accessible only to the system architect of Platform O. Post-randomisation group allocations will be stored in the Deakin data centre and accessed from the backend of Platform O with a secure login, accessible only to the system architect and a research assistant who will be responsible for maintaining a password-protected codebook in Microsoft Excel. Two weeks after self-enrolment in the study, participants will be notified of their group allocation by email, with instructions relevant to their allocation. Blinding During data collection, all members of the HypoPAST research group will be blinded, with the exception of (1) a research assistant who will maintain a password-protected codebook for the purpose of data linkage between data sources (e.g. the app, Qualtrics surveys, website analytics) and be responsible for direct contact with participants (e.g. monitoring enquiries via the HypoPAST email, inviting people to participate in adverse event/process evaluation interviews, administering e-vouchers, informing the participants’ nominated diabetes health professionals about their participation, notifying participants of their randomised group allocations); and (2) the website architect responsible for Platform O (EO). Neither will conduct data analysis. During data analysis, those analysing data for research questions 1 and 2 (ST, US, and VLV) and the investigators will remain blinded. For research question 3, the health economists (CM and MLC) will use blinded data to undertake their preliminary analyses of utility values and QALYs from the AQoL-4D, the self-reported health care resource use and lost productivity. However, the final economic evaluation will be conducted using unblinded data, as they will need to assign intervention costs to the appropriate group. Due to the nature of the intervention and control conditions, participants will be aware of their group allocation. Unblinding of the Project Manager is permissible if they are investigating a potential adverse event (see ‘ ’) via email or telephone interview. This may occur if an individual participant discloses information that identifies their group allocation. In such instances only the Project Manager will be unblinded. Breaches to blinding will be recorded and reported with the study findings. Data collection and management Plans for assessment and collection of outcomes Data will be collected in four ways: An online survey (administered via Qualtrics), to collect self-reported data on demographic and clinical characteristics; primary and secondary clinical, psychological and behavioural outcomes; and health economic outcomes. Intervention group participants will also answer questions about intervention acceptability and therapeutic alliance (mid-trial survey). The survey questions comprise a combination of validated scales and study-specific items, most of which are fixed-choice and some requiring text input (e.g. writing their age, most recent HbA1c, the number of hypos they have had in the past week, or specifying information where they have selected the ‘other’ response option) (Table and Additional File 1). At mid-trial, intervention group participants are asked two qualitative questions related to intervention acceptability (suggestions for improvement and what they would tell others about HypoPAST). At each timepoint (0, 12 and 24 weeks), the survey is anticipated to take 45 min. An ‘app’, for EMA (administered via the uMotif platform) which collects self-reported data on clinical, psychological, behavioural and health economic outcomes (Table and Additional File 1). Every day for 14 days (at two timepoints: pre-randomisation and end trial), participants will log their hypoglycaemia episodes and symptoms using the ‘motif flower’ in the app. They will complete one motif entry for each episode of hypoglycaemia and can complete the entry at any time on any day during the 2-week data collection period. Participants will also complete daily functioning ‘check-in’ survey questions twice daily (morning: between 06:00 to 12:00, and evening: 18:00 to 24:00) via the app. Each check-in will take about 5 min. Website analytics will be collected via Google Analytics and AUDCI framework, capturing user engagement with the HypoPAST intervention. Semi-structured, audio-recorded telephone interviews (approximately 30–45 min) at end-study, conducted by trained researchers with a sub-set of intervention group participants. The interviews will explore participant’s experiences using the intervention and implementing it into their daily lives, and their views about rollout of the intervention to other adults with diabetes. The interview will take place after the window for end-trial survey completion has closed (weeks 24 to 26), or the end-trial survey is returned, whichever comes first. Plans to promote participant retention and complete follow-up Participants will be offered a token of appreciation, recognising the commitment that it takes to participate in a 6-month trial. E-vouchers (AU$50) will be issued via email to participants after they complete the (1) mid-trial survey, (2) end-trial survey and EMA and (3) interview. This means, for example, an intervention group participant who takes part in both follow-ups and an interview will receive three e-vouchers (total value AU$150). Reminders will be scheduled to encourage engagement with the intervention and follow-up data return. Specifically, two email reminders (1 and 2 weeks after the initial invitation) will be automated to remind participants to complete the mid- and end-trial surveys. The app will send a daily push notification reminding participants to ‘check-in’, if their phone settings allow this. Participants will be able to withdraw from the trial, for any reason, by emailing the study team prior to completion of the 24-week follow-up survey and the closing of the dataset for the primary analysis. Data collected until that timepoint will be retained in the analysis unless otherwise requested by the participant. Due to the online nature of the trial and due to feedback from DUHREC, the study team will not proactively enquire about participant’s reasons for withdrawal or discontinued engagement with the trial (e.g. non-return of follow-up data, refusal to participate in interview); however, reasons will be recorded if they are disclosed by the participant (e.g. via email or telephone contact). Data management As per the HypoPAST data management plan, during the trial, data will be collected by and held in the following locations: Qualtrics (survey responses), uMotif (app responses), Platform O (participant contact details and web analytics), Microsoft Teams (audio recordings of interviews), a transcribing company (audio recordings and transcripts of interviews). Once each phase of data collection is complete (i.e. baseline, 12-week follow-up, 24-week follow-up), or in the case of interviews, after each interview, the database will be locked, data de-identified and downloaded (e.g. from Qualtrics, uMotif, Platform O, Microsoft Teams) to the Deakin University secure network. Subsequently, any copies of the data held elsewhere will be destroyed (i.e. electronic files deleted). Syncplicity (i.e. Sync and Share create via Deakin Research Data Store) will be used to share de-identified data with the researchers with primary responsibility for data analysis. Data will be retained on the Deakin University secure network for at least 15 years after the study findings are published, as required by law. After the study findings are published, metadata will be placed in an open-access data repository (see ‘Availability of data and materials’). Confidentiality Unique participant identification codes will be assigned to participants. Personal identifiers will be removed from the data. A password-protected codebook will be maintained by a research assistant. Individual participants will not be identified in reports of study findings, and data that could potentially be used to identify participants will not be included in the open-access meta-data or shared datasets. Statistical methods Primary and secondary outcomes Clinical, psychological and behavioural outcomes To examine the effect of the intervention on primary and secondary outcomes (collected via survey), we will use an intention to treat approach where all randomised participants will be analysed according to their study group assignment (regardless of the extent to which intervention group participants engage with the intervention). Statistical analyses will be performed using STATA MP version 17 (StataCorp). Linear mixed models using restricted maximum likelihood estimation will be used to estimate group differences in the primary (HFS-II Worry scale score) and secondary clinical, psychological and behavioural outcomes (Table ). As the primary and secondary outcomes will be measured longitudinally, the outcome at 12 and 24 weeks will be included in the model as the dependent variable. The outcome at baseline, time and an interaction between time and trial arm will be included as fixed effects in the models. Repeated outcome measures will be treated as random effects in the model and an unstructured variance–covariance structure assumed. Models will be adjusted by stratification factors: gender and glucose monitoring method. Transformations for skewed outcome measures will be considered. The estimated mean HFS-II Worry scale score at baseline, 12 and 24 weeks will be plotted for each trial arm with 95% confidence intervals. The estimated difference in mean HFS-II Worry score between arms at 12 and 24 weeks and associated 95% confidence intervals will be presented. A similar modelling approach will be used to estimate group differences in hypoglycaemic events; however, a negative binomial mixed model will be used to account for repeated count data. In secondary analyses, the primary outcome will be adjusted by age, diabetes duration, HbA1c, severe hypoglycaemia episodes in the past 6 months, Gold score and insulin administration modality. Secondary outcomes will also be adjusted by these potential confounders where relevant. A per protocol analysis for the primary outcome will be conducted to estimate the treatment effect in those who engage with the intervention (defined as using 2 or more modules). A sensitivity analysis using multiple imputation may be conducted should there be between 10 and 40% missing data in the primary outcome and auxiliary variables available in the data set to explain the missingness. A second sensitivity analysis will be performed on the primary analysis to test the robustness of the ‘data missing at random’ assumption of mixed-linear models using pattern-mixture modelling. Health economic outcomes A cost-utility analysis will be undertaken from health sector and societal perspectives. The health sector perspective includes costs borne by the government as a third-party payer in addition to out-of-pocket costs incurred by patients when accessing healthcare. Detailed costing of the HypoPAST intervention will be performed using micro-costing methods. The number and types of additional health services used by participants over the period of the trial will be collected with a resource use questionnaire, and standard Australian unit costs will be applied. Total health sector costs will be calculated as the sum of intervention delivery and additional healthcare service use costs. The societal perspective adds the cost of lost productivity (absenteeism and presenteeism) to the health sector costs. Lost productivity will be measured with questions in the resource use questionnaire and valued with the human capital approach using an average Australian wage rate plus on-costs. The AQoL-4D utility values for each participant at each timepoint will be used to calculate QALYs using the area under the curve method. The within-trial economic evaluation will measure and value any change in healthcare resource use and lost productivity and then compare any additional costs to additional QALYs through an incremental cost-effectiveness ratio. Bootstrapping will be used to determine confidence intervals for the incremental cost-effectiveness ratio and construct an acceptability curve to determine the cost-effectiveness of the intervention against the commonly used willingness to pay threshold of AU$50,000/QALY. Missing data will be explored and managed for the resource use and AQoL-4D questionnaires based on recommendations for analysis of trial-based economic evaluations with missing data . Sensitivity analyses will be undertaken to evaluate the robustness of results with changes to costing or analytical assumptions. Scale-up and implementation costs as well as longer-term cost-effectiveness will be estimated based on population-wide modelling techniques using published epidemiological data. STATA (StataCorp) will be used for these analyses. Ecological momentary assessment outcomes To examine the effect of the intervention on EMA outcomes (daily functioning, number of hypoglycaemic episodes, awareness of symptoms and hypoglycaemia burden collected via an app), we will use linear mixed models (for continuous outcomes) and mixed negative binomial models (for count outcomes). Trial arm, time of day (morning/night) and timepoint (baseline or 24 weeks) will be included as fixed effects in the models. Participants will be included as random effects in the models and an unstructured variance–covariance structure used to account for repeated measures and the correlation in outcome within individuals. A separate mixed linear model will be used to explore whether the effect of person-reported hypoglycaemia on daily functioning domains (fear of hypoglycaemia, x, y) differs between trial arms. The independent variable will be person-reported hypoglycaemia, and an interaction between this variable and trial arm (intervention or control) will be included in the model. If sufficient data, we will also assess additional models exploring effects of person-reported hypoglycaemia subtypes (i.e. how the episodes where detected and managed) and whether number of reported hypoglycaemia episodes, awareness (in terms of symptoms) and hypoglycaemia burden differ between trial arms. Models will be adjusted as for the primary outcome. Similarly, missing data will be managed as for the primary objective. Due to the question phrasing and timing of the check-ins, people who expect to primarily sleep during the day during the 2-week EMA data collection will have their EMA data excluded from analysis as it may confound the results. Data analysis will be performed in R-studio (Posit Software, PBC, Boston, MA). Reach, acceptability, usability, fidelity and sustainability Quantitative data will be analysed using descriptive statistics. This includes both survey and website analytics data, including the following: study registration rates; proportion of registrants meeting the eligibility criteria; method of referral into the study; demographic and clinical characteristics; study attrition; number of, and which modules, were accessed; study-specific survey items about intervention acceptability and sustainability. To determine socioeconomic status and geographical location, code will be generated to match postcodes against Australian Bureau of Statistics Australian Statistical Geography Standards and Index of Relative Socio-economic Advantage and Disadvantage Socio-Economic Indexes for Areas quintiles . Open-text survey responses will be collated and summarised descriptively in Microsoft Excel. Interview transcripts will be coded using NVivo and/or Microsoft Excel and analysed using thematic analysis using inductive and deductive approaches. Prior to coding the data, the researcher(s) will become familiar with the dataset by reading the transcripts and/or listening to the audio recordings. Interim and additional analyses No interim analyses are planned. No additional (e.g. subgroup) analyses are planned, but they may occur, as the data will be made available for additional research after the study findings are published (see ‘Availability of data and materials’). Plans to give access to the full protocol, participant level-data and statistical code The full protocol and data management plans will be made available upon reasonable request, in writing, to the project manager or lead researcher. The participant-level quantitative data will be made available to researchers, upon reasonable request, after the study findings are published. Participants will be able to ‘opt-out’ from having their data shared/used for future unspecified research by ticking a box on the consent form. Data of participants who tick this box will not be included in the open-access meta-data or datasets shared with non-HypoPAST researchers. Statistical code may be made available upon reasonable request. Oversight and monitoring Project coordination The study will be coordinated by research personnel from Deakin University. The lead researcher will take overall responsibility for the study, providing guidance and oversight. A programme manager will take responsibility for day-to-day project management, overseeing that all elements of the study are implemented per-protocol and in adherence with ethics principles. For example, they will oversee data monitoring, supervise HypoPAST research personnel, conduct adverse event interviews, communicate with participants (e.g. responding to queries and complaints) and maintain communication with the studies various contributors and stakeholders (e.g. investigators and steering group, website architect, funding body). A statistician/data manager will support data monitoring (e.g. rates of eligible people recruited, randomised, withdrawn, lost to follow-up) and adverse event screening. An associate research fellow will support data entry for data monitoring, adverse event screening and coordination of the HypoPAST ‘Type 1 Diabetes Lived Experience Steering Group’ (see below). A research assistant will support communication with participants (e.g. monitoring the HypoPAST email account, responding to queries and complaints), data entry for data monitoring, maintenance of the password-protected participant codebook and other administrative duties (e.g. sending letters to health professionals, administering vouchers). Communication between the research personnel will occur weekly, via meetings and email. Project oversight Two groups oversee the HypoPAST study: The HypoPAST ‘Investigator Group’ comprises 14 people, including researchers and clinicians (specialising in diabetes education, endocrinology and psychology), representatives of peak bodies (for diabetes and for health professionals), and a person with lived experience of T1D. This group meets four times per year, for project oversight and to contribute their expertise into the study implementation. The HypoPAST ‘Type 1 Diabetes Lived Experience Steering Group’ comprises eight adults with T1D. They reviewed, and informed iterative refinements of, the intervention content and design. This involved a combination of group discussion and one-to-one cognitive debriefing interviews. The Steering Group will have continued involvement throughout the duration of the study, to contribute their expertise into the study implementation. The group meets approximately four times per year. Data monitoring Data will be monitored by the HypoPAST research personnel (see Project coordination). A data monitoring committee was not deemed necessary as the study does not involve an unapproved therapeutic good requiring a Clinical Trial Notification and trial sponsorship. Adverse event reporting The survey responses (at weeks 12 and 24), EMA responses (starting at week 24) and emails from participants to project staff will be monitored for potential adverse events. Baseline survey and EMA data will not be monitored as they are collected prior to randomisation/intervention. Serious adverse events will be defined as any severe hypoglycaemic or mental health-related event requiring medical assistance for recovery (i.e. emergency call-out, emergency department attendance and/or hospital admission). Serious adverse reactions related specifically to HypoPAST are not expected, but consideration will be given to causality. Severe hypoglycaemia or mental health-related events will only be defined as ‘serious’ if they require ambulance call-out, emergency department attendance and/or hospital admission, because the person was able to recover without medical assistance. Participants reporting any event deemed as potentially adverse will be contacted by the project manager (email and/or telephone interview) for further information to clarify the nature of the event. Adverse events will be reported to DUHREC and in the publication reporting study findings. Auditing trial conduct and protocol amendments The project is subject to independent annual financial auditing and will report annually to DUHREC regarding trial implementation. Protocol changes will be communicated to DUHREC, the funders and trial register, and reported with the study findings. Funding body involvement The Medical Research Future Fund (MRFF) Targeted Translation Research Accelerator (TTRA) did not contribute to the development of this trial protocol, and will not be involved in the conduct of the trial data collection, analysis, interpretation or write-up of findings. Dissemination Study findings will be disseminated at scientific conferences and in academic journals. A plain language summary will be published in a blog (via acbrd.org.au) and promoted via social media. Participants who opted-in to future contact will be directly emailed the plain language summary of the key findings. The primary diabetes health professional of participants who opted-in to will also be notified of the published study findings. This multi-level strategy provides several opportunities for the participants and other stakeholders (e.g. health professionals, people with diabetes) to access the findings. Participants will not be identifiable in any dissemination of the research findings. Publication authorship will be defined according to the International Committee of Medical Journal Editors criteria .
The study will be conducted in Australia, using primarily online methods. Participants will use their own device(s) (i.e. laptop, desktop, smartphone, and/or tablet computer) from a physical location of their choice. Platform O, a Deakin University owned and developed e-research platform, will be used to host the online intervention and automate data collection and email reminder systems. Platform O is a widely used evidence-based research tool which provides an end-to-end e-Research solution and complies with Australian ethics requirements . It includes a dynamic intervention builder, which allows researchers to develop highly interactive contents without programming knowledge, including integration with Qualtrics (Provo, UT), Microsoft PowerPoint and Word, PDFs, Videos (You Tube and Vimeo), third party games and animations and text and images. The platform can be used on any device; however, HypoPAST participants will be encouraged to use a tablet, laptop or desktop computer to access the HypoPAST intervention, due to their larger display sizes. Survey data will be collected via Qualtrics, web analytics will be collected via Google Analytics and AUDCI framework and telephone interviews will be conducted via Microsoft Teams. EMA data will be collected via an ‘app’ using a smartphone or tablet device. The app was developed for a study of hypoglycaemia-related experiences (Hypoglycemia MEasurement, ThResholds and ImpaCtS:HypoMETRICS) among adults with T1D and insulin-treated type 2 diabetes , and adapted for HypoPAST. The app is administered via the uMotif Limited platform (umotif.com), which is used widely for EMA in person-centred data capture studies.
Eligible participants will be adults (≥ 18 years) living in Australia; with self-reported T1D; with self-reported fear of hypoglycaemia (response to a single item from the Problem Areas in Diabetes indicating that worry about low blood glucose is at least a ‘moderate problem’) scale ; and access to the internet via one of the following combinations: (1) a smartphone and tablet, (2) tablet only or (3) desktop or laptop and tablet or smartphone. Members of the HypoPAST Type 1 Diabetes Lived Experience Steering Group (comprising adults with T1D) will be excluded from participation in the trial.
Potential participants will be directed to visit the recruitment website (acbrd.org.au/take-part/hypopast-rct), which will include brief descriptions of the HypoPAST study and intervention, the plain language statement and a hyperlink to a Qualtrics survey for participant consent and eligibility screening enabling self-enrolment into the study.
Intervention description HypoPAST is an online psycho-educational training programme informed by several group-based programmes ; behavioural, psychological, educational and clinical insights from a multidisciplinary team; and lived experience insights from a Steering Group of adults with T1D. Participants allocated to the intervention group will receive email instructions about how to access the HypoPAST programme via the website (hypopast.org.au). They will be advised that HypoPAST is intended to enhance, not replace, the individualised medical advice of their diabetes health professional(s); and to continue with their usual diabetes management, clinical care and support during the trial. Participants will be advised that the recommendation is to complete all 7 modules and to do so over 4–8 weeks. This recommendation is made to provide participants with opportunity to digest the content and practice skills. In total, the programme is estimated to take 4 to 8 h (about 30 to 60 min per module), depending on the number of modules undertaken and the time an individual spends on activities and practising skills between modules. As HypoPAST is self-guided, participants will choose how many and which modules they complete, in what order, how many times and the timeframe (e.g. in one or multiple sessions, in 1 day or over multiple days/weeks). There is no limit on how often they access HypoPAST and they will have access to the programme for the full 24 weeks of the trial. Upon entry to the programme, the brief ‘Getting started’ module demonstrates how to make the most of the programme and enables participants, via self-assessment, to prioritise the modules most relevant to their needs. The HypoPAST programme comprises seven modules, covering topics such as timely and appropriate treatment of hypoglycaemia; personal risks for hypoglycaemia; recognising warning signs of hypoglycaemia; drivers of frequent hypoglycaemia; talking about hypoglycaemia with family, friends, health professionals and colleagues; hypoglycaemia while asleep; and managing worries. HypoPAST includes reflection and problem-solving activities enabling participants to discover for themselves how to strengthen their skills and develop preventative habits enabling them to reduce both their fear and their risk of hypoglycaemia (Fig. ). In a separate section of the online platform, ‘My Resources’, participants will have access to information leaflets, activity sheets and videos related to topics covered in the modules. Control description There is currently no specific pathway for managing fear of hypoglycaemia among adults with T1D in Australia. Thus, participants allocated to the control group will be advised to continue with their usual T1D management, clinical care and support, which is assumed to involve strategies for minimising problematic hypoglycaemia. This may include contact with their diabetes specialist or healthcare team and/or the National Diabetes Services Scheme (NDSS) Helpline. At trial end, they will be granted access to the HypoPAST intervention for 24 weeks. Strategies to improve adherence to interventions Intervention group participants will receive three automated email reminders to access the intervention, at 2, 11 and 23 weeks post-randomisation. Intervention uptake will be monitored during the trial. Concomitant care Concomitant care will not be prohibited during the trial. All participants will be advised to continue with their routine diabetes self-management, as discussed with their diabetes health professional. With permission of the participants, we will inform their primary diabetes health professional that they are participating in the study. Participants will be advised, in the plain language study information, to contact their diabetes health professional if they experience severe hypoglycaemia or another diabetes-related issue during the study. Provisions for post-trial care This study will not include administration, manipulation or investigation of the effects, of any pharmacological or therapeutic goods. Therefore, provisions for ancillary or post-trial care are not deemed necessary. However, adverse event monitoring will take place, per Australian Government requirements (see ‘ ’). The study is covered by Deakin University’s clinical trial insurance. Participants will be notified, in the plain language information, that they will not receive payment/reimbursement for medical expenses if they experience a severe hypoglycaemic event or another health-related issue during the study.
HypoPAST is an online psycho-educational training programme informed by several group-based programmes ; behavioural, psychological, educational and clinical insights from a multidisciplinary team; and lived experience insights from a Steering Group of adults with T1D. Participants allocated to the intervention group will receive email instructions about how to access the HypoPAST programme via the website (hypopast.org.au). They will be advised that HypoPAST is intended to enhance, not replace, the individualised medical advice of their diabetes health professional(s); and to continue with their usual diabetes management, clinical care and support during the trial. Participants will be advised that the recommendation is to complete all 7 modules and to do so over 4–8 weeks. This recommendation is made to provide participants with opportunity to digest the content and practice skills. In total, the programme is estimated to take 4 to 8 h (about 30 to 60 min per module), depending on the number of modules undertaken and the time an individual spends on activities and practising skills between modules. As HypoPAST is self-guided, participants will choose how many and which modules they complete, in what order, how many times and the timeframe (e.g. in one or multiple sessions, in 1 day or over multiple days/weeks). There is no limit on how often they access HypoPAST and they will have access to the programme for the full 24 weeks of the trial. Upon entry to the programme, the brief ‘Getting started’ module demonstrates how to make the most of the programme and enables participants, via self-assessment, to prioritise the modules most relevant to their needs. The HypoPAST programme comprises seven modules, covering topics such as timely and appropriate treatment of hypoglycaemia; personal risks for hypoglycaemia; recognising warning signs of hypoglycaemia; drivers of frequent hypoglycaemia; talking about hypoglycaemia with family, friends, health professionals and colleagues; hypoglycaemia while asleep; and managing worries. HypoPAST includes reflection and problem-solving activities enabling participants to discover for themselves how to strengthen their skills and develop preventative habits enabling them to reduce both their fear and their risk of hypoglycaemia (Fig. ). In a separate section of the online platform, ‘My Resources’, participants will have access to information leaflets, activity sheets and videos related to topics covered in the modules.
There is currently no specific pathway for managing fear of hypoglycaemia among adults with T1D in Australia. Thus, participants allocated to the control group will be advised to continue with their usual T1D management, clinical care and support, which is assumed to involve strategies for minimising problematic hypoglycaemia. This may include contact with their diabetes specialist or healthcare team and/or the National Diabetes Services Scheme (NDSS) Helpline. At trial end, they will be granted access to the HypoPAST intervention for 24 weeks.
Intervention group participants will receive three automated email reminders to access the intervention, at 2, 11 and 23 weeks post-randomisation. Intervention uptake will be monitored during the trial.
Concomitant care will not be prohibited during the trial. All participants will be advised to continue with their routine diabetes self-management, as discussed with their diabetes health professional. With permission of the participants, we will inform their primary diabetes health professional that they are participating in the study. Participants will be advised, in the plain language study information, to contact their diabetes health professional if they experience severe hypoglycaemia or another diabetes-related issue during the study.
This study will not include administration, manipulation or investigation of the effects, of any pharmacological or therapeutic goods. Therefore, provisions for ancillary or post-trial care are not deemed necessary. However, adverse event monitoring will take place, per Australian Government requirements (see ‘ ’). The study is covered by Deakin University’s clinical trial insurance. Participants will be notified, in the plain language information, that they will not receive payment/reimbursement for medical expenses if they experience a severe hypoglycaemic event or another health-related issue during the study.
Table details all outcomes, self-reported demographic and clinical characteristics to be collected, the method by which they will be collected, and the timepoint(s) at which they will be collected. Primary outcome The primary outcome is the between-group difference at 24 weeks in fear of hypoglycaemia, assessed using the 18-item Worry subscale of the Hypoglycaemia Fear Survey, version 2 (HFS-II) . We hypothesise a statistically significant difference of at least 9 points will be observed in the Worry score, favouring the intervention. Secondary outcomes Clinical, psychological and behavioural At each timepoint, several clinical, psychological and behavioural secondary outcomes are assessed using validated and study-specific survey measures. These include IAH, hypoglycaemia frequency and severity, attitudes to hypoglycaemia awareness, avoidant behaviours related to fear of hypoglycaemia, hypoglycaemia-specific quality of life, hypoglycaemia related post-traumatic stress, diabetes distress, generalised anxiety symptoms and depressive symptoms. Further details of the methods for assessment are provided in Additional File 1. We hypothesise that clinically significant between-group differences will be observed by 24 weeks in these variables, favouring the intervention. Health economics At each timepoint, participants will complete a resource use questionnaire in the online survey, to collect information about the use of other healthcare resources used and lost productivity . They will also complete a generic health-related quality of life questionnaire, the Assessment of Quality of Life 4 dimension (AQoL-4D), to assess utility values and calculate quality adjusted life years (QALYs) . We hypothesise that HypoPAST will be cost-effective from health sector and societal perspectives compared to the control (usual care), with an incremental cost-effectiveness ratio below the commonly used willingness to pay threshold of $50,000/quality-adjusted life year. Ecological momentary assessments Ecological momentary assessment (EMA) provides opportunities to collect data in ‘real time’ on experiences of hypoglycaemia (e.g. timing, glucose level, symptoms) and their impacts (e.g. on sleep quality, productivity, mood). The EMA comprises two parts, both collected via an app. The questions were previously trialled in the 10-week HypoMETRICS study of hypoglycaemia-related experiences among adults with T1D and insulin-treated type 2 diabetes . For the EMA analysis, we hypothesise that significant between-group differences at end-trial will be observed in effects of person-reported hypoglycaemia on daily functioning scores, number of hypoglycaemic episodes, awareness of symptoms and hypoglycaemia burden, all favouring intervention. Reach, acceptability, usability, fidelity and sustainability A mixed-methods approach will be used to examine the reach of HypoPAST, its acceptability and usability to adults with T1D, their experiences of integrating learnings from HypoPAST into their lives, and their views about sustainability and roll-out of HypoPAST. A mixed-methods approach enables collection of both quantitative data (e.g. intervention uptake, survey responses) and qualitative data (e.g. semi-structured interviews). This process evaluation will draw upon two highly relevant and complementary frameworks: Behaviour Interventions using Technology and Reach, Effectiveness, Adoption, Implementation, and Maintenance . Measures of reach will include study registration rates; proportion of registrants meeting the eligibility criteria; method of referral into the study; demographic and clinical characteristics; and study attrition. Additionally, various website analytics will be collected (Table ) to assess protocol fulfilment with the intervention (i.e. the proportion of participants who accessed the HypoPAST at least once). We will also examine any association(s) between participant outcome and type/duration of content accessed. Intervention group participants will complete study-specific survey items about the intervention’s acceptability and usability (e.g. reasons for accessing/not accessing modules, ease of understanding, trustworthiness, convenience, presentation, likes and dislikes, suggestions for improvement, technical problems) and sustainability (e.g. recommendations to others, suggestions for referral into the programme, potential costs for access). They will also complete the mARM to measure therapeutic alliance with the HypoPAST programme . A semi-structured interview schedule (Additional File 2) will build upon topics from the survey, with the intention to gain in-depth insights into (1) the intervention group participant’s views about the HypoPAST intervention, (2) their experiences implementing principles and strategies from the intervention into their daily lives and (3) their views about sustainability and roll-out of HypoPAST.
The primary outcome is the between-group difference at 24 weeks in fear of hypoglycaemia, assessed using the 18-item Worry subscale of the Hypoglycaemia Fear Survey, version 2 (HFS-II) . We hypothesise a statistically significant difference of at least 9 points will be observed in the Worry score, favouring the intervention.
Clinical, psychological and behavioural At each timepoint, several clinical, psychological and behavioural secondary outcomes are assessed using validated and study-specific survey measures. These include IAH, hypoglycaemia frequency and severity, attitudes to hypoglycaemia awareness, avoidant behaviours related to fear of hypoglycaemia, hypoglycaemia-specific quality of life, hypoglycaemia related post-traumatic stress, diabetes distress, generalised anxiety symptoms and depressive symptoms. Further details of the methods for assessment are provided in Additional File 1. We hypothesise that clinically significant between-group differences will be observed by 24 weeks in these variables, favouring the intervention. Health economics At each timepoint, participants will complete a resource use questionnaire in the online survey, to collect information about the use of other healthcare resources used and lost productivity . They will also complete a generic health-related quality of life questionnaire, the Assessment of Quality of Life 4 dimension (AQoL-4D), to assess utility values and calculate quality adjusted life years (QALYs) . We hypothesise that HypoPAST will be cost-effective from health sector and societal perspectives compared to the control (usual care), with an incremental cost-effectiveness ratio below the commonly used willingness to pay threshold of $50,000/quality-adjusted life year. Ecological momentary assessments Ecological momentary assessment (EMA) provides opportunities to collect data in ‘real time’ on experiences of hypoglycaemia (e.g. timing, glucose level, symptoms) and their impacts (e.g. on sleep quality, productivity, mood). The EMA comprises two parts, both collected via an app. The questions were previously trialled in the 10-week HypoMETRICS study of hypoglycaemia-related experiences among adults with T1D and insulin-treated type 2 diabetes . For the EMA analysis, we hypothesise that significant between-group differences at end-trial will be observed in effects of person-reported hypoglycaemia on daily functioning scores, number of hypoglycaemic episodes, awareness of symptoms and hypoglycaemia burden, all favouring intervention. Reach, acceptability, usability, fidelity and sustainability A mixed-methods approach will be used to examine the reach of HypoPAST, its acceptability and usability to adults with T1D, their experiences of integrating learnings from HypoPAST into their lives, and their views about sustainability and roll-out of HypoPAST. A mixed-methods approach enables collection of both quantitative data (e.g. intervention uptake, survey responses) and qualitative data (e.g. semi-structured interviews). This process evaluation will draw upon two highly relevant and complementary frameworks: Behaviour Interventions using Technology and Reach, Effectiveness, Adoption, Implementation, and Maintenance . Measures of reach will include study registration rates; proportion of registrants meeting the eligibility criteria; method of referral into the study; demographic and clinical characteristics; and study attrition. Additionally, various website analytics will be collected (Table ) to assess protocol fulfilment with the intervention (i.e. the proportion of participants who accessed the HypoPAST at least once). We will also examine any association(s) between participant outcome and type/duration of content accessed. Intervention group participants will complete study-specific survey items about the intervention’s acceptability and usability (e.g. reasons for accessing/not accessing modules, ease of understanding, trustworthiness, convenience, presentation, likes and dislikes, suggestions for improvement, technical problems) and sustainability (e.g. recommendations to others, suggestions for referral into the programme, potential costs for access). They will also complete the mARM to measure therapeutic alliance with the HypoPAST programme . A semi-structured interview schedule (Additional File 2) will build upon topics from the survey, with the intention to gain in-depth insights into (1) the intervention group participant’s views about the HypoPAST intervention, (2) their experiences implementing principles and strategies from the intervention into their daily lives and (3) their views about sustainability and roll-out of HypoPAST.
At each timepoint, several clinical, psychological and behavioural secondary outcomes are assessed using validated and study-specific survey measures. These include IAH, hypoglycaemia frequency and severity, attitudes to hypoglycaemia awareness, avoidant behaviours related to fear of hypoglycaemia, hypoglycaemia-specific quality of life, hypoglycaemia related post-traumatic stress, diabetes distress, generalised anxiety symptoms and depressive symptoms. Further details of the methods for assessment are provided in Additional File 1. We hypothesise that clinically significant between-group differences will be observed by 24 weeks in these variables, favouring the intervention.
At each timepoint, participants will complete a resource use questionnaire in the online survey, to collect information about the use of other healthcare resources used and lost productivity . They will also complete a generic health-related quality of life questionnaire, the Assessment of Quality of Life 4 dimension (AQoL-4D), to assess utility values and calculate quality adjusted life years (QALYs) . We hypothesise that HypoPAST will be cost-effective from health sector and societal perspectives compared to the control (usual care), with an incremental cost-effectiveness ratio below the commonly used willingness to pay threshold of $50,000/quality-adjusted life year.
Ecological momentary assessment (EMA) provides opportunities to collect data in ‘real time’ on experiences of hypoglycaemia (e.g. timing, glucose level, symptoms) and their impacts (e.g. on sleep quality, productivity, mood). The EMA comprises two parts, both collected via an app. The questions were previously trialled in the 10-week HypoMETRICS study of hypoglycaemia-related experiences among adults with T1D and insulin-treated type 2 diabetes . For the EMA analysis, we hypothesise that significant between-group differences at end-trial will be observed in effects of person-reported hypoglycaemia on daily functioning scores, number of hypoglycaemic episodes, awareness of symptoms and hypoglycaemia burden, all favouring intervention.
A mixed-methods approach will be used to examine the reach of HypoPAST, its acceptability and usability to adults with T1D, their experiences of integrating learnings from HypoPAST into their lives, and their views about sustainability and roll-out of HypoPAST. A mixed-methods approach enables collection of both quantitative data (e.g. intervention uptake, survey responses) and qualitative data (e.g. semi-structured interviews). This process evaluation will draw upon two highly relevant and complementary frameworks: Behaviour Interventions using Technology and Reach, Effectiveness, Adoption, Implementation, and Maintenance . Measures of reach will include study registration rates; proportion of registrants meeting the eligibility criteria; method of referral into the study; demographic and clinical characteristics; and study attrition. Additionally, various website analytics will be collected (Table ) to assess protocol fulfilment with the intervention (i.e. the proportion of participants who accessed the HypoPAST at least once). We will also examine any association(s) between participant outcome and type/duration of content accessed. Intervention group participants will complete study-specific survey items about the intervention’s acceptability and usability (e.g. reasons for accessing/not accessing modules, ease of understanding, trustworthiness, convenience, presentation, likes and dislikes, suggestions for improvement, technical problems) and sustainability (e.g. recommendations to others, suggestions for referral into the programme, potential costs for access). They will also complete the mARM to measure therapeutic alliance with the HypoPAST programme . A semi-structured interview schedule (Additional File 2) will build upon topics from the survey, with the intention to gain in-depth insights into (1) the intervention group participant’s views about the HypoPAST intervention, (2) their experiences implementing principles and strategies from the intervention into their daily lives and (3) their views about sustainability and roll-out of HypoPAST.
Self-enrolled, eligible participants will participate in 2 weeks of baseline data collection (survey plus 14-day EMA) before randomisation. Those who do not complete the baseline EMA (defined as missing ≥ 7 the 28 check-ins) will not proceed to randomisation. Those allocated to the intervention will be encouraged to complete the online HypoPAST training in the first 4 to 8 weeks, but will retain access throughout the full 24-week trial. Further data collections will take place at mid-trial (survey) and end-trial (EMA, survey, interviews). From entry to exit, participation will take ≤ 32 weeks, including all data collection. Figure shows the study flow, and Table shows the timeline of activities (e.g. enrolment, randomisation, intervention and data collection).
The RCT sample size and statistical power calculations were conducted using STATA MP (StataCorp) ‘power twomeans’ sample size estimation package. Based on previous research , a standard deviation of 17 was used to estimate a sample size of N = 196 for this study. Given there is no minimal clinically important difference in fear of hypoglycaemia reported in the literature, the anticipated effect size was estimated from previous research . To detect a between-group difference of nine points (standardised effect size of 0.5) in the primary outcome at 6 months, a total of 150 participants (75 per arm) is required. This sample size is a conservative estimate based on 1:1 group allocation for a two-sided test, at the 5% significance level, and a high level of power (90%). Allowing for 30% attrition at 24-week follow-up, the final required sample size is N = 196 participants ( N = 150 × 1.3; n = 98 per study group). For the qualitative sub-study, a purposive sample size of n = 25 was estimated to maximise diversity based on baseline survey responses (e.g. gender, age, education, glucose monitoring method, awareness of hypoglycaemia symptoms, and experience of severe hypoglycaemia).
The primary recruitment method will be via the NDSS, an initiative of the Australian government administered by Diabetes Australia. Approximately 137,000 Australian adults with T1D are registered with the NDSS. NDSS staff will email the study invitation to a random of sample of adult registrants with T1D who have consented to being contacted about research (30%). Additional NDSS emails will be sent, if needed, to ensure timely achievement of the required sample size. In addition, the study will be advertised via the e-newsletter, social media and website of the Australian Centre for Behavioural Research in Diabetes, and via direct email to adults with T1D who have previously requested to be notified of relevant research opportunities. Participant demographics will be monitored throughout recruitment to examine diversity in representation. If needed, the study may be promoted in a more targeted manner (e.g. to encourage certain demographics to participate, within the channels listed above). For the qualitative sub-study, participants will be recruited from intervention group participants using purposive sampling (see above). Engagement with the intervention will also be considered, as the interview questions require participants to comment on their experiences using and implementing the intervention into their daily lives.
Participants will be allocated at random (1:1) to the intervention or control (usual care) arm. The allocation sequence will be generated by computer (via Qualtrics/Platform O) using randomly permuted block sizes. As stratification prevents imbalance in treatment groups for important variables thought to influence the outcome, randomisation will be stratified by: Gender: men versus women. We anticipate that gender may influence the expression of emotional well-being (including the primary outcome and several secondary endpoints). If participants have a ‘non-binary’ or ‘another term’ gender identity, they will be allocated at random to either the male or female gender strata, so there is approximately equal representation of these smaller sub-groups in each arm. Glucose monitoring method: finger-prick versus CGM or intermittent-scanned CGM (isCGM, also known as ‘flash’ monitoring). We anticipate that monitoring method may be associated with fear of hypoglycaemia, frequency and severity of hypoglycaemia, IAH and/or the extent to which participants benefit from HypoPAST (given their access or not to real-time glucose data). The allocation sequence will be stored in the Deakin data centre server system with password protection, accessible only to the system architect of Platform O. Post-randomisation group allocations will be stored in the Deakin data centre and accessed from the backend of Platform O with a secure login, accessible only to the system architect and a research assistant who will be responsible for maintaining a password-protected codebook in Microsoft Excel. Two weeks after self-enrolment in the study, participants will be notified of their group allocation by email, with instructions relevant to their allocation.
During data collection, all members of the HypoPAST research group will be blinded, with the exception of (1) a research assistant who will maintain a password-protected codebook for the purpose of data linkage between data sources (e.g. the app, Qualtrics surveys, website analytics) and be responsible for direct contact with participants (e.g. monitoring enquiries via the HypoPAST email, inviting people to participate in adverse event/process evaluation interviews, administering e-vouchers, informing the participants’ nominated diabetes health professionals about their participation, notifying participants of their randomised group allocations); and (2) the website architect responsible for Platform O (EO). Neither will conduct data analysis. During data analysis, those analysing data for research questions 1 and 2 (ST, US, and VLV) and the investigators will remain blinded. For research question 3, the health economists (CM and MLC) will use blinded data to undertake their preliminary analyses of utility values and QALYs from the AQoL-4D, the self-reported health care resource use and lost productivity. However, the final economic evaluation will be conducted using unblinded data, as they will need to assign intervention costs to the appropriate group. Due to the nature of the intervention and control conditions, participants will be aware of their group allocation. Unblinding of the Project Manager is permissible if they are investigating a potential adverse event (see ‘ ’) via email or telephone interview. This may occur if an individual participant discloses information that identifies their group allocation. In such instances only the Project Manager will be unblinded. Breaches to blinding will be recorded and reported with the study findings.
Plans for assessment and collection of outcomes Data will be collected in four ways: An online survey (administered via Qualtrics), to collect self-reported data on demographic and clinical characteristics; primary and secondary clinical, psychological and behavioural outcomes; and health economic outcomes. Intervention group participants will also answer questions about intervention acceptability and therapeutic alliance (mid-trial survey). The survey questions comprise a combination of validated scales and study-specific items, most of which are fixed-choice and some requiring text input (e.g. writing their age, most recent HbA1c, the number of hypos they have had in the past week, or specifying information where they have selected the ‘other’ response option) (Table and Additional File 1). At mid-trial, intervention group participants are asked two qualitative questions related to intervention acceptability (suggestions for improvement and what they would tell others about HypoPAST). At each timepoint (0, 12 and 24 weeks), the survey is anticipated to take 45 min. An ‘app’, for EMA (administered via the uMotif platform) which collects self-reported data on clinical, psychological, behavioural and health economic outcomes (Table and Additional File 1). Every day for 14 days (at two timepoints: pre-randomisation and end trial), participants will log their hypoglycaemia episodes and symptoms using the ‘motif flower’ in the app. They will complete one motif entry for each episode of hypoglycaemia and can complete the entry at any time on any day during the 2-week data collection period. Participants will also complete daily functioning ‘check-in’ survey questions twice daily (morning: between 06:00 to 12:00, and evening: 18:00 to 24:00) via the app. Each check-in will take about 5 min. Website analytics will be collected via Google Analytics and AUDCI framework, capturing user engagement with the HypoPAST intervention. Semi-structured, audio-recorded telephone interviews (approximately 30–45 min) at end-study, conducted by trained researchers with a sub-set of intervention group participants. The interviews will explore participant’s experiences using the intervention and implementing it into their daily lives, and their views about rollout of the intervention to other adults with diabetes. The interview will take place after the window for end-trial survey completion has closed (weeks 24 to 26), or the end-trial survey is returned, whichever comes first. Plans to promote participant retention and complete follow-up Participants will be offered a token of appreciation, recognising the commitment that it takes to participate in a 6-month trial. E-vouchers (AU$50) will be issued via email to participants after they complete the (1) mid-trial survey, (2) end-trial survey and EMA and (3) interview. This means, for example, an intervention group participant who takes part in both follow-ups and an interview will receive three e-vouchers (total value AU$150). Reminders will be scheduled to encourage engagement with the intervention and follow-up data return. Specifically, two email reminders (1 and 2 weeks after the initial invitation) will be automated to remind participants to complete the mid- and end-trial surveys. The app will send a daily push notification reminding participants to ‘check-in’, if their phone settings allow this. Participants will be able to withdraw from the trial, for any reason, by emailing the study team prior to completion of the 24-week follow-up survey and the closing of the dataset for the primary analysis. Data collected until that timepoint will be retained in the analysis unless otherwise requested by the participant. Due to the online nature of the trial and due to feedback from DUHREC, the study team will not proactively enquire about participant’s reasons for withdrawal or discontinued engagement with the trial (e.g. non-return of follow-up data, refusal to participate in interview); however, reasons will be recorded if they are disclosed by the participant (e.g. via email or telephone contact). Data management As per the HypoPAST data management plan, during the trial, data will be collected by and held in the following locations: Qualtrics (survey responses), uMotif (app responses), Platform O (participant contact details and web analytics), Microsoft Teams (audio recordings of interviews), a transcribing company (audio recordings and transcripts of interviews). Once each phase of data collection is complete (i.e. baseline, 12-week follow-up, 24-week follow-up), or in the case of interviews, after each interview, the database will be locked, data de-identified and downloaded (e.g. from Qualtrics, uMotif, Platform O, Microsoft Teams) to the Deakin University secure network. Subsequently, any copies of the data held elsewhere will be destroyed (i.e. electronic files deleted). Syncplicity (i.e. Sync and Share create via Deakin Research Data Store) will be used to share de-identified data with the researchers with primary responsibility for data analysis. Data will be retained on the Deakin University secure network for at least 15 years after the study findings are published, as required by law. After the study findings are published, metadata will be placed in an open-access data repository (see ‘Availability of data and materials’). Confidentiality Unique participant identification codes will be assigned to participants. Personal identifiers will be removed from the data. A password-protected codebook will be maintained by a research assistant. Individual participants will not be identified in reports of study findings, and data that could potentially be used to identify participants will not be included in the open-access meta-data or shared datasets.
Data will be collected in four ways: An online survey (administered via Qualtrics), to collect self-reported data on demographic and clinical characteristics; primary and secondary clinical, psychological and behavioural outcomes; and health economic outcomes. Intervention group participants will also answer questions about intervention acceptability and therapeutic alliance (mid-trial survey). The survey questions comprise a combination of validated scales and study-specific items, most of which are fixed-choice and some requiring text input (e.g. writing their age, most recent HbA1c, the number of hypos they have had in the past week, or specifying information where they have selected the ‘other’ response option) (Table and Additional File 1). At mid-trial, intervention group participants are asked two qualitative questions related to intervention acceptability (suggestions for improvement and what they would tell others about HypoPAST). At each timepoint (0, 12 and 24 weeks), the survey is anticipated to take 45 min. An ‘app’, for EMA (administered via the uMotif platform) which collects self-reported data on clinical, psychological, behavioural and health economic outcomes (Table and Additional File 1). Every day for 14 days (at two timepoints: pre-randomisation and end trial), participants will log their hypoglycaemia episodes and symptoms using the ‘motif flower’ in the app. They will complete one motif entry for each episode of hypoglycaemia and can complete the entry at any time on any day during the 2-week data collection period. Participants will also complete daily functioning ‘check-in’ survey questions twice daily (morning: between 06:00 to 12:00, and evening: 18:00 to 24:00) via the app. Each check-in will take about 5 min. Website analytics will be collected via Google Analytics and AUDCI framework, capturing user engagement with the HypoPAST intervention. Semi-structured, audio-recorded telephone interviews (approximately 30–45 min) at end-study, conducted by trained researchers with a sub-set of intervention group participants. The interviews will explore participant’s experiences using the intervention and implementing it into their daily lives, and their views about rollout of the intervention to other adults with diabetes. The interview will take place after the window for end-trial survey completion has closed (weeks 24 to 26), or the end-trial survey is returned, whichever comes first.
Participants will be offered a token of appreciation, recognising the commitment that it takes to participate in a 6-month trial. E-vouchers (AU$50) will be issued via email to participants after they complete the (1) mid-trial survey, (2) end-trial survey and EMA and (3) interview. This means, for example, an intervention group participant who takes part in both follow-ups and an interview will receive three e-vouchers (total value AU$150). Reminders will be scheduled to encourage engagement with the intervention and follow-up data return. Specifically, two email reminders (1 and 2 weeks after the initial invitation) will be automated to remind participants to complete the mid- and end-trial surveys. The app will send a daily push notification reminding participants to ‘check-in’, if their phone settings allow this. Participants will be able to withdraw from the trial, for any reason, by emailing the study team prior to completion of the 24-week follow-up survey and the closing of the dataset for the primary analysis. Data collected until that timepoint will be retained in the analysis unless otherwise requested by the participant. Due to the online nature of the trial and due to feedback from DUHREC, the study team will not proactively enquire about participant’s reasons for withdrawal or discontinued engagement with the trial (e.g. non-return of follow-up data, refusal to participate in interview); however, reasons will be recorded if they are disclosed by the participant (e.g. via email or telephone contact).
As per the HypoPAST data management plan, during the trial, data will be collected by and held in the following locations: Qualtrics (survey responses), uMotif (app responses), Platform O (participant contact details and web analytics), Microsoft Teams (audio recordings of interviews), a transcribing company (audio recordings and transcripts of interviews). Once each phase of data collection is complete (i.e. baseline, 12-week follow-up, 24-week follow-up), or in the case of interviews, after each interview, the database will be locked, data de-identified and downloaded (e.g. from Qualtrics, uMotif, Platform O, Microsoft Teams) to the Deakin University secure network. Subsequently, any copies of the data held elsewhere will be destroyed (i.e. electronic files deleted). Syncplicity (i.e. Sync and Share create via Deakin Research Data Store) will be used to share de-identified data with the researchers with primary responsibility for data analysis. Data will be retained on the Deakin University secure network for at least 15 years after the study findings are published, as required by law. After the study findings are published, metadata will be placed in an open-access data repository (see ‘Availability of data and materials’).
Unique participant identification codes will be assigned to participants. Personal identifiers will be removed from the data. A password-protected codebook will be maintained by a research assistant. Individual participants will not be identified in reports of study findings, and data that could potentially be used to identify participants will not be included in the open-access meta-data or shared datasets.
Primary and secondary outcomes Clinical, psychological and behavioural outcomes To examine the effect of the intervention on primary and secondary outcomes (collected via survey), we will use an intention to treat approach where all randomised participants will be analysed according to their study group assignment (regardless of the extent to which intervention group participants engage with the intervention). Statistical analyses will be performed using STATA MP version 17 (StataCorp). Linear mixed models using restricted maximum likelihood estimation will be used to estimate group differences in the primary (HFS-II Worry scale score) and secondary clinical, psychological and behavioural outcomes (Table ). As the primary and secondary outcomes will be measured longitudinally, the outcome at 12 and 24 weeks will be included in the model as the dependent variable. The outcome at baseline, time and an interaction between time and trial arm will be included as fixed effects in the models. Repeated outcome measures will be treated as random effects in the model and an unstructured variance–covariance structure assumed. Models will be adjusted by stratification factors: gender and glucose monitoring method. Transformations for skewed outcome measures will be considered. The estimated mean HFS-II Worry scale score at baseline, 12 and 24 weeks will be plotted for each trial arm with 95% confidence intervals. The estimated difference in mean HFS-II Worry score between arms at 12 and 24 weeks and associated 95% confidence intervals will be presented. A similar modelling approach will be used to estimate group differences in hypoglycaemic events; however, a negative binomial mixed model will be used to account for repeated count data. In secondary analyses, the primary outcome will be adjusted by age, diabetes duration, HbA1c, severe hypoglycaemia episodes in the past 6 months, Gold score and insulin administration modality. Secondary outcomes will also be adjusted by these potential confounders where relevant. A per protocol analysis for the primary outcome will be conducted to estimate the treatment effect in those who engage with the intervention (defined as using 2 or more modules). A sensitivity analysis using multiple imputation may be conducted should there be between 10 and 40% missing data in the primary outcome and auxiliary variables available in the data set to explain the missingness. A second sensitivity analysis will be performed on the primary analysis to test the robustness of the ‘data missing at random’ assumption of mixed-linear models using pattern-mixture modelling. Health economic outcomes A cost-utility analysis will be undertaken from health sector and societal perspectives. The health sector perspective includes costs borne by the government as a third-party payer in addition to out-of-pocket costs incurred by patients when accessing healthcare. Detailed costing of the HypoPAST intervention will be performed using micro-costing methods. The number and types of additional health services used by participants over the period of the trial will be collected with a resource use questionnaire, and standard Australian unit costs will be applied. Total health sector costs will be calculated as the sum of intervention delivery and additional healthcare service use costs. The societal perspective adds the cost of lost productivity (absenteeism and presenteeism) to the health sector costs. Lost productivity will be measured with questions in the resource use questionnaire and valued with the human capital approach using an average Australian wage rate plus on-costs. The AQoL-4D utility values for each participant at each timepoint will be used to calculate QALYs using the area under the curve method. The within-trial economic evaluation will measure and value any change in healthcare resource use and lost productivity and then compare any additional costs to additional QALYs through an incremental cost-effectiveness ratio. Bootstrapping will be used to determine confidence intervals for the incremental cost-effectiveness ratio and construct an acceptability curve to determine the cost-effectiveness of the intervention against the commonly used willingness to pay threshold of AU$50,000/QALY. Missing data will be explored and managed for the resource use and AQoL-4D questionnaires based on recommendations for analysis of trial-based economic evaluations with missing data . Sensitivity analyses will be undertaken to evaluate the robustness of results with changes to costing or analytical assumptions. Scale-up and implementation costs as well as longer-term cost-effectiveness will be estimated based on population-wide modelling techniques using published epidemiological data. STATA (StataCorp) will be used for these analyses. Ecological momentary assessment outcomes To examine the effect of the intervention on EMA outcomes (daily functioning, number of hypoglycaemic episodes, awareness of symptoms and hypoglycaemia burden collected via an app), we will use linear mixed models (for continuous outcomes) and mixed negative binomial models (for count outcomes). Trial arm, time of day (morning/night) and timepoint (baseline or 24 weeks) will be included as fixed effects in the models. Participants will be included as random effects in the models and an unstructured variance–covariance structure used to account for repeated measures and the correlation in outcome within individuals. A separate mixed linear model will be used to explore whether the effect of person-reported hypoglycaemia on daily functioning domains (fear of hypoglycaemia, x, y) differs between trial arms. The independent variable will be person-reported hypoglycaemia, and an interaction between this variable and trial arm (intervention or control) will be included in the model. If sufficient data, we will also assess additional models exploring effects of person-reported hypoglycaemia subtypes (i.e. how the episodes where detected and managed) and whether number of reported hypoglycaemia episodes, awareness (in terms of symptoms) and hypoglycaemia burden differ between trial arms. Models will be adjusted as for the primary outcome. Similarly, missing data will be managed as for the primary objective. Due to the question phrasing and timing of the check-ins, people who expect to primarily sleep during the day during the 2-week EMA data collection will have their EMA data excluded from analysis as it may confound the results. Data analysis will be performed in R-studio (Posit Software, PBC, Boston, MA). Reach, acceptability, usability, fidelity and sustainability Quantitative data will be analysed using descriptive statistics. This includes both survey and website analytics data, including the following: study registration rates; proportion of registrants meeting the eligibility criteria; method of referral into the study; demographic and clinical characteristics; study attrition; number of, and which modules, were accessed; study-specific survey items about intervention acceptability and sustainability. To determine socioeconomic status and geographical location, code will be generated to match postcodes against Australian Bureau of Statistics Australian Statistical Geography Standards and Index of Relative Socio-economic Advantage and Disadvantage Socio-Economic Indexes for Areas quintiles . Open-text survey responses will be collated and summarised descriptively in Microsoft Excel. Interview transcripts will be coded using NVivo and/or Microsoft Excel and analysed using thematic analysis using inductive and deductive approaches. Prior to coding the data, the researcher(s) will become familiar with the dataset by reading the transcripts and/or listening to the audio recordings. Interim and additional analyses No interim analyses are planned. No additional (e.g. subgroup) analyses are planned, but they may occur, as the data will be made available for additional research after the study findings are published (see ‘Availability of data and materials’).
Clinical, psychological and behavioural outcomes To examine the effect of the intervention on primary and secondary outcomes (collected via survey), we will use an intention to treat approach where all randomised participants will be analysed according to their study group assignment (regardless of the extent to which intervention group participants engage with the intervention). Statistical analyses will be performed using STATA MP version 17 (StataCorp). Linear mixed models using restricted maximum likelihood estimation will be used to estimate group differences in the primary (HFS-II Worry scale score) and secondary clinical, psychological and behavioural outcomes (Table ). As the primary and secondary outcomes will be measured longitudinally, the outcome at 12 and 24 weeks will be included in the model as the dependent variable. The outcome at baseline, time and an interaction between time and trial arm will be included as fixed effects in the models. Repeated outcome measures will be treated as random effects in the model and an unstructured variance–covariance structure assumed. Models will be adjusted by stratification factors: gender and glucose monitoring method. Transformations for skewed outcome measures will be considered. The estimated mean HFS-II Worry scale score at baseline, 12 and 24 weeks will be plotted for each trial arm with 95% confidence intervals. The estimated difference in mean HFS-II Worry score between arms at 12 and 24 weeks and associated 95% confidence intervals will be presented. A similar modelling approach will be used to estimate group differences in hypoglycaemic events; however, a negative binomial mixed model will be used to account for repeated count data. In secondary analyses, the primary outcome will be adjusted by age, diabetes duration, HbA1c, severe hypoglycaemia episodes in the past 6 months, Gold score and insulin administration modality. Secondary outcomes will also be adjusted by these potential confounders where relevant. A per protocol analysis for the primary outcome will be conducted to estimate the treatment effect in those who engage with the intervention (defined as using 2 or more modules). A sensitivity analysis using multiple imputation may be conducted should there be between 10 and 40% missing data in the primary outcome and auxiliary variables available in the data set to explain the missingness. A second sensitivity analysis will be performed on the primary analysis to test the robustness of the ‘data missing at random’ assumption of mixed-linear models using pattern-mixture modelling. Health economic outcomes A cost-utility analysis will be undertaken from health sector and societal perspectives. The health sector perspective includes costs borne by the government as a third-party payer in addition to out-of-pocket costs incurred by patients when accessing healthcare. Detailed costing of the HypoPAST intervention will be performed using micro-costing methods. The number and types of additional health services used by participants over the period of the trial will be collected with a resource use questionnaire, and standard Australian unit costs will be applied. Total health sector costs will be calculated as the sum of intervention delivery and additional healthcare service use costs. The societal perspective adds the cost of lost productivity (absenteeism and presenteeism) to the health sector costs. Lost productivity will be measured with questions in the resource use questionnaire and valued with the human capital approach using an average Australian wage rate plus on-costs. The AQoL-4D utility values for each participant at each timepoint will be used to calculate QALYs using the area under the curve method. The within-trial economic evaluation will measure and value any change in healthcare resource use and lost productivity and then compare any additional costs to additional QALYs through an incremental cost-effectiveness ratio. Bootstrapping will be used to determine confidence intervals for the incremental cost-effectiveness ratio and construct an acceptability curve to determine the cost-effectiveness of the intervention against the commonly used willingness to pay threshold of AU$50,000/QALY. Missing data will be explored and managed for the resource use and AQoL-4D questionnaires based on recommendations for analysis of trial-based economic evaluations with missing data . Sensitivity analyses will be undertaken to evaluate the robustness of results with changes to costing or analytical assumptions. Scale-up and implementation costs as well as longer-term cost-effectiveness will be estimated based on population-wide modelling techniques using published epidemiological data. STATA (StataCorp) will be used for these analyses. Ecological momentary assessment outcomes To examine the effect of the intervention on EMA outcomes (daily functioning, number of hypoglycaemic episodes, awareness of symptoms and hypoglycaemia burden collected via an app), we will use linear mixed models (for continuous outcomes) and mixed negative binomial models (for count outcomes). Trial arm, time of day (morning/night) and timepoint (baseline or 24 weeks) will be included as fixed effects in the models. Participants will be included as random effects in the models and an unstructured variance–covariance structure used to account for repeated measures and the correlation in outcome within individuals. A separate mixed linear model will be used to explore whether the effect of person-reported hypoglycaemia on daily functioning domains (fear of hypoglycaemia, x, y) differs between trial arms. The independent variable will be person-reported hypoglycaemia, and an interaction between this variable and trial arm (intervention or control) will be included in the model. If sufficient data, we will also assess additional models exploring effects of person-reported hypoglycaemia subtypes (i.e. how the episodes where detected and managed) and whether number of reported hypoglycaemia episodes, awareness (in terms of symptoms) and hypoglycaemia burden differ between trial arms. Models will be adjusted as for the primary outcome. Similarly, missing data will be managed as for the primary objective. Due to the question phrasing and timing of the check-ins, people who expect to primarily sleep during the day during the 2-week EMA data collection will have their EMA data excluded from analysis as it may confound the results. Data analysis will be performed in R-studio (Posit Software, PBC, Boston, MA). Reach, acceptability, usability, fidelity and sustainability Quantitative data will be analysed using descriptive statistics. This includes both survey and website analytics data, including the following: study registration rates; proportion of registrants meeting the eligibility criteria; method of referral into the study; demographic and clinical characteristics; study attrition; number of, and which modules, were accessed; study-specific survey items about intervention acceptability and sustainability. To determine socioeconomic status and geographical location, code will be generated to match postcodes against Australian Bureau of Statistics Australian Statistical Geography Standards and Index of Relative Socio-economic Advantage and Disadvantage Socio-Economic Indexes for Areas quintiles . Open-text survey responses will be collated and summarised descriptively in Microsoft Excel. Interview transcripts will be coded using NVivo and/or Microsoft Excel and analysed using thematic analysis using inductive and deductive approaches. Prior to coding the data, the researcher(s) will become familiar with the dataset by reading the transcripts and/or listening to the audio recordings.
To examine the effect of the intervention on primary and secondary outcomes (collected via survey), we will use an intention to treat approach where all randomised participants will be analysed according to their study group assignment (regardless of the extent to which intervention group participants engage with the intervention). Statistical analyses will be performed using STATA MP version 17 (StataCorp). Linear mixed models using restricted maximum likelihood estimation will be used to estimate group differences in the primary (HFS-II Worry scale score) and secondary clinical, psychological and behavioural outcomes (Table ). As the primary and secondary outcomes will be measured longitudinally, the outcome at 12 and 24 weeks will be included in the model as the dependent variable. The outcome at baseline, time and an interaction between time and trial arm will be included as fixed effects in the models. Repeated outcome measures will be treated as random effects in the model and an unstructured variance–covariance structure assumed. Models will be adjusted by stratification factors: gender and glucose monitoring method. Transformations for skewed outcome measures will be considered. The estimated mean HFS-II Worry scale score at baseline, 12 and 24 weeks will be plotted for each trial arm with 95% confidence intervals. The estimated difference in mean HFS-II Worry score between arms at 12 and 24 weeks and associated 95% confidence intervals will be presented. A similar modelling approach will be used to estimate group differences in hypoglycaemic events; however, a negative binomial mixed model will be used to account for repeated count data. In secondary analyses, the primary outcome will be adjusted by age, diabetes duration, HbA1c, severe hypoglycaemia episodes in the past 6 months, Gold score and insulin administration modality. Secondary outcomes will also be adjusted by these potential confounders where relevant. A per protocol analysis for the primary outcome will be conducted to estimate the treatment effect in those who engage with the intervention (defined as using 2 or more modules). A sensitivity analysis using multiple imputation may be conducted should there be between 10 and 40% missing data in the primary outcome and auxiliary variables available in the data set to explain the missingness. A second sensitivity analysis will be performed on the primary analysis to test the robustness of the ‘data missing at random’ assumption of mixed-linear models using pattern-mixture modelling.
A cost-utility analysis will be undertaken from health sector and societal perspectives. The health sector perspective includes costs borne by the government as a third-party payer in addition to out-of-pocket costs incurred by patients when accessing healthcare. Detailed costing of the HypoPAST intervention will be performed using micro-costing methods. The number and types of additional health services used by participants over the period of the trial will be collected with a resource use questionnaire, and standard Australian unit costs will be applied. Total health sector costs will be calculated as the sum of intervention delivery and additional healthcare service use costs. The societal perspective adds the cost of lost productivity (absenteeism and presenteeism) to the health sector costs. Lost productivity will be measured with questions in the resource use questionnaire and valued with the human capital approach using an average Australian wage rate plus on-costs. The AQoL-4D utility values for each participant at each timepoint will be used to calculate QALYs using the area under the curve method. The within-trial economic evaluation will measure and value any change in healthcare resource use and lost productivity and then compare any additional costs to additional QALYs through an incremental cost-effectiveness ratio. Bootstrapping will be used to determine confidence intervals for the incremental cost-effectiveness ratio and construct an acceptability curve to determine the cost-effectiveness of the intervention against the commonly used willingness to pay threshold of AU$50,000/QALY. Missing data will be explored and managed for the resource use and AQoL-4D questionnaires based on recommendations for analysis of trial-based economic evaluations with missing data . Sensitivity analyses will be undertaken to evaluate the robustness of results with changes to costing or analytical assumptions. Scale-up and implementation costs as well as longer-term cost-effectiveness will be estimated based on population-wide modelling techniques using published epidemiological data. STATA (StataCorp) will be used for these analyses.
To examine the effect of the intervention on EMA outcomes (daily functioning, number of hypoglycaemic episodes, awareness of symptoms and hypoglycaemia burden collected via an app), we will use linear mixed models (for continuous outcomes) and mixed negative binomial models (for count outcomes). Trial arm, time of day (morning/night) and timepoint (baseline or 24 weeks) will be included as fixed effects in the models. Participants will be included as random effects in the models and an unstructured variance–covariance structure used to account for repeated measures and the correlation in outcome within individuals. A separate mixed linear model will be used to explore whether the effect of person-reported hypoglycaemia on daily functioning domains (fear of hypoglycaemia, x, y) differs between trial arms. The independent variable will be person-reported hypoglycaemia, and an interaction between this variable and trial arm (intervention or control) will be included in the model. If sufficient data, we will also assess additional models exploring effects of person-reported hypoglycaemia subtypes (i.e. how the episodes where detected and managed) and whether number of reported hypoglycaemia episodes, awareness (in terms of symptoms) and hypoglycaemia burden differ between trial arms. Models will be adjusted as for the primary outcome. Similarly, missing data will be managed as for the primary objective. Due to the question phrasing and timing of the check-ins, people who expect to primarily sleep during the day during the 2-week EMA data collection will have their EMA data excluded from analysis as it may confound the results. Data analysis will be performed in R-studio (Posit Software, PBC, Boston, MA).
Quantitative data will be analysed using descriptive statistics. This includes both survey and website analytics data, including the following: study registration rates; proportion of registrants meeting the eligibility criteria; method of referral into the study; demographic and clinical characteristics; study attrition; number of, and which modules, were accessed; study-specific survey items about intervention acceptability and sustainability. To determine socioeconomic status and geographical location, code will be generated to match postcodes against Australian Bureau of Statistics Australian Statistical Geography Standards and Index of Relative Socio-economic Advantage and Disadvantage Socio-Economic Indexes for Areas quintiles . Open-text survey responses will be collated and summarised descriptively in Microsoft Excel. Interview transcripts will be coded using NVivo and/or Microsoft Excel and analysed using thematic analysis using inductive and deductive approaches. Prior to coding the data, the researcher(s) will become familiar with the dataset by reading the transcripts and/or listening to the audio recordings.
No interim analyses are planned. No additional (e.g. subgroup) analyses are planned, but they may occur, as the data will be made available for additional research after the study findings are published (see ‘Availability of data and materials’).
The full protocol and data management plans will be made available upon reasonable request, in writing, to the project manager or lead researcher. The participant-level quantitative data will be made available to researchers, upon reasonable request, after the study findings are published. Participants will be able to ‘opt-out’ from having their data shared/used for future unspecified research by ticking a box on the consent form. Data of participants who tick this box will not be included in the open-access meta-data or datasets shared with non-HypoPAST researchers. Statistical code may be made available upon reasonable request.
Project coordination The study will be coordinated by research personnel from Deakin University. The lead researcher will take overall responsibility for the study, providing guidance and oversight. A programme manager will take responsibility for day-to-day project management, overseeing that all elements of the study are implemented per-protocol and in adherence with ethics principles. For example, they will oversee data monitoring, supervise HypoPAST research personnel, conduct adverse event interviews, communicate with participants (e.g. responding to queries and complaints) and maintain communication with the studies various contributors and stakeholders (e.g. investigators and steering group, website architect, funding body). A statistician/data manager will support data monitoring (e.g. rates of eligible people recruited, randomised, withdrawn, lost to follow-up) and adverse event screening. An associate research fellow will support data entry for data monitoring, adverse event screening and coordination of the HypoPAST ‘Type 1 Diabetes Lived Experience Steering Group’ (see below). A research assistant will support communication with participants (e.g. monitoring the HypoPAST email account, responding to queries and complaints), data entry for data monitoring, maintenance of the password-protected participant codebook and other administrative duties (e.g. sending letters to health professionals, administering vouchers). Communication between the research personnel will occur weekly, via meetings and email. Project oversight Two groups oversee the HypoPAST study: The HypoPAST ‘Investigator Group’ comprises 14 people, including researchers and clinicians (specialising in diabetes education, endocrinology and psychology), representatives of peak bodies (for diabetes and for health professionals), and a person with lived experience of T1D. This group meets four times per year, for project oversight and to contribute their expertise into the study implementation. The HypoPAST ‘Type 1 Diabetes Lived Experience Steering Group’ comprises eight adults with T1D. They reviewed, and informed iterative refinements of, the intervention content and design. This involved a combination of group discussion and one-to-one cognitive debriefing interviews. The Steering Group will have continued involvement throughout the duration of the study, to contribute their expertise into the study implementation. The group meets approximately four times per year. Data monitoring Data will be monitored by the HypoPAST research personnel (see Project coordination). A data monitoring committee was not deemed necessary as the study does not involve an unapproved therapeutic good requiring a Clinical Trial Notification and trial sponsorship. Adverse event reporting The survey responses (at weeks 12 and 24), EMA responses (starting at week 24) and emails from participants to project staff will be monitored for potential adverse events. Baseline survey and EMA data will not be monitored as they are collected prior to randomisation/intervention. Serious adverse events will be defined as any severe hypoglycaemic or mental health-related event requiring medical assistance for recovery (i.e. emergency call-out, emergency department attendance and/or hospital admission). Serious adverse reactions related specifically to HypoPAST are not expected, but consideration will be given to causality. Severe hypoglycaemia or mental health-related events will only be defined as ‘serious’ if they require ambulance call-out, emergency department attendance and/or hospital admission, because the person was able to recover without medical assistance. Participants reporting any event deemed as potentially adverse will be contacted by the project manager (email and/or telephone interview) for further information to clarify the nature of the event. Adverse events will be reported to DUHREC and in the publication reporting study findings. Auditing trial conduct and protocol amendments The project is subject to independent annual financial auditing and will report annually to DUHREC regarding trial implementation. Protocol changes will be communicated to DUHREC, the funders and trial register, and reported with the study findings. Funding body involvement The Medical Research Future Fund (MRFF) Targeted Translation Research Accelerator (TTRA) did not contribute to the development of this trial protocol, and will not be involved in the conduct of the trial data collection, analysis, interpretation or write-up of findings. Dissemination Study findings will be disseminated at scientific conferences and in academic journals. A plain language summary will be published in a blog (via acbrd.org.au) and promoted via social media. Participants who opted-in to future contact will be directly emailed the plain language summary of the key findings. The primary diabetes health professional of participants who opted-in to will also be notified of the published study findings. This multi-level strategy provides several opportunities for the participants and other stakeholders (e.g. health professionals, people with diabetes) to access the findings. Participants will not be identifiable in any dissemination of the research findings. Publication authorship will be defined according to the International Committee of Medical Journal Editors criteria .
The study will be coordinated by research personnel from Deakin University. The lead researcher will take overall responsibility for the study, providing guidance and oversight. A programme manager will take responsibility for day-to-day project management, overseeing that all elements of the study are implemented per-protocol and in adherence with ethics principles. For example, they will oversee data monitoring, supervise HypoPAST research personnel, conduct adverse event interviews, communicate with participants (e.g. responding to queries and complaints) and maintain communication with the studies various contributors and stakeholders (e.g. investigators and steering group, website architect, funding body). A statistician/data manager will support data monitoring (e.g. rates of eligible people recruited, randomised, withdrawn, lost to follow-up) and adverse event screening. An associate research fellow will support data entry for data monitoring, adverse event screening and coordination of the HypoPAST ‘Type 1 Diabetes Lived Experience Steering Group’ (see below). A research assistant will support communication with participants (e.g. monitoring the HypoPAST email account, responding to queries and complaints), data entry for data monitoring, maintenance of the password-protected participant codebook and other administrative duties (e.g. sending letters to health professionals, administering vouchers). Communication between the research personnel will occur weekly, via meetings and email.
Two groups oversee the HypoPAST study: The HypoPAST ‘Investigator Group’ comprises 14 people, including researchers and clinicians (specialising in diabetes education, endocrinology and psychology), representatives of peak bodies (for diabetes and for health professionals), and a person with lived experience of T1D. This group meets four times per year, for project oversight and to contribute their expertise into the study implementation. The HypoPAST ‘Type 1 Diabetes Lived Experience Steering Group’ comprises eight adults with T1D. They reviewed, and informed iterative refinements of, the intervention content and design. This involved a combination of group discussion and one-to-one cognitive debriefing interviews. The Steering Group will have continued involvement throughout the duration of the study, to contribute their expertise into the study implementation. The group meets approximately four times per year.
Data will be monitored by the HypoPAST research personnel (see Project coordination). A data monitoring committee was not deemed necessary as the study does not involve an unapproved therapeutic good requiring a Clinical Trial Notification and trial sponsorship.
The survey responses (at weeks 12 and 24), EMA responses (starting at week 24) and emails from participants to project staff will be monitored for potential adverse events. Baseline survey and EMA data will not be monitored as they are collected prior to randomisation/intervention. Serious adverse events will be defined as any severe hypoglycaemic or mental health-related event requiring medical assistance for recovery (i.e. emergency call-out, emergency department attendance and/or hospital admission). Serious adverse reactions related specifically to HypoPAST are not expected, but consideration will be given to causality. Severe hypoglycaemia or mental health-related events will only be defined as ‘serious’ if they require ambulance call-out, emergency department attendance and/or hospital admission, because the person was able to recover without medical assistance. Participants reporting any event deemed as potentially adverse will be contacted by the project manager (email and/or telephone interview) for further information to clarify the nature of the event. Adverse events will be reported to DUHREC and in the publication reporting study findings.
The project is subject to independent annual financial auditing and will report annually to DUHREC regarding trial implementation. Protocol changes will be communicated to DUHREC, the funders and trial register, and reported with the study findings.
The Medical Research Future Fund (MRFF) Targeted Translation Research Accelerator (TTRA) did not contribute to the development of this trial protocol, and will not be involved in the conduct of the trial data collection, analysis, interpretation or write-up of findings.
Study findings will be disseminated at scientific conferences and in academic journals. A plain language summary will be published in a blog (via acbrd.org.au) and promoted via social media. Participants who opted-in to future contact will be directly emailed the plain language summary of the key findings. The primary diabetes health professional of participants who opted-in to will also be notified of the published study findings. This multi-level strategy provides several opportunities for the participants and other stakeholders (e.g. health professionals, people with diabetes) to access the findings. Participants will not be identifiable in any dissemination of the research findings. Publication authorship will be defined according to the International Committee of Medical Journal Editors criteria .
This study will provide high-quality evidence regarding the effectiveness, cost-effectiveness and acceptability of a novel, online psycho-educational programme, called HypoPAST, which is designed to reduce fear of hypoglyceamia among adults with T1D. HypoPAST draws upon considerable evidence from group-based hypoglycaemia-specific psycho-educational programmes. As it will be delivered fully online, it is expected to involve minimal cost to the health service, and enable nationally, consistent delivery and equitable access, with potential for global reach (following cultural adaptation and translations, beyond the scope of this study). As no health economic analyses have been published of group-based programmes for reducing fear of hypoglycaemia, our study will be the first to provide cost-effectiveness data, which has implications for decision-making regarding efficient resource allocation. HypoPAST has a strong value proposition. Our work and other published research demonstrate that people with T1D (and their families) bear the daily burden of both hypoglycaemia and fear of hypoglycaemia . They manage self-treated and severe episodes and live with the ongoing anxiety related to the risk of such episodes, as well as the often-overlooked impacts on their productivity, sleep and quality of life , as well as on their family members and relationships . We expect that the self-paced learning and experimentation approach will connect directly with participants’ real-world experiences of hypoglycaemia and fear of hypoglycaemia. Online delivery enables easy and convenient access to the programme, with no barriers in terms of time and place of delivery, which could mean broader reach for equitable and effective access and associated value, particularly for those living in rural and remote areas where internet access allows. For health professionals, HypoPAST seeks to address a complex gap in clinical T1D care. Fear of hypoglycaemia is rarely addressed in clinical practice , partly because hypoglycaemia has not been a major clinical focus until recent years. Importantly, a global study (24 countries, > 27,000 participants) shows that hypoglycaemia is under-reported and its impact under-estimated . Face-to-face programmes are resource intensive and have not been implemented routinely, and diabetes technologies do not necessarily reduce fear of hypoglycaemia and can increase the psychological burden . Thus, health professionals do not have appropriate solutions to offer. If this programme were to be delivered face-to-face, access would likely be limited to standard ‘9-to-5’ working hours in specialist diabetes centres in metropolitan areas, and subject to waiting lists. It may also require the individual with diabetes to incur ‘out-of-pocket’ expenses to access the programme, due to the expense involved in delivering it. Our solution, if found to be effective and cost-effective, proposes maximum reach with minimal cost, as it is designed with scalability in mind. A further potential benefit of online delivery includes consistent delivery of content, reducing issues of facilitator fidelity to the curriculum, and ensuring that the programme is delivered as originally developed and intended, with minimal cost to health services. Finally, for national diabetes organisations, HypoPAST may provide an accessible solution that they could offer direct to people with T1D to support them to live well every day with diabetes. If effective, and with appropriate implementation and roll-out, HypoPAST may enable national consistency, reach and equitable access, which is particularly important in countries such as Australia, where there is inequitable access to specialist diabetes care for people living in regional/rural communities .
This study was prospectively registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12623000894695) on 21 August 2023. The study will be conducted in compliance with this protocol (Version 1.1; 24 January 2024). Participant recruitment commenced 29 January 2024 and is expected to be completed by 30 September.
Supplementary Material 1. Supplementary Material 2. Supplementary Material 3.
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Effect of Combining Exercise with Adipose-Derived Mesenchymal Stem Cells in Muscle Atrophy Model of Sarcopenia | 8b39d240-5d38-4ec9-bb16-e61cda84be37 | 11764817 | Surgical Procedures, Operative[mh] | Muscle atrophy is primarily defined as a reduction in muscle mass, whereas sarcopenia is characterized by a progressive decline in muscle mass, muscle strength, and physical function . Sarcopenia is categorized into two types. Primary sarcopenia results from age-related factors, while secondary sarcopenia results from other diseases or pathological conditions with or without the involvement of aging . As the global elderly population steadily increases annually, sarcopenia is becoming an increasingly severe concern. Although primarily observed in the elderly, sarcopenia can also affect middle-aged individuals due to various factors . Additionally, recent meta-analyses have identified sarcopenia as a significant predictor of increased all-cause mortality . The etiology of sarcopenia encompasses reduced protein synthesis, chronic inflammation, oxidative stress, impaired function of satellite cells, hormonal deficiencies, nutritional deficiencies, and a lack of physical activity . These factors cause various diseases and increase multiple risk factors , thereby restricting daily activities, causing functional impairments, and diminishing both the quality of life and physical activity levels in the elderly. This highlights the urgent need for treatments to manage sarcopenia . Previous studies have demonstrated the benefits of exercise and adequate nutrient intake in managing sarcopenia. Specifically, exercise reduces the incidence of chronic diseases, prevents muscle loss, and extends lifespan, making it an effective strategy for improving the health of the elderly. The impact of exercise on muscle structure and function in sarcopenia is substantial . While research on pharmacological treatments continues to be conducted in an effort to discover a fundamental cure for sarcopenia, an effective pharmacological solution has yet to be established . Consequently, it is crucial to identify treatments that halt the pathological progression of muscle degradation in sarcopenia and enhance muscle regeneration. Recent studies have shown that mesenchymal stem cells (MSCs) possess therapeutic potential for treating sarcopenia . MSC-based therapies were reported to promote skeletal muscle regeneration in both animal and cellular models . Among MSCs, adipose-derived MSCs (ADMSCs) offer several advantages: they can be easily obtained in large quantities through minimally invasive procedures and are plentiful in various tissues, making autologous transplantation a viable option. ADMSCs also exhibit exceptional potential to differentiate into various cell types, providing significant prospects for tissue repair and reinforcement . Research indicates that ADMSCs undergo myogenic differentiation when co-cultured with satellite cells or myoblasts , with one study demonstrating muscle recovery in a muscle atrophy model . However, the therapeutic effects of ADMSCs in aged rats with sarcopenia have not been definitively established in previous studies. While individual treatments for sarcopenia often focus on exercise or MSC-based therapies, the combined effects of exercise and MSCs on sarcopenia have not been extensively explored. Therefore, this study examines the effects of exercise only, ADMSC injection only, and the combination of both on the treatment of sarcopenia in an aged rat model.
2.1. Effect of ADMSCs and Exercise on Body and Gastrocnemius (GCM) Muscle Weights in Immobilized Induced Atrophy Models After 28 d, no differences in body weight were observed among the experimental groups ( A). However, the GCM muscle weight increased in both the Ex and MSC groups (333.6 ± 11.7 and 326.4 ± 8.8, respectively), with the MSC + Ex group showing a more substantial increase compared to the control (349.2 ± 7.8 vs. 276.5 ± 11.8, respectively) ( B). Since we hypothesized that injecting stem cells or engaging in exercise might have a beneficial impact on muscle repair, we measured the myofiber cross-sectional area (CSA) in GCM muscles, and the mean myofiber CSA in GCM muscles did not show statistically significant differences between groups . Thus, the distribution of myofiber sizes shifted toward larger sizes in the Ex and MSC + Ex groups compared to the control ( C,D). 2.2. Behavioral Assessments To determine whether changes in muscle mass impacted motor and exercise performance, we used the rotarod, hot plate, and beam walk tests. After 28 d, the latency to fall from the rotarod significantly increased in the MSC + Ex group compared to the control (129.6 ± 10.0 vs. 40.1 ± 14.5, respectively), indicating enhanced performance ( A). Although not statistically significant, the latency to fall also increased in both the Ex and MSC groups (79.0 ± 26.4 and 93.9 ± 20.4, respectively) ( A). In the hot plate test, the response times to the thermal stimulus decreased in both the Ex and MSC groups, with the fastest response being observed in the MSC + Ex group, indicating improved escape behavior ( B). In the beam walk test, although the results did not reach statistical significance, a decreasing trend in the time to cross the beam was observed (10.3 ± 1.7 for control, 8.6 ± 1.0 for MSC, and 6.2 ± 0.3 for MSC + Ex) ( C). 2.3. Altered GCM Muscle Inflammatory Cytokine Expression The mRNA expression levels of six candidate genes ( Tnf , Il10 , Il6 , Il1β , Ccl2 , and Mif ) of inflammatory markers were measured using Quantitative Reverse Transcription PCR (qPCR) in GCM muscles. The expression levels of Tnf were significantly reduced in the Ex, MSC, and MSC + Ex groups compared to the control (0.53 ± 0.11 for Ex, 0.55 ± 0.08 for MSC, and 0.44 ± 0.03 for MSC + Ex vs. 1.13 ± 0.24 for control) ( A). Il10 expression was notably lower in the MSC group compared to the control (0.30 ± 0.03 vs. 0.86 ± 0.12, respectively) ( B). Il6 expression also showed a decreasing trend in the Ex, MSC, and MSC + Ex groups (0.73 ± 0.17 for Ex, 0.46 ± 0.07 for MSC, and 0.48 ± 0.15 for MSC + Ex vs. 1.36 ± 0.37 for control) ( C). The expression levels of Il1β , Ccl2 , and Mif did not exhibit substantial differences across all groups ( D–F). Furthermore, TNF protein expression in GCM muscles was diminished in both the Ex and MSC groups (0.56 ± 0.05 for Ex and 0.48 ± 0.05 for MSC), with an even greater reduction observed in the MSC + Ex group compared to the control (0.41 ± 0.08 for MSC + Ex vs. 1.0 ± 0.14 for control; G). 2.4. Effect of Intramuscular Injection of MSC and Exercise on AMP-Activated Protein Kinase (AMPK) Phosphorylation Skeletal muscle regeneration is influenced by the survival and proliferation of MSCs. Previous studies have indicated that AMPK plays a crucial role in regulating the self-renewal and proliferation of stem cells . We assessed the protein levels of AMPK after 28 d. Following 2 weeks of exercise and ADMSC injections in rats with immobilization-induced atrophy, a statistically significant decrease in AMPK phosphorylation was observed in the GCM muscles (0.52 ± 0.13 for Ex and 0.24 ± 0.09 for MSC) . Additionally, a further reduction was evident in the MSC + Ex group compared to the control (0.20 ± 0.02 for MSC + Ex vs. 1.0 ± 0.09 for control; ).
After 28 d, no differences in body weight were observed among the experimental groups ( A). However, the GCM muscle weight increased in both the Ex and MSC groups (333.6 ± 11.7 and 326.4 ± 8.8, respectively), with the MSC + Ex group showing a more substantial increase compared to the control (349.2 ± 7.8 vs. 276.5 ± 11.8, respectively) ( B). Since we hypothesized that injecting stem cells or engaging in exercise might have a beneficial impact on muscle repair, we measured the myofiber cross-sectional area (CSA) in GCM muscles, and the mean myofiber CSA in GCM muscles did not show statistically significant differences between groups . Thus, the distribution of myofiber sizes shifted toward larger sizes in the Ex and MSC + Ex groups compared to the control ( C,D).
To determine whether changes in muscle mass impacted motor and exercise performance, we used the rotarod, hot plate, and beam walk tests. After 28 d, the latency to fall from the rotarod significantly increased in the MSC + Ex group compared to the control (129.6 ± 10.0 vs. 40.1 ± 14.5, respectively), indicating enhanced performance ( A). Although not statistically significant, the latency to fall also increased in both the Ex and MSC groups (79.0 ± 26.4 and 93.9 ± 20.4, respectively) ( A). In the hot plate test, the response times to the thermal stimulus decreased in both the Ex and MSC groups, with the fastest response being observed in the MSC + Ex group, indicating improved escape behavior ( B). In the beam walk test, although the results did not reach statistical significance, a decreasing trend in the time to cross the beam was observed (10.3 ± 1.7 for control, 8.6 ± 1.0 for MSC, and 6.2 ± 0.3 for MSC + Ex) ( C).
The mRNA expression levels of six candidate genes ( Tnf , Il10 , Il6 , Il1β , Ccl2 , and Mif ) of inflammatory markers were measured using Quantitative Reverse Transcription PCR (qPCR) in GCM muscles. The expression levels of Tnf were significantly reduced in the Ex, MSC, and MSC + Ex groups compared to the control (0.53 ± 0.11 for Ex, 0.55 ± 0.08 for MSC, and 0.44 ± 0.03 for MSC + Ex vs. 1.13 ± 0.24 for control) ( A). Il10 expression was notably lower in the MSC group compared to the control (0.30 ± 0.03 vs. 0.86 ± 0.12, respectively) ( B). Il6 expression also showed a decreasing trend in the Ex, MSC, and MSC + Ex groups (0.73 ± 0.17 for Ex, 0.46 ± 0.07 for MSC, and 0.48 ± 0.15 for MSC + Ex vs. 1.36 ± 0.37 for control) ( C). The expression levels of Il1β , Ccl2 , and Mif did not exhibit substantial differences across all groups ( D–F). Furthermore, TNF protein expression in GCM muscles was diminished in both the Ex and MSC groups (0.56 ± 0.05 for Ex and 0.48 ± 0.05 for MSC), with an even greater reduction observed in the MSC + Ex group compared to the control (0.41 ± 0.08 for MSC + Ex vs. 1.0 ± 0.14 for control; G).
Skeletal muscle regeneration is influenced by the survival and proliferation of MSCs. Previous studies have indicated that AMPK plays a crucial role in regulating the self-renewal and proliferation of stem cells . We assessed the protein levels of AMPK after 28 d. Following 2 weeks of exercise and ADMSC injections in rats with immobilization-induced atrophy, a statistically significant decrease in AMPK phosphorylation was observed in the GCM muscles (0.52 ± 0.13 for Ex and 0.24 ± 0.09 for MSC) . Additionally, a further reduction was evident in the MSC + Ex group compared to the control (0.20 ± 0.02 for MSC + Ex vs. 1.0 ± 0.09 for control; ).
Following the induction of muscle atrophy through hindlimb immobilization and aging, both exercise and ADMSC treatments showed increases in muscle mass and improvements in muscle function. Notably, the combined therapy of exercise and ADMSC injections demonstrated the highest indicators of muscle mass increase and functional enhancement. The combined therapy group exhibited the highest performance in the rotarod test and the fastest escape in the hot plate test. Furthermore, the combined therapy group presented the lowest TNF level and activated AMPK, which were possibly related to the pathogenesis of sarcopenia. A previous similar study demonstrated that in rats with muscle atrophy induced by spinal cord injury, the combination of exercise and bone marrow stromal cell transplantation had the potential to accelerate protein synthesis and hypertrophy . This was a denervation-induced muscle atrophy model. However, immobilization-induced muscle atrophy involves a physiologically different mechanism for decreasing muscle mass compared to nerve injuries like spinal cord injury. The present study reports that the combined therapy of exercise and ADMSC injections was the most efficacious and synergistic intervention for the model of muscle atrophy induced by immobilization and aging. Muscle atrophy-induced sarcopenia is characterized by declines in muscle mass and function. In our study, the Ex, MSC, and MSC + Ex groups showed an increase in the GCM muscle mass compared to the control. Although statistical significance was not achieved, the distribution of the CSA in the GCM muscle indicates that the Ex and MSC + Ex groups had a higher proportion of fibers of larger size compared to the control group. These findings align with recent studies demonstrating that MSCs and exercise therapy increase muscle mass and the CSA. Wang et al. demonstrated that the intravenous injection of ADMSCs in dexamethasone-induced sarcopenic rats improved body lean mass, muscle mass, and the CSA of the GCM muscle. Another recent study showed that in sarcopenic mice, the injection of umbilical cord-derived MSCs either via the tail vein or through local intramuscular administration resulted in improved muscle mass, CSA of the GCM muscle, and overall muscle strength . Herein, we conducted a muscle function assessment using the rotarod and hot plate tests, revealing that the Ex, MSC, and MSC + Ex groups exhibited enhanced balance and rapid escape performance compared to the control group. Notably, the MSC + Ex group demonstrated the most significant improvements. Therefore, using ADMSCs in conjunction with exercise interventions effectively improved both muscle mass and function. TNF is known to play a significant role in the pathogenesis of sarcopenia . Previous studies have reported that TNF promotes inflammatory responses and increases protein degradation in skeletal muscle, decreasing muscle mass and impairing function. Therefore, the decreased levels of TNF, a representative pro-inflammatory cytokine, in the Ex, MSC, and MSC + Ex groups compared to the control indicate that exercise and ADMSC injections suppress inflammation, thereby facilitating the mitigation of sarcopenia. Previous animal model studies support our current results, showing that treadmill exercise often leads to decreased TNF levels . Additionally, a recent study demonstrated that the local injection of ADMSCs into the midpoint of the GCM muscle induced reductions in TNF and IL-6 levels in dystrophin-deficient mice . In the present study, we observed that the TNF levels were the lowest in the MSC + Ex group, indicating a synergistic effect of the combination of exercise and ADMSCs in decreasing inflammation associated with sarcopenia. AMPK regulates both protein synthesis and degradation in muscle growth and is involved in various processes, such as muscle size in a basal state, muscle hypertrophy, muscle atrophy, and muscle regeneration . Bolster et al. demonstrated that subcutaneous injection of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) in rats resulted in a 45% reduction in protein synthesis rates in skeletal muscle compared to the control group . However, the role of AMPK in muscle atrophy remains uncertain. Studies on rodent skeletal muscle subjected to 1–4 weeks of hindlimb unloading have reported conflicting results: some reported an increase in AMPK phosphorylation , whereas others observed a decrease . Additionally, other research has found no effect of hindlimb unloading on AMPK activity or the phosphorylation of acetyl-CoA carboxylase, a target and biomarker of AMPK . Furthermore, previous studies have shown that activated AMPK plays a crucial role in reducing the self-renewal and proliferation of MSCs in vitro and ex vivo . This supports the findings of the present study, where the p-AMPK/AMPK ratio was decreased in the Ex, MSC, and MSC + Ex groups compared to the control. These results suggest that a reduction in AMPK phosphorylation at Thr-172 may lead to increased muscle mass and enhanced MSC self-renewal and proliferation, potentially alleviating sarcopenia. However, there are several limitations to consider. First, the present study did not include muscle strength as an outcome measure. Although grip strength is an important marker of sarcopenia, its validity in rats was unsatisfactory as the strength values varied among individual rats during our pilot study. Second, muscle atrophy was induced through immobilization and aging to establish a sarcopenia model. Even though there are previous reports that showed this model being used as a sarcopenic rat model , this approach may not fully demonstrate a true sarcopenia model, highlighting the need for a more appropriate and refined model representing primary sarcopenia. Third, ultrasound-guided intramuscular injection was performed to ensure that ADMSCs were accurately injected into the targeted regions of the gastrocnemius muscle. However, tracking their homing and visualizing them post-injection proved challenging, indicating the need for additional strategies, such as cell staining or labeling. Fourth, the dosage of MSCs was not determined. We based our MSC transplant design on a previous study , but further research is needed to determine the optimal concentration of MSCs for injection according to age and to identify potential side effects. Moreover, optimizing variables such as the route of administration, timing, and cell condition that affect MSCs’ survival and homing is crucial for maximizing therapeutic efficacy . Fifth, it is necessary to assess the most effective exercise regimen when combining MSC injection with exercise. Sixth, while we employed immunohistochemistry to assess the cross-sectional area (CSA) of myofibers, myonuclear staining was not included in this study. According to previous studies , myonuclear staining is not deemed essential for CSA analysis. However, incorporating myonuclear staining in future investigations could provide additional insights into myofiber structure. Finally, this study was conducted with an evaluation period of only 28 days, which is relatively short. A follow-up study is necessary to assess the long-term effects.
4.1. Animals and Muscle Tissue Preparation For this muscle atrophy-induced sarcopenia study, experiments were conducted on 18-month-old male rats (DBL, Chungcheongbuk-do, Republic of Korea). The rats were randomly divided into four groups, each comprising six rats: control (control), treadmill exercise (Ex), ADMSC injection (MSC), and ADMSC injection with treadmill (MSC + Ex) groups. After the experiments, the rats were anesthetized with 5% isoflurane and euthanized in a carbon dioxide chamber, and GCM muscle samples were collected. The GCM muscles from both hindlimbs were excised and weighed. One hindlimb was immediately frozen in liquid nitrogen and stored at −80 °C, while the other was used to create a formalin-fixed, paraffin-embedded block for a muscle histology analysis. All animal experiments received approval from the Kangbuk Samsung Hospital Research Institute’s Animal Care Committee (Approval ID: 202011133; approval date: 12 October 2020). 4.2. Velcro Immobilization The hindlimb Velcro immobilization method was adopted to induce a muscle atrophy model . The procedure was conducted under anesthesia with 2% isoflurane. The rats’ hindlimbs and surrounding fur were shaved. Each hindlimb was kept in an extended position to maintain the knee and ankle joints in a stretched state, secured with surgical and Velcro tapes around the limb, and then reinforced with adhesive plaster. In the initial stage of the experiment, all groups of rats were subjected to Velcro immobilization for 14 d, during which they were able to freely move within the cage using their forelimbs and had access to food and water. After the Velcro was removed, the treadmill exercises, ADMSC injections, and combined treatments were initiated to enhance muscle regeneration and promote functional improvement in the muscles ( A). 4.3. ADMSC Preparation and Ultrasound-Guided Intramuscular Injection ADMSCs at passage 1, which were isolated from interscapular brown fat tissue of rats, were obtained from iXCells Biotechnologies (10RA-002, San Diego, CA, USA). The cells were cultured in adipose-derived stem cell growth medium (MD-0003, iXCells Biotechnologies, San Diego, CA, USA), which includes 500 mL of Basal Medium, 50 mL of FBS, 1 mL of adipose-derived stem cell growth supplement, and 5 mL of antibiotic-antimycotic (100X), in a humidified 5% CO 2 incubator at 37 °C according to the manufacturer’s protocol. ADMSCs were assessed based on their passage number, morphology, and stem cell potency with reference to previous studies . The purchased cells (at passage 1) were counted and seeded to 5 × 10 3 cell/cm 2 . When the cells reached 70–80% confluency, they were sub-cultured at a density of 5 × 10 3 cells/cm 2 . Flow cytometry analysis was conducted on cells at passage 2 to confirm stem cell identity before creating a stock, and they tested positive for stem cell marker CD90 and negative for CD18 and CD45 . During the immobilization period, frozen cells were thawed and allowed to recover. Once they reached passage 3, the cells were dissociated into single cells and stained with trypan blue, and only the viable cells were used for the experiments. Following the removal of the Velcro, rats in both the MSC and MSC + Ex groups received injections of 1.0 × 10 6 cells in Phosphate-Buffered Saline (PBS) into their right and left GCM muscles, respectively. Meanwhile, the control and Ex groups were injected with the same volume of PBS as the ADMSC injection groups . The injections were performed under ultrasound guidance to ensure accurate delivery into the GCM muscle, specifically targeting the intersection point of the x -axis and y -axis at the thickest portions of the muscle. 4.4. Ultrasound Assessment Ultrasound evaluations of the GCM muscle were performed in triplicate: prior to Velcro immobilization, during the ultrasound-guided injection of ADMSCs and PBS ( B), and following the recovery period. The measurements taken included the distance (mm), circumference (cm), and CSA (cm 2 ) of the GCM muscle under ultrasound guidance . 4.5. Exercise Intervention To explore the relationship between ADMSC injection and exercise during the recovery period in rats with muscle atrophy, treadmill exercise was performed (DJ-344, Daejonglab, Seoul, Republic of Korea) . Rats in the Ex and MSC + Ex groups underwent a 3 d adaptation period before Velcro immobilization. On day 1, rats adapted to the treadmill for 20 min. On day 2, they exercised at a speed of 10 m/min for 20 min, and on day 3, the duration was extended to 40 min at the same speed. Following Velcro removal, during the recovery period, the rats engaged in training sessions of 60 min at 10 m/min, 5 d a week, for 2 weeks. 4.6. Functional Tests Functional tests, including the rotarod, beam walk, and hot plate tests, were conducted on days 0 and 28 of the experimental period. The rotarod test (DJ-345, Daejonglab, Seoul, Republic of Korea) assessed muscle function by gradually increasing the speed from 4 to 40 rpm over 300 s, with three trials being conducted with 15 min intervals between each. The average value was recorded. The beam walk assessed motor balance and coordination by timing how long it took each rat to traverse a beam measuring 60 mm in width and 120 cm in length . The journey from the starting point to a dark box (25 × 25 × 25 cm) at the opposite end was timed three times and averaged. The hot plate test (JD-A-10A, Jeongdo Bio, Seoul, Republic of Korea) evaluated sensory and motor functions by measuring the time taken for specific responses to occur, such as climbing, jumping, stamping, and licking of the forelimbs and hindlimbs . Rats were placed on a hot plate maintained at a temperature in the range of 50–55 °C, and the reaction times were recorded. 4.7. Immunohistochemistry The following antibodies and reagents were used to determine fiber size: (1) rabbit polyclonal anti-laminin primary antibody (L9393, Sigma-Aldrich, St. Louis, MO, USA); (2) donkey anti-rabbit IgG (H + L) secondary antibody, Alexa Fluor488 (A-21206, Thermo Fisher Scientific, Waltham, MA, USA); (3) vibrance antifade mounting medium with DAPI (H-1800, Vector Laboratories, Newark, CA, USA); and (4) pepsin (P6887, Sigma-Aldrich, St. Louis, MO, USA). The GCM muscles were fixed in 10% buffered formalin for 48 h and then embedded in paraffin blocks. Prior to staining, slides were prepared from the central region of the GCM injection site and sectioned at a thickness of six micrometers. The slides were deparaffinized, rehydrated, and incubated for 1 h with pepsin (4 mg/mL in 0.01 M HCl, pH approximately 2.0) at 37 °C in a humidified chamber for antigen retrieval. Afterward, the slides were incubated in a blocking solution (5% Bovine Serum Albumin with 0.1% Triton X-100 in PBS) for 1 h at room temperature (22–24 °C). Following blocking, the sections were incubated overnight with laminin antibody (diluted 1:25) at 4 °C. The slides were then washed three times with PBS for 10 min each, and secondary antibodies (diluted 1:1000) were applied for 1 h at 37 °C in a humidified chamber. Following this, the slides were washed three times with PBS and mounted with 4′,6-diamidino-2-phenylindole-containing medium. The slides were left to dry at room temperature or at 4 °C. Five sections from the central region of each stained slide were randomly selected and imaged at 20× magnification using a confocal microscope (STELLARIS 5, Leica Microsystems, Deerfield, IL, USA) with Leica LAS X imaging software version 3.7.6.25997. The CSA was analyzed using ImageJ software version 1.54. 4.8. RNA Extraction and Quantitative Reverse Transcription PCR Total RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). mRNA was reverse-transcribed using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using specific primers and SYBR Green on an LC480 instrument (Roche, Welwyn Garden City, UK). A list of the primers used is provided in . The relative mRNA expression levels were analyzed using the 2 −ΔΔCt method with GAPDH serving as the reference gene. 4.9. Western Blotting Total protein from GCM muscle tissues was homogenized using RIPA lysis buffer (Thermo Fisher Scientific) containing a protease inhibitor, followed by centrifugation at 13,500× g at 4 °C for 15 min to extract the proteins. The proteins were then separated on Sodium Dodecyl Sulfate–polyacrylamide gels and transferred for immunoblotting using specific antibodies, including GAPDH (#2118, Danvers, MA, USA), α/β-Tubulin (#2148, Danvers, MA, USA), AMPK (#5831, Danvers, MA, USA), phosphorylated AMPK (#2535, Danvers, MA, USA), anti-rabbit IgG HRP-linked antibody (#7074, Danvers, MA, USA), anti-mouse IgG HRP-linked antibody (#7076, Cell Signaling Technology, Danvers, MA, USA), and TNF-α (#SC-52746, Santa Cruz, Dallas, TX, USA). Primary antibodies were diluted to a ratio of 1:1000 and secondary antibodies to a ratio of 1:10,000. Protein detection was conducted using Enhanced Chemiluminescence solution and imaged with a ChemiDoc system and then quantified using FUSION FX software version 18.10. 4.10. Statistical Analysis After data collection, statistical analysis was performed using GraphPad Prism version 10.0.3. For comparisons between groups subjected to different treatments such as behavioral tests, qPCR, and Western blotting, a one-way analysis of variance (ANOVA) was conducted, followed by Tukey’s multiple comparisons test. For analyses of CSA differences between groups, a two-way ANOVA was utilized, also followed by Tukey’s multiple comparisons test. All data are presented as the mean ± standard error of the mean. Statistical significance was established at a p -value < 0.05.
For this muscle atrophy-induced sarcopenia study, experiments were conducted on 18-month-old male rats (DBL, Chungcheongbuk-do, Republic of Korea). The rats were randomly divided into four groups, each comprising six rats: control (control), treadmill exercise (Ex), ADMSC injection (MSC), and ADMSC injection with treadmill (MSC + Ex) groups. After the experiments, the rats were anesthetized with 5% isoflurane and euthanized in a carbon dioxide chamber, and GCM muscle samples were collected. The GCM muscles from both hindlimbs were excised and weighed. One hindlimb was immediately frozen in liquid nitrogen and stored at −80 °C, while the other was used to create a formalin-fixed, paraffin-embedded block for a muscle histology analysis. All animal experiments received approval from the Kangbuk Samsung Hospital Research Institute’s Animal Care Committee (Approval ID: 202011133; approval date: 12 October 2020).
The hindlimb Velcro immobilization method was adopted to induce a muscle atrophy model . The procedure was conducted under anesthesia with 2% isoflurane. The rats’ hindlimbs and surrounding fur were shaved. Each hindlimb was kept in an extended position to maintain the knee and ankle joints in a stretched state, secured with surgical and Velcro tapes around the limb, and then reinforced with adhesive plaster. In the initial stage of the experiment, all groups of rats were subjected to Velcro immobilization for 14 d, during which they were able to freely move within the cage using their forelimbs and had access to food and water. After the Velcro was removed, the treadmill exercises, ADMSC injections, and combined treatments were initiated to enhance muscle regeneration and promote functional improvement in the muscles ( A).
ADMSCs at passage 1, which were isolated from interscapular brown fat tissue of rats, were obtained from iXCells Biotechnologies (10RA-002, San Diego, CA, USA). The cells were cultured in adipose-derived stem cell growth medium (MD-0003, iXCells Biotechnologies, San Diego, CA, USA), which includes 500 mL of Basal Medium, 50 mL of FBS, 1 mL of adipose-derived stem cell growth supplement, and 5 mL of antibiotic-antimycotic (100X), in a humidified 5% CO 2 incubator at 37 °C according to the manufacturer’s protocol. ADMSCs were assessed based on their passage number, morphology, and stem cell potency with reference to previous studies . The purchased cells (at passage 1) were counted and seeded to 5 × 10 3 cell/cm 2 . When the cells reached 70–80% confluency, they were sub-cultured at a density of 5 × 10 3 cells/cm 2 . Flow cytometry analysis was conducted on cells at passage 2 to confirm stem cell identity before creating a stock, and they tested positive for stem cell marker CD90 and negative for CD18 and CD45 . During the immobilization period, frozen cells were thawed and allowed to recover. Once they reached passage 3, the cells were dissociated into single cells and stained with trypan blue, and only the viable cells were used for the experiments. Following the removal of the Velcro, rats in both the MSC and MSC + Ex groups received injections of 1.0 × 10 6 cells in Phosphate-Buffered Saline (PBS) into their right and left GCM muscles, respectively. Meanwhile, the control and Ex groups were injected with the same volume of PBS as the ADMSC injection groups . The injections were performed under ultrasound guidance to ensure accurate delivery into the GCM muscle, specifically targeting the intersection point of the x -axis and y -axis at the thickest portions of the muscle.
Ultrasound evaluations of the GCM muscle were performed in triplicate: prior to Velcro immobilization, during the ultrasound-guided injection of ADMSCs and PBS ( B), and following the recovery period. The measurements taken included the distance (mm), circumference (cm), and CSA (cm 2 ) of the GCM muscle under ultrasound guidance .
To explore the relationship between ADMSC injection and exercise during the recovery period in rats with muscle atrophy, treadmill exercise was performed (DJ-344, Daejonglab, Seoul, Republic of Korea) . Rats in the Ex and MSC + Ex groups underwent a 3 d adaptation period before Velcro immobilization. On day 1, rats adapted to the treadmill for 20 min. On day 2, they exercised at a speed of 10 m/min for 20 min, and on day 3, the duration was extended to 40 min at the same speed. Following Velcro removal, during the recovery period, the rats engaged in training sessions of 60 min at 10 m/min, 5 d a week, for 2 weeks.
Functional tests, including the rotarod, beam walk, and hot plate tests, were conducted on days 0 and 28 of the experimental period. The rotarod test (DJ-345, Daejonglab, Seoul, Republic of Korea) assessed muscle function by gradually increasing the speed from 4 to 40 rpm over 300 s, with three trials being conducted with 15 min intervals between each. The average value was recorded. The beam walk assessed motor balance and coordination by timing how long it took each rat to traverse a beam measuring 60 mm in width and 120 cm in length . The journey from the starting point to a dark box (25 × 25 × 25 cm) at the opposite end was timed three times and averaged. The hot plate test (JD-A-10A, Jeongdo Bio, Seoul, Republic of Korea) evaluated sensory and motor functions by measuring the time taken for specific responses to occur, such as climbing, jumping, stamping, and licking of the forelimbs and hindlimbs . Rats were placed on a hot plate maintained at a temperature in the range of 50–55 °C, and the reaction times were recorded.
The following antibodies and reagents were used to determine fiber size: (1) rabbit polyclonal anti-laminin primary antibody (L9393, Sigma-Aldrich, St. Louis, MO, USA); (2) donkey anti-rabbit IgG (H + L) secondary antibody, Alexa Fluor488 (A-21206, Thermo Fisher Scientific, Waltham, MA, USA); (3) vibrance antifade mounting medium with DAPI (H-1800, Vector Laboratories, Newark, CA, USA); and (4) pepsin (P6887, Sigma-Aldrich, St. Louis, MO, USA). The GCM muscles were fixed in 10% buffered formalin for 48 h and then embedded in paraffin blocks. Prior to staining, slides were prepared from the central region of the GCM injection site and sectioned at a thickness of six micrometers. The slides were deparaffinized, rehydrated, and incubated for 1 h with pepsin (4 mg/mL in 0.01 M HCl, pH approximately 2.0) at 37 °C in a humidified chamber for antigen retrieval. Afterward, the slides were incubated in a blocking solution (5% Bovine Serum Albumin with 0.1% Triton X-100 in PBS) for 1 h at room temperature (22–24 °C). Following blocking, the sections were incubated overnight with laminin antibody (diluted 1:25) at 4 °C. The slides were then washed three times with PBS for 10 min each, and secondary antibodies (diluted 1:1000) were applied for 1 h at 37 °C in a humidified chamber. Following this, the slides were washed three times with PBS and mounted with 4′,6-diamidino-2-phenylindole-containing medium. The slides were left to dry at room temperature or at 4 °C. Five sections from the central region of each stained slide were randomly selected and imaged at 20× magnification using a confocal microscope (STELLARIS 5, Leica Microsystems, Deerfield, IL, USA) with Leica LAS X imaging software version 3.7.6.25997. The CSA was analyzed using ImageJ software version 1.54.
Total RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). mRNA was reverse-transcribed using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using specific primers and SYBR Green on an LC480 instrument (Roche, Welwyn Garden City, UK). A list of the primers used is provided in . The relative mRNA expression levels were analyzed using the 2 −ΔΔCt method with GAPDH serving as the reference gene.
Total protein from GCM muscle tissues was homogenized using RIPA lysis buffer (Thermo Fisher Scientific) containing a protease inhibitor, followed by centrifugation at 13,500× g at 4 °C for 15 min to extract the proteins. The proteins were then separated on Sodium Dodecyl Sulfate–polyacrylamide gels and transferred for immunoblotting using specific antibodies, including GAPDH (#2118, Danvers, MA, USA), α/β-Tubulin (#2148, Danvers, MA, USA), AMPK (#5831, Danvers, MA, USA), phosphorylated AMPK (#2535, Danvers, MA, USA), anti-rabbit IgG HRP-linked antibody (#7074, Danvers, MA, USA), anti-mouse IgG HRP-linked antibody (#7076, Cell Signaling Technology, Danvers, MA, USA), and TNF-α (#SC-52746, Santa Cruz, Dallas, TX, USA). Primary antibodies were diluted to a ratio of 1:1000 and secondary antibodies to a ratio of 1:10,000. Protein detection was conducted using Enhanced Chemiluminescence solution and imaged with a ChemiDoc system and then quantified using FUSION FX software version 18.10.
After data collection, statistical analysis was performed using GraphPad Prism version 10.0.3. For comparisons between groups subjected to different treatments such as behavioral tests, qPCR, and Western blotting, a one-way analysis of variance (ANOVA) was conducted, followed by Tukey’s multiple comparisons test. For analyses of CSA differences between groups, a two-way ANOVA was utilized, also followed by Tukey’s multiple comparisons test. All data are presented as the mean ± standard error of the mean. Statistical significance was established at a p -value < 0.05.
In conclusion, this study demonstrates that the combination of exercise plus MSC injection significantly enhances muscle mass and function in a model of muscle atrophy induced by immobilization and aging. This finding is supported by the observed reduction in TNF levels and the activation of AMPK. Thus, these findings provide a foundation for further clinical trials investigating the use of MSCs as a treatment for sarcopenia. In the long term, they may represent a potential therapeutic approach for patients suffering from musculoskeletal diseases.
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Out of the laboratory, into the field: perspectives on social, ethical and regulatory challenges in UK wildlife research | eef0a85f-3183-469a-91e3-996aaf0ee0b6 | 8237164 | Physiology[mh] | . Introduction Laboratory–field borders have long interested scholars in the humanities and social sciences. Attention has focused in particular on how laboratories and fields were historically constructed in opposition to one another and, in some ways, have competed for scientific legitimacy. While laboratories are often presented as stripped-down ‘placeless places’ that produce knowledge which can be universally applied across contexts, fields promise greater realism at the expense of control . Of course, it is widely acknowledged that there is not a firm distinction between laboratories and fields, as exemplified by hybrid spaces such as ‘semifield stations’ and mesocosms, and the incorporation of laboratory-like elements into the field and vice versa such as in ‘natural experiments’ . Still, laboratory–field comparisons are useful for exploring how spatial and social elements can alter the practice and representation of science . In this paper, we draw on this tradition of using insights from the social sciences to consider how science works differently in laboratories versus fields. However, we are less concerned with the legitimacy and kind of scientific knowledge produced in these sites than with the social, ethical and regulatory challenges scientists face in the field compared with the laboratory, and what these challenges mean for the health and well-being of animals and ecosystems. In particular, we show that laboratory–field comparisons are valuable for gaining insight into other key themes in social studies of science and conservation, namely: transparency and openness in animal research ; affective relationships between researchers and wildlife study subjects ; conflicts between conservation and animal welfare goals ; negotiating expertise between professional scientists and other knowledge-holders ; and the relationship between animal welfare law and good care in practice . We explore these ethical, social and regulatory elements of field-based animal research through the lens of work covered by the UK's Animals (Scientific Procedures) Act (1986). This research typically involves activities such as sedation, the collection of blood samples or the attachment of tracking devices to free-ranging animals. Our goal is to prompt reflection among researchers and others involved in this work (e.g. vets, regulators) about the unique challenges posed by field-based animal research, and to propose some ways that these parties might work together to improve care for animals and ecosystems. While we draw on qualitative research with animal research communities in the UK, we propose that many of these lessons are broadly applicable to international contexts. In particular, we focus on four key challenges faced by researchers working in the field, which relate to the management of (i) relationships with publics and stakeholders, who may be present at field sites or crucial to research success; (ii) ethical considerations not present in the laboratory, such as the impacts of research on populations and ecosystems; (iii) working under an array of regulations, which may operate in accordance with competing ethical principles or objectives; and (iv) relationships with regulators (especially vets), which may involve disagreements over ethics and expertise, especially because regulators are likely to be more accustomed to overseeing research in the laboratory than the field. We conclude by arguing that flexibility and mutual respect emerged as two key ways of managing ethical challenges; fostering good relationships between researchers, stakeholders and regulators; and improving care for individual animals and ecosystems. While these might be difficult to implement in practice, and fundamental differences (e.g. in ethical stances) might prove irresolvable, we propose that where possible researchers and others involved in field-based animal research aspire towards these dispositions in order to improve outcomes for animals and ecosystems under study.
. Methods This paper draws on qualitative social science research undertaken as part of ‘The Animal Research Nexus’ programme (AnNex: https://animalresearchnexus.org/ ): a collaborative, interdisciplinary project which aims to highlight historical and emerging challenges in the UK animal research sector, and encourage communication across and beyond the communities of animal research . We draw on one specific strand of the AnNex project focusing on sites classed as Places Other than Licenced Establishments (or POLEs) under the Animals (Scientific Procedures) Act (A[SP]A, 1986), which regulates invasive animal research in the UK. POLEs may include wildlife field sites, fisheries, farms, zoos or veterinary clinics. The POLEs strand of the AnNex project involved semi-structured interviews with 30 people and 24 lengthy informal conversations with others. Together, 21 (38%) of these conversations focused primarily on wildlife research and 5 (9%) on fisheries. A further 10 (19%) conversations were conducted with named veterinary surgeons (NVSs) and former and current A(SP)A regulators and focused on a broad range of sites. Participant-observation was conducted during visits of 1–2 days to five non-laboratory research projects (three of which involved tracking of free-ranging animals), and during shorter site visits, a wildlife research training course and relevant conferences. Notes derived from participant observation and informal conversations are referred to as ‘field notes’ throughout this paper. This qualitative research was not intended to capture views representative of wildlife researchers and regulators, but rather to explore perspectives in-depth and identify key emerging themes. In addition to this qualitative research, the POLEs strand of AnNex involved running a stakeholder workshop on non-laboratory research and a panel discussion on the regulation of wildlife-focused citizen science . Analysis of interview transcripts, field notes and relevant documents was conducted using the qualitative data analysis software NVivo. A policy of de-contextualization has been adopted due to the sensitive nature of the topic; all names presented in this paper are pseudonyms. We also refer only to the class of animal under study (e.g. mammal, fish and bird) rather than the species, given the small number of UK-based wildlife researchers working on a given species. All interviews were conducted with written consent from participants. Ethical approval for this research was granted by the Central University Research Ethics Committee of the University of Oxford (reference number: SOGE 18A-7).
. Relationships with publics and stakeholders As social scientists have observed, laboratories tend to be spaces inhabited only by researchers and others involved in research support and animal husbandry; by contrast, fields are potentially used by multiple groups . Thus, unlike researchers working in laboratory settings (though see for an exception of patient tours of laboratories), those working with free-ranging or agricultural animals may need to negotiate relationships with members of the public and stakeholders, who may also be present at field sites . Several kinds of encounters with publics and stakeholders may occur at field sites. First, members of the public may come across research activities incidentally, such as while out for a walk. Though these encounters are not necessarily adversarial, they may be disruptive. For example, Annika, whose research involves the use of biologging technologies in fish, noted that she has had curious members of the public come by to ask what her team is doing. Sometimes this happens when the team has been undertaking invasive procedures. Annika reflected that while she always tries to engage with people and explain the research, she would never do this at the expense of the fish: she would always ask people to wait if speaking with them would compromise fish welfare (field notes, 15 April 2019). Wild mammal researcher Geoff described how public encounters had become sufficiently disruptive to the research process that his team moved their work from outdoors to an indoor location in a field station (field notes, 7 September 2018). For these reasons, researchers may be explicitly advised to be as discreet as possible when undertaking research in the field (field notes during training course, 11 March 2019). While this advice might appear to run counter to proposals to make research more transparent, public encounters with field research can pose risks to the process of science and the welfare of animals under study. Researchers working with animals sometimes perceive a personal risk associated with transparency, the concern being that the public may misunderstand their work or animal rights activists will threaten them . This may in some cases be less of a risk for wildlife researchers given that public perceptions of such work are often, as researcher Genevieve put it, ‘Disneyesque’ and shaped by positive portrayals of such work in film and other media (interview 21 June 2019; see for further discussion). But the public may conflate wildlife research with practices they object to. For example, deliberate sabotage of traps during the hunting season can occur when anti-hunting activists fail to distinguish between traps set for research purposes and those set by hunters (field notes during training course, 11 March 2019). Some wildlife research is also in itself controversial, such as with animals whose management is subject to debate (e.g. invasive species and badgers). This can pose challenges for researchers if they require permission from members of the public to undertake research—such as landowners, companies, game and fisheries trusts and even local wildlife groups—who may in practice act as gatekeepers to research in a particular area. In some countries, indigenous communities may be added to this list. Where stakeholders take issue with research practices or ethics, conflicts can emerge. For example, a researcher involved in a long-running study of free-ranging mammals explained that recently two farmers had retracted permission for the researchers to work on their land, meaning that the researchers immediately lost access to a subset of their study population (field notes, 20 August 2019). Disagreements about ethics can also go in the other direction, such that researchers may collaborate with stakeholders with whom they disagree. It is important to recognize that approaches to conservation and animal welfare are not universal within social, cultural and ethnic groups. For example, Western conservation approaches based on preservation and protection may be at odds with indigenous groups' traditional practices and customary hunting rights , and Western and non-Western approaches to animal welfare and ethics may differ . Among our participants, fish researcher Greg's work requires collaborating with anglers, who catch fish for use in his research. Yet Greg admitted to having ‘preconceptions’ about working with anglers, because ‘I want [to work with] people that show high levels of empathy and I don't equate fishing with empathy’. In an effort to recruit anglers who would act as ‘more than human winches'—who would catch fish in a manner sensitive to animal welfare—Greg designed a set of questions aimed at assessing anglers' empathy. He explained that, somewhat to his surprise, some anglers' responses strongly implied empathy and concern for fish welfare. For example, some anglers explained their motivations for participating in Greg's project as ‘I am spellbound by these animals’ or similar. Greg added that through this and other similar projects ‘you kind of see empathy in people build’, meaning that interaction with the research may change stakeholders' ethics (interview, 1 October 2019; see also ). This example implies that even if researchers and stakeholders start out thinking that they disagree about fundamental ethics, these disagreements can potentially be resolved if both parties take the time to learn from each other's approaches and find areas of agreement. Still, this may not always be possible, in which case researchers may need to consider whether research should proceed if it requires collaborating with stakeholders whose approaches are, in the researchers' eyes, ethically problematic. Disagreements between researchers and stakeholders may also relate to personal interests. For example, fish researcher Gavin explained that anglers and fisheries management groups may be reluctant to permit research that involves killing fish, or invasive procedures that could harm the welfare or survival of the fish, since they perceive such work as a threat to their stocks (interview with Gavin, 16 January 2019). The issue is therefore not that fisheries managers object to the killing of fish in general, but that they want to ensure that killing only occurs when it serves their interests. When this occurs, a strategy for building good relationships may be to give something back to stakeholders so they feel the research is to their benefit. For example, fish researcher Annika observed that it makes a big difference to relationships when they visit local angling groups and speak with them about the goals of the research, since this makes people feel that they have a stake in the research and would benefit from its results. This approach responds to calls to rethink science–society relationships to make scientific research and outputs more accessible and directly applicable to problems encountered by the public, as part of a broader commitment to ‘Mode 2’ knowledge production that encourages scientific research to be produced with a high degree of social accountability and reflexivity . Annika added that she also draws on anglers' expertise, not only to make them feel included but also because it benefits her research, since anglers often hold detailed knowledge of where fish are located in their local area. This reflects how in field science it is not just professional scientists who may possess the expertise of value to scientific research; various other groups may also acquire knowledge (including technical knowledge) through means other than scientific education, such as everyday work and traditional practices, another key dimension of Mode 2 science . This observation is the foundation of the field of ethnobiology, which is premised on the idea that indigenous and local knowledge of the environment is not only valuable in its own right, but can also be usefully employed for managing resources and ecosystems . Similarly, citizen scientists—who may also be present at wildlife field sites through acting as skilled volunteers—may hold considerable expertise on catching and handling animals; collaboration with experienced citizen scientists may therefore allow researchers to substantially speed up data collection and even to learn from their collaborators. An important caveat to such alliances, however, is to recognize that citizen scientists and other volunteers may not hold as much experience as researchers, meaning that training or oversight could be required for successful collaboration. This in turn means volunteers could also personally gain from participating in research . A key lesson for researchers is therefore that the knowledge of volunteers and other stakeholders should be gauged, acknowledged, and where possible put to use for the benefit of the research, and the humans and animals it involves.
. Controllability and ethics Social scientists have observed that laboratories are often regarded as ‘placeless places’ in that they are intentionally stripped of unique environmental features in order to generate universally applicable findings. By contrast, fields tend to be heterogeneous and distinctive, with this distinctiveness very often the subject of field research . This means that while field research can serve as a more accurate reflection of the real world, it can also (rightly or wrongly) be perceived as less replicable and universally relevant than laboratory-based research . As summarized by fish researcher Gordon, his field research involves ‘so many uncontrolled variables that it's […] very difficult to manage. But you have a real life, wild situation so you have a kind of a serious reality check’ (interview, 15 January 2019). In field-based animal research specifically, sources of variation may arise from genetically heterogeneous populations (potentially necessitating larger sample sizes than in the laboratory), intraspecific variation in behaviour and physiology, and non-standardized environmental conditions . The reduced controllability of fields compared with laboratories can have important practical, regulatory, ethical and animal welfare implications. From a practical perspective, field-based research can be physically challenging for researchers, which can in turn affect animals' experiences. For example, AP's visit to Annika's fisheries-based research project occurred during very cold weather and required the researchers to at one point cover their work station (which was set up on the back of a truck) with a tarpaulin. Despite this, the poor weather prompted Annika and the other researchers to compare stories of even more challenging weather conditions in which they had previously worked (field notes, 15–16 April 2019). Furthermore, as researcher Hugh noted, the field researcher's equipment is limited to ‘what's in the van’ (field notes, 29 October 2018). Some exceptions to animal welfare standards are therefore explicitly made for wildlife research in A(SP)A guidance. For example, given that wildlife researchers in the field ‘may not have immediate access to drugs, equipment or experienced staff’ as would be expected in a laboratory, in ‘emergency situations' a method of killing not specified by the A(SP)A might be used instead in order to ensure animal suffering is minimized . In other words, animal care may need to be compromised due to difficult research conditions in the field. However, researcher Hugh argued emphatically that this should not lead wildlife researchers to cut corners on animal care. A balance must therefore be struck in negotiations between researchers and regulators, where the limitations of animal care in the field are acknowledged but not used as an excuse for poor standards. A second challenge deriving from uncontrollability in the field is that ‘you don't know what you're going to catch’ (interview with Genevieve, 21 June 2019), such that researchers may inadvertently catch non-target animals in traps set for research animals. Various steps can be taken to mitigate this risk; for example, one research group AP visited sets traps for nocturnal animals late in the afternoon or evening to avoid catching diurnal species (field notes, 20 August 2019). However, bycatch may be unavoidable and may result in harm to the bycaught animal, if for example animals are injured as a result of traps being designed for a different species. Non-target species may even need to be killed. As researcher Geoff observed, his days occasionally begin with the unpleasant task of killing grey squirrels ( Sciurus carolinensis ), which are unintentionally caught in his team's traps, as EU invasive alien species regulations (1143/2014) dictate that grey squirrels must be killed if caught (field notes, 13 September 2018). Non-target members of the study species may also be inadvertently caught, such as lactating females, which researchers may wish to avoid disturbing due to the knock-on effects on dependent young (interview with Genevieve; for more on the above, see ). Bycatch is therefore one way in which wildlife research can affect the welfare not only of research animals, but also other animals living in the same area. As Geoff's example illustrates, these encounters with bycatch can pose an ethical and emotional challenge for researchers. The conditions under which animals live in the wild are variable and potentially difficult, which means that researchers may also catch animals that are in poor physical condition. Guidance on the A(SP)A indicates that animals taken from the wild found to be ‘injured or in poor health’ should not be ‘subjected to a regulated procedure unless and until it has been examined by a veterinary surgeon or other competent person; and, unless the Secretary of State has agreed otherwise, action has been taken to minimize the suffering of the animal’ . What this means in practice is that researchers may release captured animals that are in poor condition before undertaking research, to avoid noncompliance with research animal welfare regulation. For example, Gordon explained that his team excludes from their study any fish they catch that look especially thin. However, Gordon also explained that doing so suits his research, which focuses on migration tracking: ‘We wouldn't want to use animals which we thought were in some way giving us poor information because they're already in poor condition’. Furthermore, this approach still comes with risks to animal welfare, with many wildlife researchers observing that capture is usually the most stressful part of research for a wild animal (see also ). Decisions may become trickier when researchers require animals in poor condition for their study. Greg, for example, explained that his research involves a difficult balance between sampling all of his target animals, which includes those in poor health condition when caught, and avoiding causing excessive suffering by tagging and releasing visibly unwell animals. Thus, he explained, ‘I do not really want to [tag and release an unwell animal] but actually I probably should’. Again, Greg's comment implies an ethical and emotional challenge for wildlife researchers posed by catching unwell animals and speaks to a broader problem across all animal research: that harm to animals should be minimized, but not if doing so invalidates the research and therefore means that animals used in the study suffered for no reason. Further complicating such assessments is researchers' inability to intervene if animals were to suffer or die after release. Ordinarily under the A(SP)A, the personal licence holder's role involves ‘being responsible for the welfare of the animals you have performed procedures on and ensuring that they are properly monitored and cared for’, including ‘making sure that any animal that is in severe pain or severe distress, which cannot be alleviated, is painlessly killed using an appropriate method’ . In wildlife research, animals can potentially be both living freely in the wild and simultaneously under the controls of the A(SP)A, and therefore technically entitled to monitoring, care and euthanasia if they experience severe pain or distress. For example, this occurs when animals are part of an ongoing research project in which they are trapped and released repeatedly, or when they are released with tracking devices. There is therefore a tension whereby the A(SP)A ordinarily assumes that researchers can carefully control the experiences of their research animals to minimize suffering, but such control is impossible in the field, meaning that animal welfare is often unknown and difficult to control. For this and various other reasons, research participants regularly complained that the A(SP)A was not written with field research in mind, and in some ways is an awkward fit. Similar views have been expressed by researchers working in the USA, who have proposed that Institutional Animal Care and Use Committees (IACUCs) are not oriented towards field-based animal research, and may fail to comprehend the important differences between research in the laboratory and field . The idea that animal welfare regulations are an awkward fit for field research may refer not only just to practical matters, but also to ethical principles. For example, many research participants referred to the principle of avoiding perturbation as justification for why they would treat any illnesses or injuries that they had clearly caused (e.g. trap-related injuries), but would avoid euthanizing or treating animals where the problems were due to ‘natural’ causes, as this could alter the functioning of the population and ecosystem under study and thereby pose both a scientific and ethical challenge. As well as reflecting an ethical commitment associated with conservation ethics to minimize human interference in the natural world , this approach also reflects what anthropologist Matei Candea calls ‘inter-patience’: a seemingly paradoxical relationship between researchers and the wildlife they study in which both parties agree to avoid interfering with each other's lives . Of course, the line between ‘natural’ and ‘unnatural’ ailments and injuries may be subjective and unclear, and several participants acknowledged that they have set the principles of non-perturbation and inter-patience aside when they felt emotionally compelled to intervene to avoid visible animal suffering (see also ). For example, researcher Graham acknowledged, ‘I confess I've found lizards with ticks on them and I've pulled them off. But that's wearing my compassionate hat; if I'm wearing my hard-headed ecologist hat I'd say let them go’ (interview, 14 September 2018). Concerns about perturbation reflect the idea that wildlife researchers must consider not only just the effects of their research on the welfare of individual research subjects, but also on wider populations and ecosystems. For this reason, researcher Geoff suggested that an alternative ethic for wildlife research might simply be to ‘do no harm’ by ensuring that ‘the welfare of the animal is not made worse by what you did’ (interview, 7 September 2018). However, it is unclear when, if ever, this could be achieved given the stress potentially caused even just by the capture of free-ranging animals . In a similar vein, researcher Graham argued that assessments of harms to animal welfare in wildlife research should consider whether researchers are ‘stressing [free-ranging animals] more than they would be stressed in the wild’. In other words, researchers may feel that welfare assessments and ethical principles should account for the uncontrollability of the field, the difficult conditions that many free-ranging animals experience in their daily lives and the undesirability of perturbation. Some have also proposed that field research ethics should explicitly consider the risks to populations and ecosystems, and animal welfare harms to non-research animals living in the same area. For example, environmental philosopher Howard Curzer and others have proposed that the 3Rs framework—which is commonly applied to animal research around the world and emphasizes replacing animals with other methods of testing, reducing the number of animals used in research and refining procedures to minimize suffering—should be replaced with 9Rs for wildlife research, which prompt consideration of not only individual animal welfare but also the effects of research on populations and ecosystems . This suggestion echoes broader conversations about integrating animal welfare with conservation, such as via a ‘compassionate conservation’ approach, which encourages consideration of both the interests of individual animals and ecosystems in conservation decision-making . This proposal is made in light of the fact that individual animals are often harmed for the sake of ecosystem or population health (e.g. non-native or overly abundant animals are often culled), which social scientists have identified as the problem of ‘violent care’ inherent in conservation . However, neither 9Rs nor compassionate conservation fully resolves these dilemmas, since while both frameworks encourage attending to the interests of both individual animals and broader populations or ecosystems, they do not specify which interests should prevail where they are in conflict . Field-based animal research therefore perhaps inevitably involves tensions between care for individual animals and ecosystems, which researchers and regulators should thoroughly assess and consider.
. Legal complexities Field research is often governed by a broader range of laws and regulations than laboratory research. Thus, wildlife researchers in the USA have expressed a feeling of ‘running the permit maze’ . We found a similar sense among many wildlife researchers in the UK; some key laws governing UK wildlife research are described in (see for the original table on which this is based). For example, if a researcher were to undertake a study of a free-ranging non-native bird species such as the Egyptian goose ( Alopochen aegyptiaca ), they may require licences from the Home Office (which oversees the A[SP]A), a Statutory Nature Conservation Organisation (SNCO, e.g. Natural England, Scottish Natural Heritage, which oversee the Wildlife and Countryside Act [WCA]), the British Trust for Ornithology (BTO) and the Animal and Plant Health Agency (APHA). However, other tagging of free-ranging animals may require no licences at all, depending on whether the species is protected under the WCA and other wildlife laws, and whether the research is determined to meet the criteria for inclusion under the A(SP)A, such as whether it is conducted for a ‘scientific purpose’ and exceeds the threshold of invasiveness as defined in the Act. As we have discussed elsewhere, both science- and invasiveness-based criteria are somewhat flexible and in some cases difficult to assess, with the attachment of tracking devices one example of a complex regulatory area . The various laws under which field-based researchers work sometimes present different advice or requirements for undertaking the same activity. For instance, researcher Geoff observed that when wildlife vaccination is undertaken for animal health, it falls under DEFRA and is licensed by SNCOs, and involves quite specific requirements for animal care, and treatment frequency and dosage. When wildlife vaccination is undertaken for science, it falls under the A(SP)A, which allows researchers more flexibility in creating and justifying their own animal care and treatment procedures to suit the context. This is in line with the commonly made observation that A(SP)A is a deliberately flexible law, which is one of its key strengths. Laws may also operate in accordance with competing ethical principles or objectives. Several participants highlighted what they perceived as insufficient attention to individual animal welfare in the WCA, with animal welfare advocate Gareth observing that not all trapping of animals is regulated under the WCA, and where trapping is regulated there are not always clear requirements around what kind of traps may be used and how trapping is conducted (interview, 27 February 2019). This may be considered part of a broader international trend in which wild animal welfare is viewed as neglected compared with the welfare of captive animals, in philosophy , animal research , wildlife law and conservation . By contrast, the A(SP)A features considerable focus on individual animal welfare, as demonstrated by its emphasis on the 3Rs and euthanasia as a tool for minimizing animal suffering. Genevieve related her personal experience of Home Office inspectors (HOIs), who oversee implementation of the A(SP)A, as follows: I would say personally that they are just focused on your subject animal. We've never had, we've never—I don't think—ever had much comeback on non-targets or on conspecifics or anything like that from the Home Office. So yeah, they are very much targeted on the welfare of that individual. That said, Genevieve acknowledged that her organization— where many experienced wildlife researchers work—extensively considers ecosystem-level effects in their internal ethical review, which occurs before licence applications are sent to the Home Office. It is therefore possible that inspectors feel that ecosystem-level questions have already been well addressed by Genevieve's research group, and that they would ask more questions in other contexts. Furthermore, wildlife-specific guidance on the A(SP)A, which was released in 2016, explicitly highlights the potential environmental consequences of capturing and removing animals from ecosystems, and emphasizes that research with free-ranging animals should be both ‘humane and environmentally sensitive’ . In Genevieve's opinion, this is a good example of how there are ‘definitely moves for the Home Office to at least understand it [wildlife research] a bit more, which is good’, and efforts to outline how the law works differently in the laboratory and field. At the same time, the application of an explicitly animal welfare-focused law like the A(SP)A to wildlife research could be perceived as helping to address the minimal legal attention paid to wild animal welfare. It was for this reason that animal welfare advocate Gareth proposed that the A(SP)A should be extended to cover wildlife capture when undertaken for science. While he acknowledged that this would lead to inconsistency in the regulation of wildlife capture depending on why it was being done, Gareth argued that it would signal the crucial need to consider animal welfare and justifications for trapping in other contexts.
. Regulatory oversight and expertise The difficulty of balancing risks to animal welfare and ecosystems is sometimes shown through disagreements between researchers and vets involved in oversight and regulation. HOIs, who are primarily responsible for overseeing research under the A(SP)A, are typically vets by training, although a few are medics (interview with HOI Gail, 15 May 2019). Named veterinary surgeons (NVSs) also play an important role at a local level, being responsible for overseeing animal health and welfare within institutions . As in other countries like the USA, relationships between vets and researchers are therefore important for effective regulation, but potentially characterized by lack of understanding or conflict . Disagreements between researchers and vets involved in oversight may derive from disciplinary training and ethical focus. This is demonstrated through perspectives on when to treat or euthanize suffering wild animals involved in research, with vets erring on the side of euthanasia and researchers preferring to avoid this. For instance, NVS Gretchen described a disagreement with a group of wildlife researchers over euthanizing or releasing an unwell animal: I made very clear under certain circumstances they needed to consider euthanasia. As soon as I mentioned euthanasia, everybody shut down. And I find the concept of, that you'd rather release an animal that is potentially suffering acceptable in comparison to kill it and have no suffering, I find-, it's a completely different approach. (Interview, 25 May 2019) Similarly, a researcher described disagreeing with an NVS's proposal of euthanizing a nocturnal animal that was behaving unusually by ranging during the day (field notes, 20 August 2019). It is important to stress that in other cases vets and researchers agreed over these matters, with several vets for example observing that it is crucial to avoid perturbation in wildlife research. Still, cases of disagreement imply that researchers and vets involved in research oversight may hold differing opinions on what wildlife research ethics ought to involve, with vets tending to focus more on individual animal welfare, and wildlife researchers more concerned with ecosystem-level impacts and avoiding perturbation. Disagreements between researchers and vets may also arise from perceived differences in expertise, especially in relation to fieldwork. As summarized by researcher Genevieve, I think a lot of the inspectors come from a lab animal background, so actually understanding this [field research] is quite difficult for them. I don't mean that to sound patronising but I mean I think if you've not experienced doing this kind of work it's really difficult for them to understand the difficulties behind it. Some HOIs develop a degree of specialism throughout their careers, with a small team within the Home Office specializing in wildlife research (interview with HOI Gail, 15 May 2019); similarly, some NVSs possess expertise in wildlife medicine. However, one such NVS, who has considerable experience with wildlife research, described himself as a ‘rare beast’ in acknowledgement of his unusual skillset (interview with Guy, 8 April 2019). A researcher who works in South Asia similarly noted that the primary vet involved in overseeing her research lacks experience with wildlife (including her study species), implying that this issue could extend to multiple international contexts (interview with Caroline, 11 August 2020). Thus, researchers sometimes described vets' suggestions as odd or ill-informed. For example, Geoff described an HOI's efforts to replace the homemade tables at Geoff's field station (on which samples from sedated animals were collected) with more hygienic stainless-steel benches. However, Geoff objected that due to the lack of heating in the field station, during winter metal benches would be freezing cold. He additionally rejected the inspector's suggestion of placing absorbent pads under the animals to keep them warm, as animals could carry disease and urinate on the pads, thereby generating a large amount of biohazardous waste (interview, 7 September 2019). In short, strategies for improving animal welfare and reducing risks (e.g. of biohazards) in the laboratory do not necessarily work in the field, and researchers with extensive fieldwork experience may be in the best position to identify when this is the case. Geoff added that the potential for misunderstandings about the realities of fieldwork can be exacerbated because ‘[w]e don't get inspected as often as a laboratory would’. Other participants made similar observations, pointing to the difficulty of arranging for HOIs and NVSs to visit field sites given the short duration of fieldwork seasons, remote locations of field sites, variable and condition-dependent fieldwork schedules, and limited time and resources available to vets. In particular, Geoff noted that his current HOI has never visited his field site in person. For this reason, Geoff suggested, ‘I don't think they understand in real terms what goes on. I don't think they've pictured what we're doing quite the way that we do it. […] Because they've not physically visited the site’. Geoff here suggests that the unique feel of a field space, which shapes relationships between humans and animals , needs to be experienced first-hand for field research to be fully understood. Thus, misunderstandings may be exacerbated by vets' limited direct experience of visiting field sites. This situation is by no means unique to the UK; off-site work often receives less scrutiny and oversight than laboratory-based research in other countries , and some have argued that communication and understanding could be facilitated by IACUC vets visiting field research sites in the USA . For their part, vets sometimes also acknowledged the value and importance of field visits, with former HOI Craig observing that through field visits ‘[y]ou develop a relationship with them [the researchers]’ (25 June 2019). However, because building these relationships may take time, changes of inspectors (which several participants observed has become more common in recent years) can be disruptive. Researcher Evan, for example, explained that it is frustrating when inspectors change because you build up a relationship, but you also I think build up knowledge with your inspector. They learn to know what you're doing with the kind of rather strange animals that we sometimes work with, and then you're back to square one with a new one. (15 January 2019) Alongside visits to the field, and trust gained from long-term relationships, researchers and vets alike highlighted two additional important features of good working relationships: flexibility and mutual respect. The A(SP)A and its associated guidance already contain a certain degree of flexibility, as illustrated by guidance allowing field researchers to use non-standard euthanasia methods in emergencies (see §4). Researchers and vets also often spoke of the importance of both vets and researchers employing ‘flexibility’ or ‘common sense’ in applying and implementing the law. For instance, HOI Gail observed that it may be possible to negotiate compromises for simultaneously minimizing research animal suffering and ecosystem perturbation. Gail described how in a case when sick fish were shedding bacteria, a compromise involved euthanizing unwell fish but putting their bodies back into the water to mitigate the ecological impacts of removing them from the population. Thus, good care in practice—for animal research subjects and ecosystems—may differ to, or go beyond, formal laws and regulations , highlighting the difficulty of creating universally applicable rules about good animal care across contexts . Mutual respect was also highlighted as an important feature of a positive vet–researcher relationship. HOIs and NVSs very often acknowledged researchers as the greatest experts on their study subject and species, and some also noted that they themselves lacked the expertise of wildlife research compared with laboratory work. Former HOI Craig, for example, noted that if I went to a laboratory animal facility as a Home Office inspector, I felt I was quite comfortable and I was in my environment. If I was out at a POLE then I felt I was probably more in the hands of the researchers in terms of, you know, their views in terms of doing things. Yet vets also argued that they could make important contributions, in particular because the greater isolation of field researchers from research support communities can impede the updating of animal welfare practices (interview with former HOI Heather, 17 January 2019). Vets also felt that they can make valuable contributions by drawing on their general, non-species-specific veterinary knowledge (interview with NVS Grace, 28 January 2019) and by asking intelligent questions that prompt researchers to reflect on their practices. For example, NVS Elaine argued that to build constructive and effective relationships with researchers, vets must avoid saying, ‘“I know best; do what I say’’. And actually, you need to come from, […] ‘‘You are the expert here. I'm going to ask you lots of questions, so that I can reflect on whether what you're suggesting makes sense”’ (interview, 11 January 2019). For their part, some researchers described finding such constructive questioning and support invaluable. Hugh, for example, noted that both his NVS and HOI acknowledge and respect his species-specific expertise, but he appreciates the ‘challenge’ presented by having someone around to ask intelligent questions (field notes, 29 October 2018).
. Conclusion and recommendations As we have demonstrated, invasive research with free-ranging animals involves a range of different social, ethical and regulatory challenges compared with laboratory research. While not all of these challenges are resolvable, our qualitative research suggests that flexibility—in policy, personal relationships and animal care practices—and mutual respect between researchers, stakeholders and regulators are important aspirations, which when achieved can help ensure that research with free-ranging animals supports positive outcomes for ecosystems and animals used in research. (a) Flexibility The first key lesson from our qualitative research is that flexibility can play an important role in managing social, ethical and regulatory challenges encountered in field-based animal research. The ethical challenges presented in fields may be perceived as different to those in laboratories, due for example to a lack of control over animals' experiences, and the risk of perturbation. Participants expressed the idea that UK animal research law was not written with these unique challenges in mind and is therefore in some respects an awkward fit for field research. Similar views may arise in other countries, such as the USA. However, there are signs that in the UK this is changing through the development of field-specific guidelines, which was perceived as a positive development . Thus, flexibility can be demonstrated at the level of regulation itself through the development and dissemination of field-specific advice. Furthermore, the A(SP)A was sometimes described as more flexible than other laws affecting wildlife, and that its flexibility is one of its greatest strengths as it enables regulators and researchers to modify animal care practices to best suit the context, and to navigate tensions between individual animal welfare and risks to populations and ecosystems. Participants additionally highlighted the importance of flexibility at a personal level in how researchers, regulators and stakeholders collectively interpret and apply regulation and guidance when managing animal care and ethical issues. These messages highlight how good care in practice in wildlife research—for individual animals, and for broader populations and ecosystems—requires being flexible (see for related arguments). We therefore recommend that both researchers and other stakeholders working in field-based animal research, rather than sticking to universally applied rules, remain flexible in their approach to applying regulations and guidelines and recognize this may involve going above and beyond legal requirements. (b) Mutual respect The second key theme highlighted by our qualitative research is the importance of mutual respect in interactions between researchers, stakeholders and regulators. Stakeholders (e.g. landowners, anglers, farmers, citizen scientists and indigenous communities in many countries) may play a crucial role in enabling research to proceed. They can also, if brought on board, bring valuable skills (e.g. experience capturing and handling the study species) and knowledge (e.g. location of study species in the local area) that can facilitate and improve research and the experiences of research animals. As well as seeking to make research appealing and valuable to stakeholders—thereby helping to make science more publicly accountable—acknowledging and drawing on stakeholders’ expertise could therefore serve as an important strategy for building positive relationships and promoting good care for animal welfare and ecosystems. Such a move also resonates with the broader shift towards Mode-2 knowledge production and participatory research . Similar lessons apply in relationships with regulators. While vets involved in regulatory oversight may lack expertise on field conditions and the species under study, they can develop this knowledge over time, especially via field visits. Furthermore, they can make valuable contributions to research practices and animal care from their more general expertise and ability to ask challenging and intelligent questions of researchers. Participants highlighted the importance of researchers recognizing the importance of vets' contributions, and in turn the need for vets to recognize the species- and field-specific expertise of researchers. In some ways, flexibility and mutual respect must go together. If, for example, regulators employ flexibility in implementing the A(SP)A but are not respected by researchers, researchers may feel that regulators’ recommendations and decisions are arbitrary (see ). Furthermore, both flexibility and mutual respect are to some extent products of long-term relationships and efforts to understand each other's points of view. An important take-home message from our research is therefore that long-term relationships, and other tools for building understanding such as field visits by regulators, should be fostered where possible. However, these steps may be out of the hands of anyone directly involved in research; for example, it may be dictated by institutional policy. There are other factors further limiting the extent to which flexibility and mutual respect can be built. For example, parties involved in research may disagree about fundamental ethical principles, although these disagreements may subside where people make concerted efforts to understand each other's perspectives or reach a mutually acceptable compromise. Where possible we therefore recommended that steps be taken by all those involved in field-based animal research to acknowledge and respect contrary ethical perspectives, in the hope that doing so will benefit the animals used in research, and the populations and ecosystems of which they are part. Furthermore, such efforts to foster mutual respect between researchers and other stakeholders form a key dimension of the ‘culture of care’ in animal research, with implications for the well-being of both the humans and animals involved .
Flexibility The first key lesson from our qualitative research is that flexibility can play an important role in managing social, ethical and regulatory challenges encountered in field-based animal research. The ethical challenges presented in fields may be perceived as different to those in laboratories, due for example to a lack of control over animals' experiences, and the risk of perturbation. Participants expressed the idea that UK animal research law was not written with these unique challenges in mind and is therefore in some respects an awkward fit for field research. Similar views may arise in other countries, such as the USA. However, there are signs that in the UK this is changing through the development of field-specific guidelines, which was perceived as a positive development . Thus, flexibility can be demonstrated at the level of regulation itself through the development and dissemination of field-specific advice. Furthermore, the A(SP)A was sometimes described as more flexible than other laws affecting wildlife, and that its flexibility is one of its greatest strengths as it enables regulators and researchers to modify animal care practices to best suit the context, and to navigate tensions between individual animal welfare and risks to populations and ecosystems. Participants additionally highlighted the importance of flexibility at a personal level in how researchers, regulators and stakeholders collectively interpret and apply regulation and guidance when managing animal care and ethical issues. These messages highlight how good care in practice in wildlife research—for individual animals, and for broader populations and ecosystems—requires being flexible (see for related arguments). We therefore recommend that both researchers and other stakeholders working in field-based animal research, rather than sticking to universally applied rules, remain flexible in their approach to applying regulations and guidelines and recognize this may involve going above and beyond legal requirements.
Mutual respect The second key theme highlighted by our qualitative research is the importance of mutual respect in interactions between researchers, stakeholders and regulators. Stakeholders (e.g. landowners, anglers, farmers, citizen scientists and indigenous communities in many countries) may play a crucial role in enabling research to proceed. They can also, if brought on board, bring valuable skills (e.g. experience capturing and handling the study species) and knowledge (e.g. location of study species in the local area) that can facilitate and improve research and the experiences of research animals. As well as seeking to make research appealing and valuable to stakeholders—thereby helping to make science more publicly accountable—acknowledging and drawing on stakeholders’ expertise could therefore serve as an important strategy for building positive relationships and promoting good care for animal welfare and ecosystems. Such a move also resonates with the broader shift towards Mode-2 knowledge production and participatory research . Similar lessons apply in relationships with regulators. While vets involved in regulatory oversight may lack expertise on field conditions and the species under study, they can develop this knowledge over time, especially via field visits. Furthermore, they can make valuable contributions to research practices and animal care from their more general expertise and ability to ask challenging and intelligent questions of researchers. Participants highlighted the importance of researchers recognizing the importance of vets' contributions, and in turn the need for vets to recognize the species- and field-specific expertise of researchers. In some ways, flexibility and mutual respect must go together. If, for example, regulators employ flexibility in implementing the A(SP)A but are not respected by researchers, researchers may feel that regulators’ recommendations and decisions are arbitrary (see ). Furthermore, both flexibility and mutual respect are to some extent products of long-term relationships and efforts to understand each other's points of view. An important take-home message from our research is therefore that long-term relationships, and other tools for building understanding such as field visits by regulators, should be fostered where possible. However, these steps may be out of the hands of anyone directly involved in research; for example, it may be dictated by institutional policy. There are other factors further limiting the extent to which flexibility and mutual respect can be built. For example, parties involved in research may disagree about fundamental ethical principles, although these disagreements may subside where people make concerted efforts to understand each other's perspectives or reach a mutually acceptable compromise. Where possible we therefore recommended that steps be taken by all those involved in field-based animal research to acknowledge and respect contrary ethical perspectives, in the hope that doing so will benefit the animals used in research, and the populations and ecosystems of which they are part. Furthermore, such efforts to foster mutual respect between researchers and other stakeholders form a key dimension of the ‘culture of care’ in animal research, with implications for the well-being of both the humans and animals involved .
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Significantly increased bone volume in a critical-sized defect model in the rat animal model by transplantation of a stand-alone vascularized periosteal flap | e88b15f8-f84a-4370-943a-c96a7a5f80bd | 11870878 | Surgical Procedures, Operative[mh] | Successful treatment of bony non-unions is challenging in orthopedic-trauma surgery. Bony nonunions can be observed in high- middle- and low-income countries after high energy trauma and it further seems an overall ubiquitous problem in fracture care . The economy and patient’s financial situation play a significant role in the treatment and outcome of non-unions, affecting everything from access to healthcare and the quality of treatment options to patient compliance and recovery. Addressing these discrepancies requires a multifaceted approach, including policy interventions, insurance reforms, targeted research and developmental efforts to ensure suitable access for an effective treatment. Multiple techniques and concepts have been described, from very basic to sophisticated surgical approaches for the successful treatment of non-unions . The use of periosteal flaps (PF) for treating non-unions in bone represent a specific technique to promote fracture healing. This method works by activating the biological tissues surrounding the fracture. The local blood flow to the fracture site is enhanced, which is crucial because increased blood flow delivers essential nutrients and osteogenic elements directly to the site of the non-union. This approach distinguishes itself by directly targeting and improving the conditions needed for bone healing . The osteogenic potential of the periosteum is known for its abundant amount of vascular supply in the outer fibrous and its inner cambium layer containing mesenchymal stem cells (MSC) and other factors promoting osteogenesis by intramembranous- and endochondral ossification . PF have already been used successfully clinically and in animal models . While its clinical application is mostly described in published case series, studies in rodents paradoxically combine the PF with other osteogenic matrices (scaffolds) instead of testing it alone . The question remains how potent the PF is, as a stand-alone technique. This consideration gains importance given that the introduction of additive materials, often inert, can potentially increase the risk of infection. This is particularly pertinent in cases of bony non-unions, where an underlying infection is frequently a contributing factor . As a bony non-union model is validated by creating a critical-size defect (CSD) in a rat femur and PF have shown to be practicable in rats, they seem to be an ideal setting for this study . The aim of this present study was to investigate the bone forming capacity of a stand-alone PF in bony non-unions by CSD models in a rat femur. Further, PFs were tested in a persisting CSD after 5 weeks to determine its osteogenic potential in a persisting non-union. Finally, the potential requirement of a vascular supply of the PF was evaluated by comparing two treatment groups; one with a pedicled periosteal flap and the other one with a ligated one.
Experimental animals All animal experiments and conducted procedures were in accordance with the law on animal experimentation and are approved by the regulatory authorities of the province of Salzburg, Austria (Permit No. 20901-TVG/134/11-2021 from jan. 19th 2021). The study meets the ARRIVE criteria. A total of thirty-six Sprague Dawley rats (12 weeks old, weighing approximately 475–550 g; Janvier Labs SAS, France) were used for this study. The animals were randomly assigned to either one of four experimental groups. All animals were kept under standard housing conditions (2–3 rats per cage) with free access to food and water. Post-operatively the same animals were kept in groups of 2 to 3 rats per cage. Rooms were maintained at 25 ± 2 °C and a 12:12 h light/dark cycle, light on at 07:00 h. Animal study design The study design incorporated 4 groups to which the animals were randomly assigned to as demonstrated in Fig. . Group I – PF group: Incorporated a Critical-size defect (CSD), bridged with a plate (P) and the Periosteal flap (PF) which covered the bony non-union. Group II - Crossover group: First a CSD was created and stabilized with a plate (P). After 5 weeks, the PF was transplanted through a second surgery. Group III - Ligated PF (PFx) group: Incorporated a CSD, bridged with a plate (P) and a ligated Periosteal flap (PFx) covered the bony non-union. Group IV - Control group: Consisted of CSD, which was bridged with a Plate (P). The aim of the selective group division was that animals in the PF group should induce bony healing within the CSD. The crossover group should allow for evaluation of the efficiency of bony healing in a persistent non-union. The PFx group should assess the necessity of a vascular pedicle and the control group should represent a plate treatment only. Surgical procedures After an appropriate acclimatization phase, all rats underwent surgery on the right femur. Thirty minutes preoperatively the rats received 0,03 mg/kg buprenorphine and antibiotics (Clindamycin, 45 mg/kg) subcutaneously (s.c.). Anesthesia was induced in an airtight box with 4% (v/v) isoflurane in oxygen and maintained at 2% (v/v) with a flow rate of 500 ml/min. During surgery, the animals were placed on an electric heating pad to prevent hypothermia (Harvard Apparatus, Holliston, MA, USA). Following shaving and sterile preparation of the surgical area, a skin incision measuring 4–5 cm was made medially along the right femur. A 5-hole angle-stable plate (1.5 mm Aptus titanium locking plate, Medartis, Basel, Switzerland) was attached to the distal femur by drilling 4 holes with a 1.1 mm drill bit (Gebrüder Brasseler, Lemgo, Germany). The central hole was intentionally not secured, leaving it unlocked. A 5 mm CSD was created using a 0.44 mm Gigli wire saw (RISystem AG, Lanquart, Switzerland) distal to the central plate hole. The removed bone fragment was measured using a caliper and the defect site rinsed with sterile saline solution. Depending on the group, a PF was elevated from the medial condyle with a blade after its vascular pedicle was isolated and then rotated into the CSD. The PF was sutured with a single stitch onto the central plate hole. In the PFx group the vascular pedicle was ligated. In the crossover group, the PF was prepared during a second follow-up surgery after 5 weeks. Within the control group, only a CSD was stabilized with a plate (Fig. ). The wound was shut by sutures in layers. Wound clips were used for closure and removed after 7 days. Magnifying glasses (4x-500, Zeiss, Oberkochen, Germany) were used during the entire operative procedures. The postoperative analgetic treatment consisted of 0.03 mg/kg buprenorphine s.c. twice a day for three days and 20 mg/kg tramadol-hydrochloride once a day through drinking water for 5 consecutive days. The animals had ad libitum access to food and water and were frequently monitored for complications, weight loss or abnormal behavior. Rats were euthanized (Pentobarbital-Natrium 300 mg/mL; Release; intraperitoneal injection) 10 weeks postoperatively (first surgery) and the femurs harvested for µCT and histological analysis. X-Ray control and Microcomputed tomography X-Ray images, antero-posterior and lateral views, were performed under general anesthesia immediately postoperatively, after 1 and 2 weeks and then in 2-week intervals until the endpoint (10 weeks) by two independent surgeons. The positioning of the rat femur, to obtain replicable images, was constant due to rod fixations of the right leg at the same angle. (X-ray source: Orange 9020HF (EcoRay Co.,Ltd.; Seoul, Korea) Detector: Flat Panel Detector FPD2P(Venu1417P) (iRayTechnology Ltd., Shanghai, China) Software: Conaxx2 VET (PROTEC GmbH & Co. KG, Oberstenfeld, Germany) X-ray source settings: 44 kV, 20mAs). After 10 weeks, femoral bones and the covering muscles were exarticulated in the hip and knee joint and then placed into 4% paraformaldehyde (PFA) in PBS (phosphate buffered saline). After 24 h, the PFA concentration was reduced to 2% (2 g paraformaldehyde powder (Sarstedt AG and Co, Nümbrecht, Germany) to 100 mL of 1x PBS. µ-CT scans were performed on all samples in this solution. During the scanning procedure the samples were stored in polymer sample tubes filled with PFA to prevent dehydration, scanning three samples at a time. The samples were scanned at a resolution of 35 μm isometric voxel size using a RayScan 250E cone beam XCT device equipped with a Perkin Elmer flat panel detector (2048 × 2048 pixels with a pixel size 200 μm) and a Viscom 225 kV microfocus X-Ray tube. The X-Ray scanning parameters were set to 180 kV and 200 µA with an integration time of 999 ms. A 0.5 mm physical copper filter-plate was applied to prevent beam hardening artefacts. Reconstruction of the acquired data was performed using X-AID Software (MITOS GmbH, Garching, Germany) involving a beam hardening correction. Post processing was performed in VGSTUDIOMAX 3.5 (Volume Graphics GmbH, Heidelberg, Germany). Firstly, image data was aligned so that the X-, Y- and Z-axes of the software coordinate system coincide with the frontal, sagittal, and transversal plane respectively. This was done for all samples in order to enable the establishment of a comparable Region of Interest (ROI) throughout the complete datasets. This ROI was chosen to be of cylindrical shape in the center of the diaphysis of the femur. For this purpose, a circle with the diameter of the inner sample tube surface was drawn in the transversal plane and then extruded along the vertical axis. The height of the individual cylinder for each sample was limited by the distance between the two screws nearest to the fracture site. The heads of the two screws were included in the ROI and thus mark the vertical ROI-cylinder boundaries. Subsequently, the region growing tool provided by VGSTUDIOMAX 3.5 was utilized to segment the implant and the screws. This segmentation was then subtracted from the ROI in order to be eliminated from the evaluation. Afterwards, the ROI was extracted and a median filter with a size of 3 × 3 × 3 voxels was applied for the purpose of denoising the images. The region growing tool was then used to segment the bone structure in the ROI. In this tool, a grey value (GV) threshold, which depended on the grey value distribution of the individual measurement, was set with the purpose of excluding liquid areas with GVs similar to bone GVs caused by BH artefacts from the selection. However, this approach was not sufficient to exclude all of the liquid areas. The remaining areas with BH artefacts were subsequently processed manually for each image stack. The finished evaluation contained the bone volume separated from the formalin liquid in the ROI. VGSTUDIOMAX 3.5 provides morphometric data for the evaluation in form of a ratio between Bone Volume and Tissue Volume (BV/TV) in percent, which was chosen as result parameter for the ROI . For the samples containing broken implants, an adapted evaluation process was established. After the anatomical alignment of the images, the polyline tool was used to create two three-dimensional ROIs containing the proximal and the distal part of the femur respectively. The proximal femur part was aligned to the distal part using the flat surfaces of the broken implant as reference. After the alignment, the same evaluation process as described above was carried out. Histological examination and staining After the µ-CT scans, the samples were decalcified in 2% PFA/12.5% EDTA solution (pH = 7.5). After a minimum of 7 weeks the plates were removed and the femora were processed for paraffin embedding and 7 μm sections were deparaffinized using Roti ® -Histol (Carl Roth, Germany), rehydrated in a graded alcohol series and stained either with Masson-Goldner trichrome, Alcianblue Nuclear Fast Red or Movat’s pentachrome stain (MORPHISTPO, Offenbach, Germany). Digital high-resolution images were acquired with focusing on the CSD area, using a Olympus slide scanner VS 120 (Olympus/Evident, Hamburg Germany). In addition, histological sections of periosteal flaps of the medial femoral condyle of the opposite side were prepared and stained by Movat´s Pentachrome staining (Fig. ). Statistical methods For comparison of the defect bone volumes determined by µCT analysis (mean ± SD; mm 3 ), a One-way ANOVA test with post-hoc pairwise comparisons (Tukey´s) were performed. Samples were tested for normal distribution using the Shapiro Wilk test. For comparison of two groups the unpaired t-test with Welch’s correction was used. Significance was set at α = 0.05. All tests were performed using GraphPad Prism v. 9.02 (La Jolla, CA, USA).
All animal experiments and conducted procedures were in accordance with the law on animal experimentation and are approved by the regulatory authorities of the province of Salzburg, Austria (Permit No. 20901-TVG/134/11-2021 from jan. 19th 2021). The study meets the ARRIVE criteria. A total of thirty-six Sprague Dawley rats (12 weeks old, weighing approximately 475–550 g; Janvier Labs SAS, France) were used for this study. The animals were randomly assigned to either one of four experimental groups. All animals were kept under standard housing conditions (2–3 rats per cage) with free access to food and water. Post-operatively the same animals were kept in groups of 2 to 3 rats per cage. Rooms were maintained at 25 ± 2 °C and a 12:12 h light/dark cycle, light on at 07:00 h.
The study design incorporated 4 groups to which the animals were randomly assigned to as demonstrated in Fig. . Group I – PF group: Incorporated a Critical-size defect (CSD), bridged with a plate (P) and the Periosteal flap (PF) which covered the bony non-union. Group II - Crossover group: First a CSD was created and stabilized with a plate (P). After 5 weeks, the PF was transplanted through a second surgery. Group III - Ligated PF (PFx) group: Incorporated a CSD, bridged with a plate (P) and a ligated Periosteal flap (PFx) covered the bony non-union. Group IV - Control group: Consisted of CSD, which was bridged with a Plate (P). The aim of the selective group division was that animals in the PF group should induce bony healing within the CSD. The crossover group should allow for evaluation of the efficiency of bony healing in a persistent non-union. The PFx group should assess the necessity of a vascular pedicle and the control group should represent a plate treatment only.
After an appropriate acclimatization phase, all rats underwent surgery on the right femur. Thirty minutes preoperatively the rats received 0,03 mg/kg buprenorphine and antibiotics (Clindamycin, 45 mg/kg) subcutaneously (s.c.). Anesthesia was induced in an airtight box with 4% (v/v) isoflurane in oxygen and maintained at 2% (v/v) with a flow rate of 500 ml/min. During surgery, the animals were placed on an electric heating pad to prevent hypothermia (Harvard Apparatus, Holliston, MA, USA). Following shaving and sterile preparation of the surgical area, a skin incision measuring 4–5 cm was made medially along the right femur. A 5-hole angle-stable plate (1.5 mm Aptus titanium locking plate, Medartis, Basel, Switzerland) was attached to the distal femur by drilling 4 holes with a 1.1 mm drill bit (Gebrüder Brasseler, Lemgo, Germany). The central hole was intentionally not secured, leaving it unlocked. A 5 mm CSD was created using a 0.44 mm Gigli wire saw (RISystem AG, Lanquart, Switzerland) distal to the central plate hole. The removed bone fragment was measured using a caliper and the defect site rinsed with sterile saline solution. Depending on the group, a PF was elevated from the medial condyle with a blade after its vascular pedicle was isolated and then rotated into the CSD. The PF was sutured with a single stitch onto the central plate hole. In the PFx group the vascular pedicle was ligated. In the crossover group, the PF was prepared during a second follow-up surgery after 5 weeks. Within the control group, only a CSD was stabilized with a plate (Fig. ). The wound was shut by sutures in layers. Wound clips were used for closure and removed after 7 days. Magnifying glasses (4x-500, Zeiss, Oberkochen, Germany) were used during the entire operative procedures. The postoperative analgetic treatment consisted of 0.03 mg/kg buprenorphine s.c. twice a day for three days and 20 mg/kg tramadol-hydrochloride once a day through drinking water for 5 consecutive days. The animals had ad libitum access to food and water and were frequently monitored for complications, weight loss or abnormal behavior. Rats were euthanized (Pentobarbital-Natrium 300 mg/mL; Release; intraperitoneal injection) 10 weeks postoperatively (first surgery) and the femurs harvested for µCT and histological analysis.
X-Ray images, antero-posterior and lateral views, were performed under general anesthesia immediately postoperatively, after 1 and 2 weeks and then in 2-week intervals until the endpoint (10 weeks) by two independent surgeons. The positioning of the rat femur, to obtain replicable images, was constant due to rod fixations of the right leg at the same angle. (X-ray source: Orange 9020HF (EcoRay Co.,Ltd.; Seoul, Korea) Detector: Flat Panel Detector FPD2P(Venu1417P) (iRayTechnology Ltd., Shanghai, China) Software: Conaxx2 VET (PROTEC GmbH & Co. KG, Oberstenfeld, Germany) X-ray source settings: 44 kV, 20mAs). After 10 weeks, femoral bones and the covering muscles were exarticulated in the hip and knee joint and then placed into 4% paraformaldehyde (PFA) in PBS (phosphate buffered saline). After 24 h, the PFA concentration was reduced to 2% (2 g paraformaldehyde powder (Sarstedt AG and Co, Nümbrecht, Germany) to 100 mL of 1x PBS. µ-CT scans were performed on all samples in this solution. During the scanning procedure the samples were stored in polymer sample tubes filled with PFA to prevent dehydration, scanning three samples at a time. The samples were scanned at a resolution of 35 μm isometric voxel size using a RayScan 250E cone beam XCT device equipped with a Perkin Elmer flat panel detector (2048 × 2048 pixels with a pixel size 200 μm) and a Viscom 225 kV microfocus X-Ray tube. The X-Ray scanning parameters were set to 180 kV and 200 µA with an integration time of 999 ms. A 0.5 mm physical copper filter-plate was applied to prevent beam hardening artefacts. Reconstruction of the acquired data was performed using X-AID Software (MITOS GmbH, Garching, Germany) involving a beam hardening correction. Post processing was performed in VGSTUDIOMAX 3.5 (Volume Graphics GmbH, Heidelberg, Germany). Firstly, image data was aligned so that the X-, Y- and Z-axes of the software coordinate system coincide with the frontal, sagittal, and transversal plane respectively. This was done for all samples in order to enable the establishment of a comparable Region of Interest (ROI) throughout the complete datasets. This ROI was chosen to be of cylindrical shape in the center of the diaphysis of the femur. For this purpose, a circle with the diameter of the inner sample tube surface was drawn in the transversal plane and then extruded along the vertical axis. The height of the individual cylinder for each sample was limited by the distance between the two screws nearest to the fracture site. The heads of the two screws were included in the ROI and thus mark the vertical ROI-cylinder boundaries. Subsequently, the region growing tool provided by VGSTUDIOMAX 3.5 was utilized to segment the implant and the screws. This segmentation was then subtracted from the ROI in order to be eliminated from the evaluation. Afterwards, the ROI was extracted and a median filter with a size of 3 × 3 × 3 voxels was applied for the purpose of denoising the images. The region growing tool was then used to segment the bone structure in the ROI. In this tool, a grey value (GV) threshold, which depended on the grey value distribution of the individual measurement, was set with the purpose of excluding liquid areas with GVs similar to bone GVs caused by BH artefacts from the selection. However, this approach was not sufficient to exclude all of the liquid areas. The remaining areas with BH artefacts were subsequently processed manually for each image stack. The finished evaluation contained the bone volume separated from the formalin liquid in the ROI. VGSTUDIOMAX 3.5 provides morphometric data for the evaluation in form of a ratio between Bone Volume and Tissue Volume (BV/TV) in percent, which was chosen as result parameter for the ROI . For the samples containing broken implants, an adapted evaluation process was established. After the anatomical alignment of the images, the polyline tool was used to create two three-dimensional ROIs containing the proximal and the distal part of the femur respectively. The proximal femur part was aligned to the distal part using the flat surfaces of the broken implant as reference. After the alignment, the same evaluation process as described above was carried out.
After the µ-CT scans, the samples were decalcified in 2% PFA/12.5% EDTA solution (pH = 7.5). After a minimum of 7 weeks the plates were removed and the femora were processed for paraffin embedding and 7 μm sections were deparaffinized using Roti ® -Histol (Carl Roth, Germany), rehydrated in a graded alcohol series and stained either with Masson-Goldner trichrome, Alcianblue Nuclear Fast Red or Movat’s pentachrome stain (MORPHISTPO, Offenbach, Germany). Digital high-resolution images were acquired with focusing on the CSD area, using a Olympus slide scanner VS 120 (Olympus/Evident, Hamburg Germany). In addition, histological sections of periosteal flaps of the medial femoral condyle of the opposite side were prepared and stained by Movat´s Pentachrome staining (Fig. ).
For comparison of the defect bone volumes determined by µCT analysis (mean ± SD; mm 3 ), a One-way ANOVA test with post-hoc pairwise comparisons (Tukey´s) were performed. Samples were tested for normal distribution using the Shapiro Wilk test. For comparison of two groups the unpaired t-test with Welch’s correction was used. Significance was set at α = 0.05. All tests were performed using GraphPad Prism v. 9.02 (La Jolla, CA, USA).
A total of 43 rats were included in this study. Eleven animals were excluded due to implant failure with plate osteosynthesis fracture observed during routine radiographic examinations (PF Group 2, PFx Group 4, Control Group 4, and Crossover Group 1). X-Ray X-Ray evaluations were carried out to determine plate stabilization and fracture healing progression. Overall, four rats suffered implant failure through a plate breakage within the first 8 weeks postoperatively. Affected animals were euthanized and excluded from the final evaluation. Observed plate breakages in the last radiographs (10 weeks) were included in the study. They were evenly distributed across the 4 treatment groups and the rats´ behavior and gait pattern were normal. Radiographic evaluation by X-Ray showed no bridging callus in any group. BV/TV Thirty-two rats were evaluated and the results are summarized in Table . Newly formed bone tissue was measured by BV/TV. The highest BV/TV ratio was observed in the PF group (18.77%; after 10 weeks, Fig. ). This was significantly higher than in the crossover- (11.28%; p = 0.0436), the PFx- (10,98%; p = 0.0411) or the control group (10.47%; p = 0.0293). Histological analysis Figure shows representative histological sections of osteotomy zones of all four treatment groups. Analysed specimens within the PF group showed primary fibrotic tissue inside the CSD with partial calcification. This represents immature woven bone. The specimens of the PFx treated femur showed “capping” at the end of the defected ends. Here the medullary cavity was closed with compact bone. The central region displayed fibrotic tissue. Samples of the crossover group showed “capping” as well. The CSD was filled with fibrotic tissue, which was partially calcified but did not correspond to immature woven bone. Empty defect models presented with “capping” and fibrotic tissue inside the defect zone.
X-Ray evaluations were carried out to determine plate stabilization and fracture healing progression. Overall, four rats suffered implant failure through a plate breakage within the first 8 weeks postoperatively. Affected animals were euthanized and excluded from the final evaluation. Observed plate breakages in the last radiographs (10 weeks) were included in the study. They were evenly distributed across the 4 treatment groups and the rats´ behavior and gait pattern were normal. Radiographic evaluation by X-Ray showed no bridging callus in any group.
Thirty-two rats were evaluated and the results are summarized in Table . Newly formed bone tissue was measured by BV/TV. The highest BV/TV ratio was observed in the PF group (18.77%; after 10 weeks, Fig. ). This was significantly higher than in the crossover- (11.28%; p = 0.0436), the PFx- (10,98%; p = 0.0411) or the control group (10.47%; p = 0.0293).
Figure shows representative histological sections of osteotomy zones of all four treatment groups. Analysed specimens within the PF group showed primary fibrotic tissue inside the CSD with partial calcification. This represents immature woven bone. The specimens of the PFx treated femur showed “capping” at the end of the defected ends. Here the medullary cavity was closed with compact bone. The central region displayed fibrotic tissue. Samples of the crossover group showed “capping” as well. The CSD was filled with fibrotic tissue, which was partially calcified but did not correspond to immature woven bone. Empty defect models presented with “capping” and fibrotic tissue inside the defect zone.
The goal of the study was to evaluate the bone forming potential of periosteal flaps without using any additional matrices. The bone volume in the PF group was significantly higher than in all other groups. After creation of the defect by osteotomy in the femur, the PF induced osteogenesis and increased bone volume quantitatively in comparison to the other groups. Histologic analysis shows capping in the control, the ligated periosteal flap and the crossover group, however does not reflect the significant advantage of the PF, potentially due to the cutting plane and the irregular structure of the tissue. This osteogenic potential of the PFs was described before, but never as a stand-alone technique . The PF in our study didn’t need a carrier material to enable the bone formation process, which was contrary to other studies. We fixed the PF with a single suture to the plate without any additional fixation after positioning it right onto the CSD. To our knowledge, no other study has shown the potential of bone regeneration by PFs alone without additives. Our results indicated that the bone forming process doesn’t necessarily need a scaffold or other similar structures, rather demonstrated that adequate blood flow to the fracture site as well as hematoma formation are sufficient for new bone formation to occur. It could hence be observed that the novel bone formation through PFs occured by intramembranous- and endochondral ossification . In the crossover group, BV/TV values were higher compared to both the PFx group and the control group (11.28% versus 10.98% and 10.47%, respectively). This marginal, not significant difference may be attributed to the timing of the periosteal flap transplantation, which in the crossover group was performed 5 weeks after the primary surgery. The difference between the PF- and the crossover-group (18.77% versus 11.28%) could be explained by the different time interval, as the time of insertion for the periosteal flap in the crossover-group occurred 5 weeks after the primary surgery. It seems plausible that the periosteal flap, despite its delayed transplantation, contributed to improved bone healing, although significantly less than in the initial PF group. Through a series of X-rays over time it could be observed that the processes of bone formation stopped after 5 weeks and it could not be re-initiated by periosteal flap implantation alone. The reason for this could be explained through the mechanism of healing, which has already ceased. The ligated periosteal flap itself was not able to induce new bone. Hence, bone healing is a process over time and is especially advanced within the first few weeks. It cannot be supported or induced by a stand-alone periosteal flap. Possibly, the formed hemorrhage at the fracture site attracts mesenchymal stem cells of the cambium layer of the periosteal flap by chemotaxis. Contrary to an initial osteotomy, a 5 weeks old osteotomy doesn’t form a hemorrhage anymore and the chemotactic stimulus might be missing. Further, other osteogenic stimuli might decrease after this time period (5 weeks). Factors like low oxygen, low intensity pulse, and bone morphogenetic protein (BMP) probably be present initially but are not as abundant anymore after 6 weeks . The PF was sutured directly to the plate without any tension to protect the vascular pedicle. It has been shown that tension on the periosteum activates the Wnt- and BMP- signaling pathway to activate the differentiation of osteoblasts . All those theories combined might explain that bony healing has ceased after 6 weeks. In an additional/future study to enhance the result of our findings, a second osteotomy of the already osteotomized edges could be carried out to induce a new bone forming process. This might support the surgical technique of decorticating the non-union ends before initiating treatment with periosteal flaps. Resecting the bone until it bleeds to observe the “paprika sign”, is commonly accepted in the surgical treatment. The PFx group showed no significant potential to increase the bone volume. Contrary to the PF group, the blood supply provided by the pedicle was cut. Therefore, the overall blood supply provided by the vascular pedicle seems to be essential for the bone formation process . This necessity of a vascular pedicle is supported by other researchers where the periosteal flap was used with a β-TCP scaffold . Their research also suggests, that only cells of the periosteal flap are not sufficient to increase bone volume. In our study samples of the PFx group showed the least osteogenic potential measured by BV/TV. This is interesting so far, as the induced membrane in the Masquelet technique (which is the use of a temporary cement spacer followed by staged bone grafting), is a recent treatment strategy to manage a posttraumatic bone defect. It provides additional osteogenic cells and factors, although the defect was continually filled with cancellous bone . The expectation of an osteogenic potential between the control- and PF-ground was not met. The control group demonstrated the expected lack of bony healing as it has been previously published . The research available for periosteal flaps in rodents mostly demonstrated bone forming potential of periosteal flaps in combination with different matrices, such as biodegradable scaffolds, bone marrow derived stem cells, allografts, or bioactive glass. In our study this bone forming potential was solely dependent on the periosteal flap itself. The reason for this was that, in our opinion, it was the most often used technique for bone defects in surgical procedures to stabilize non-unions. In such cases, it is often not advised to add inert carrying materials due to the increased risk of infection, as bone defects mainly result from severe soft tissue trauma such as high graded- closed or open fractures. The periosteal flap might even be beneficial for the treatment of infection as it increases local blood supply and administers the necessary immune blood cells . The bone forming potential of the vascularized periosteal flap only, suffices to promote a bony consolidation in a CSD in rats, without any accessory adjuncts. In contrast to our approach, most of the published studies focused on bone defect reconstruction using 3 columns: 3D-scaffolds in combination with osteogenic factors and the PF as an enhancer . In these studies, the PF showed an additional benefit to the bone forming capacity of the other factors, scaffolds and cytokines or cells but not as a stand-alone treatment. Vögelin emphasized the advantage of “biological containment and concentration of BMP within the osseous defect” by the PF but no bone formation of the PF with Orthogonal PolyLactic Acid (PLA) and HYaluronic acid (HY) (OPLA-HY) alone in the CSD after 8 weeks . OPLA-HY is a composite material used in bone regeneration research. This material is designed to support the growth and healing of bone tissue. It is often combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) and used with a vascularized periosteal flap to enhance bone formation in critical-sized defects, such as those in the femur of rats. More recent studies combined the PF with MSCs (Mesenchymal Stem Cells) and EPCs (Endothelial Progenitor Cells) and found similar enhancing capabilities of the PF, However, the latter studies corroborate our findings that the PF has bone forming capabilities alone with a scaffold only and without the other factors like MSC and EPC . Article focus Newly formed bone in a 5 mm critical-sized defect in the rat femur diaphysis. Pedicled periosteal flap have the highest bone forming capacity. Key messages Newly formed bone tissue measured by BV/TV was the highest in the PF group. PFs to form new bone do not need a scaffold or other similar carrying structures. Strengths and limitations The present study had several limitations. First, the animals were euthanized 10 weeks post-femoral surgery, and therefore it was not possible to determine the long-term outcome of the treatments on bone remodeling. The underlying cellular and molecular mechanisms driving the synergistic effects of bone formation with a PF remain unclear.
Newly formed bone in a 5 mm critical-sized defect in the rat femur diaphysis. Pedicled periosteal flap have the highest bone forming capacity.
Newly formed bone tissue measured by BV/TV was the highest in the PF group. PFs to form new bone do not need a scaffold or other similar carrying structures.
The present study had several limitations. First, the animals were euthanized 10 weeks post-femoral surgery, and therefore it was not possible to determine the long-term outcome of the treatments on bone remodeling. The underlying cellular and molecular mechanisms driving the synergistic effects of bone formation with a PF remain unclear.
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Adoption, implementation, definitions, and future of blockchain technology in ophthalmology | fe1eda9c-d99b-4cfc-9b17-84a304248f9b | 10229909 | Ophthalmology[mh] | A lot of ophthalmologists depend on ASCRS intraocular lens (IOL) calculators for the calculation of IOL power. Having such data on the server can be a potential target of cybercrime and malware and may result in data modification without the users’ knowledge. To avoid this problem, blockchain technology has emerged as a possible solution. This technology could also assist in maintaining an extensive database of A constants worldwide. The pre and postoperative refractive surgery data may not always be available for IOL power calculation. By using blockchain technology, this data can be stored and later can be retrieved in the future. The stored data will also be protected from cyber-attack or criminal corruption. A patient’s genome can be secured with the help of blockchain technology, which can be of academic support in future applications. Genecoin is a company that helps store a patient’s genome with blockchain technology. Blockchain technology could also revolutionize international data collection of various private and government institutes aimed at better patient care and isolating epidemiological data. The prime examples include endophthalmitis, toxic anterior segment syndrome, and cataract surgical outcomes. Blockchain technology has been utilized in bitcoins and cryptocurrencies as a method of payment. They may be used to accept payments for elective surgeries like LASIK, cataracts, pterygium, etc. A large number of retinal AI clinical trials are being conducted across the world. These trials require automated analysis of retinal images and optical coherence tomography (OCT) scans, database preservation, data security, confidentiality, application of deep learning, a firm belief among different organizations for data transfer, and building relationships with a collaborative effort. This has been possible only through the concept of blockchain technology. The global coronavirus pandemic gave birth to multiple epidemics and pandemics, such as mucormycosis, mask-associated dry eyes, and digital eye strain, leading to the global myopia pandemic. Tan et al . designed a blockchain-enabled platform using hyperledger fabric to tackle the global myopia pandemic by developing deep learning algorithms for the same. They proved that blockchain technology helps secure data transfer, sharing, and validation of models, testing for three separate sites in two countries. They developed a deep learning module for the automated detection of myopic changes in the retina by analyzing the retinal changes. COVID-19 vaccines for ophthalmology patients, total parenteral nutrition for Xerophthalmia, intravitreal anti- Vascular Endothelial Growth Factor (VEGF), autologous serum, blood products, and biologics require stringent storage conditions. They are expensive products mandating good supply chain management. Wu et al . proposed the concept of online pharmacy for ophthalmology patients using blockchain technology. They suggested that prescriptions can be monitored, drugs can be delivered to remote locations, and, if needed, can also be renewed. Blockchain technology can also monitor and increase patient compliance with drugs and treatment. This will revolutionize patient treatment with costly medicines. To conclude, the adoption and implementation of blockchain technology have revolutionized the ophthalmological network ensuring transparent, large-volume, efficient data-sharing across digital platforms and maintaining integrity. Knowledge of AI and blockchain technology has helped maintain data integrity, an international collection and sharing of data along various platforms with accountability, validity, and transparency. Novel uses like IOL power calculation and refractive surgery have aroused interest in data annotators for rapid implementation of this technology. The characteristics of blockchain networking, such as consensus, provenance, immutability, and finality, have ensured the smooth flow of data, sharing of the patient database, and use by the patient themselves. This has also paved the way for future research for the development of newer protocols and algorithms for the widespread implementation of AI in ophthalmology. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. Nil. There are no conflicts of interest. |
Health Advertising on Short-Video Social Media: A Study on User Attitudes Based on the Extended Technology Acceptance Model | 4f1a79d2-d682-41a5-8c63-97b21cb0c6e3 | 7084731 | Health Communication[mh] | Social media is one of the most promising tools in the digital advertising environment . Since 2016, short video has become an important field for the expansion of social media such as Tik Tok, Facebook live, YouTube, Snapchat, Instagram, Douyin (Chinese), and Douyu (Chinese). These real-time interactive information platforms provide a free and easy-to-access online resource for live videos for anchors and viewers. For example, shows a typical short video (15 s by default) on the largest Chinese live-video platform Douyin ( https://www.douyin.com/ ), where people can use self-generated videos to share with others something interesting, such as new products and nice places. In , the user is introducing a new Swisse product from Australia. So far, short video has gradually developed into a popular social media channel. For example, a recent report showed that the number of short-video users in China had reached 422 million . The rapid development of short-video platforms introduces new opportunities for health promotion, e.g., health-related advertising and recommendation. Compared with traditional social media, short-video platforms have little time delay and can support one-on-one as well as many-to-many social interaction by videos and the barrage (scrolling texts on the screen). For example, the largest Chinese live-video platform Douyin ( https://www.douyin.com/ ) provides one-on-one video interaction that allows one user discusses some topics with another user through live video. At the same time, other users can watch the live video and interact with others via barrages, text reviews, emojis, and images. This new form of social interaction on short-video social network platforms has been demonstrated to be an effective way of advertising . However, in health-related short-video platforms, an important issue is to know users’ intentions of accepting health-related short-video advertisements. Therefore, this study attempts to investigate whether and how short-video ads affect users’ purchase intentions on health-related products. Particularly, we take the Technology Acceptance Model (TAM) as the base model and extend social interaction, intrusiveness, informativeness, and relevance as four new antecedents to the perceived usefulness of health-related short-video ads. Then, we set perceived usefulness and attitude as two mediate factors and validate their mediating effect in the model. Finally, we conduct an empirical analysis of questionnaire data from the Internet and short-video social network platforms based on the structural equation model. Briefly, following the research framework of the TAM model, we aim to study the following research questions: RQ1: What are the key factors that affect the perceived usefulness of health-related short-video ads? RQ2: How do the factors affect the perceived usefulness of health-related ads? RQ3: How do perceived usefulness and perceived ease of use affect user attitudes and purchase intention on health-related short-video ads? RQ4: What kind of relationship exists between user attitudes toward health-related short-video ads and users’ purchase intention on health-related products? RQ5:What is the mediating effect of perceived usefulness and user attitudes to users’ purchase intention on health-related products? In summary, we make the following contributions in this paper: (1) We extend the Technology Acceptance Model (TAM) with new antecedents including social interaction, intrusiveness, informativeness, and relevance to analyze the users’ response to health-related short-video ads. To the best of our knowledge, this is the first study that extends the TAM model to analyze user acceptance of health-related short-video ads. (2) We conduct a survey on the Internet and short-video social network platforms and perform systematical data analysis over the surveyed data. The data analysis consists of many aspects, including measurement model evaluation, structural model evaluation, and mediating effect analysis. The results show that social interaction, intrusiveness, informativeness, relevance, and the TAM factors (perceived usefulness and perceived ease of use) have a significant influence on user acceptance of health-related short-video ads. Further, we find that users’ perceived ease of use on health-related short-video ads cannot significantly predict users’ attitudes toward ads. This is a new finding that is contrary to previous studies in social media-oriented ads. (3) We integrate the empirical findings and present reasonable suggestions for advertisers and marketers to better develop health-related short-video ads. The remainder of this paper is structured as follows. describes the related work and the differences between previous studies and this paper. presents the research model as well as the hypothesis propositions. describes the data collection and measurements. presents the results of data analysis. discusses the research implications and suggestions. Finally, in , we conclude the entire paper.
2.1. Short-Video Advertising Digital advertising significantly influences users’ attitudes and intentions to purchase . There are many studies about the factors that influence the effectiveness of online advertising , video advertising , and social media advertising . Although short-video ads are a promising form of online advertising, this ad form has not been adequately examined in the research. The core of short-video ads is “video + sociality”, and it has become an important field for the expansion of social media. Therefore, we study our questions mainly based on the research of social media advertising. Some quote video studies, of course. There have been some existing studies on informativeness and intrusiveness and how such concerns may influence users’ attitudes toward advertising and product purchase intentions . Generally, informativeness is defined as the information value provided by media like texts, images, or videos and intrusiveness is defined as the degree to which people deem the presentation of information as contrary to their goals . Both informativeness and intrusiveness have been regarded as key factors to predict the helpfulness of messages in the research of social networks. For example, Dehghani et al. conducted an empirical study on YouTube ads and found that a high value of informativeness led to a high level of user acceptance of YouTube ads, meaning that informativeness is a positive factor affecting users’ attitude toward YouTube ads. Meanwhile, they found intrusiveness is a negative factor, meaning that a high value of intrusiveness led to a low level of user acceptance of YouTube ads. Lee and Hong empirically investigated that informativeness has a potential contribution to a positive online behavior based on the theory of reasoned action and the social influence theory . Jung found perceived ad relevance influences advertising effectiveness while it could increase privacy concerns, which ultimately raise ad avoidance in social media . Relevance is another factor that may affect user acceptance of advertising. Relevance has been widely adopted in web search and online recommendation . For instance, a web search engine can rank the searching results according to the relevance between the user query and the returned web pages. In the online recommendation in E-commerce, the relevance between the user profile and products is usually used to predict the items that may fit within the users’ interests. Similarly, the relevance between a user’s interest and short-video advertisements may also affect the user acceptance of short-video ads . Therefore, we will also consider relevance as a construct in the research model of this study, which will be detailed in . On the other hand, according to the social influence theory , a high level of social interactions can enhance the feelings of affection, trust, belongingness, and warmth. Currently, most people are engaged in short videos by mobile apps. The mobile short-video apps offer people real-time interaction with other ones. In addition, platforms like Douyin and Tik Tok allow users to interact with short videos. For example, users can click the product link and fill a form to deliver their purchase interest to vendors. Therefore, social interaction can be a key factor affecting user acceptance of short-video ads. So far, short-video ads are still in the early developing stage. Existing work has paid little attention to how the new features of short videos impact users’ attitudes toward purchase behavior. Based on previous experiences in social media ads, many researchers claimed that two aspects of information should be considered in the context of social media ads . The first one, product information, focuses on informativeness, relevance, and intrusiveness of information . The second one, personal interaction, refers to social interaction, which reflects the impact of social identity and group norms on community users’ group intention of accepting advertising . Windels et al. examined the differences between native advertisements and friend referrals on social networking sites. They found that social relationships did not always work . In this paper, we consider these related studies as well as the features of short-video ads to design antecedents toward the perceived usefulness of users and the attitude to short-video ads. As a result, four antecedents including informativeness, relevance, intrusiveness, and social interaction are designed in our study. 2.2. Technology Adoption Model (TAM) The Technology Acceptance Model (TAM) was developed to describe users’ behavior to accept or reject the use of new technologies . The TAM model defines two variables, namely perceived usefulness and perceived ease of use, to quantify user attitude to information technology, which in turn can be used to measure user acceptance of information technologies. Although the TAM model is initially designed to explain and predict the behavior of individuals on the use of information systems, it has been used in many studies . Previous studies have demonstrated the applicability of the TAM model to online advertising. For example, Demangeot and Broderick examined a modified technology acceptance model (TAM) that was developed to test the intention to use SMS advertising . Based on the ability of TAM and short video including a wide variety of online media services and SNS, it is a reasonable choice to consider the TAM model to model and quantify user acceptance of short-video ads. The TAM model has been widely adopted in many existing studies. In this paper, we also adopt the TAM model as the basic research model. There are two reasons. First, many studies in social media have applied the TAM model to analyze the acceptance factors of online advertising and social network advertising. The research scope of this paper, i.e., short video, has the characteristics of both online media and social networks; thus, it is a reasonable choice to select the TAM model. Second, the basic factors in the TAM model, namely perceived usefulness (PU) and perceptual ease of use (PEOU), are suitable to distinguish the usefulness of live advertising and the impact of live-video technologies. On the other hand, differing from the traditional TAM model, we integrate social interaction, intrusiveness, informativeness, and relevance with TAM and propose to use perceived usefulness as the mediate factor, forming an augmented TAM model that is more suitable for studying the intact path of short-video ads. Even though research integrating these external variables with the TAM is limited, available empirical findings generally support the influence of these factors on perceptions about users’ ad attributes and adoption intentions. In addition to the TAM model, another widely used model for predicting user acceptance of short-video ads is the Unified Theory of Acceptance and Use of Technology (UTAUT) model , which was proposed to incorporate various models of human behavior theory. The UTAUT model was constructed by extracting three variables that affect users’ behavioral intentions, one variable that influences action, and four moderators that mediate the effects of the process. Some of the variables had similar concepts with variables that construct the TAM model. One major difference between UTAUT and TAM was that UTAUT proposed four control variables (i.e., gender, age, experience, and voluntariness) to further enhance the predictive power of the model. Based on UTAUT, Venkatesh et al. incorporated three other constructs into UTAUT, namely hedonic motivation, price value, and habit, extending UTAUT into UTAUT 2 . Compared to UTAUT, the extensions proposed in UTAUT2 produced a substantial improvement in the variance explained in behavioral intention. As a result, both UTAUT and UTAUT 2 have been actively used in predicting users’ purchase behavior in many areas like healthcare systems and social-network-based advertising . However, as suggested by Venkatesh et al. , in order to increase the applicability of the original UTAUT as well as the UTAUT 2 model, other relevant factors are usually needed to be extended to meet the specific requirements of different applications, technologies, countries, etc. In summary, both TAM and UTAUT are actively utilized in the research of user adoption of new technologies. Whether or not to use TAM or UTAUT mainly depends on the particular type of the application. In this paper, we select TAM rather than UTAUT as the basic model for analyzing user acceptance of health-related short-video ads for the following reasons. First, UTAUT is a behavioral model aiming to explain the behavior of people in the use of information systems , but in this study, the content of short-videos, as measured by the antecedents named informativeness, relevance, and intrusiveness in our model, is also an important factor. Second, the UTAUT model is more complex than TAM; thus, extending TAM is easier than extending UTAUT. Nevertheless, as Venkatesh et al. and Dwivedi et al. reported, most studies only employed a subset of the UTAUT model, and the moderators were typically removed. To this end, the TAM model can have similar predictive power as the UTAUT model without moderators. However, it is still worth investigating the extension of the UTAUT or UTAUT 2 model to predict the user adoption of health-related short-video ads in the future.
Digital advertising significantly influences users’ attitudes and intentions to purchase . There are many studies about the factors that influence the effectiveness of online advertising , video advertising , and social media advertising . Although short-video ads are a promising form of online advertising, this ad form has not been adequately examined in the research. The core of short-video ads is “video + sociality”, and it has become an important field for the expansion of social media. Therefore, we study our questions mainly based on the research of social media advertising. Some quote video studies, of course. There have been some existing studies on informativeness and intrusiveness and how such concerns may influence users’ attitudes toward advertising and product purchase intentions . Generally, informativeness is defined as the information value provided by media like texts, images, or videos and intrusiveness is defined as the degree to which people deem the presentation of information as contrary to their goals . Both informativeness and intrusiveness have been regarded as key factors to predict the helpfulness of messages in the research of social networks. For example, Dehghani et al. conducted an empirical study on YouTube ads and found that a high value of informativeness led to a high level of user acceptance of YouTube ads, meaning that informativeness is a positive factor affecting users’ attitude toward YouTube ads. Meanwhile, they found intrusiveness is a negative factor, meaning that a high value of intrusiveness led to a low level of user acceptance of YouTube ads. Lee and Hong empirically investigated that informativeness has a potential contribution to a positive online behavior based on the theory of reasoned action and the social influence theory . Jung found perceived ad relevance influences advertising effectiveness while it could increase privacy concerns, which ultimately raise ad avoidance in social media . Relevance is another factor that may affect user acceptance of advertising. Relevance has been widely adopted in web search and online recommendation . For instance, a web search engine can rank the searching results according to the relevance between the user query and the returned web pages. In the online recommendation in E-commerce, the relevance between the user profile and products is usually used to predict the items that may fit within the users’ interests. Similarly, the relevance between a user’s interest and short-video advertisements may also affect the user acceptance of short-video ads . Therefore, we will also consider relevance as a construct in the research model of this study, which will be detailed in . On the other hand, according to the social influence theory , a high level of social interactions can enhance the feelings of affection, trust, belongingness, and warmth. Currently, most people are engaged in short videos by mobile apps. The mobile short-video apps offer people real-time interaction with other ones. In addition, platforms like Douyin and Tik Tok allow users to interact with short videos. For example, users can click the product link and fill a form to deliver their purchase interest to vendors. Therefore, social interaction can be a key factor affecting user acceptance of short-video ads. So far, short-video ads are still in the early developing stage. Existing work has paid little attention to how the new features of short videos impact users’ attitudes toward purchase behavior. Based on previous experiences in social media ads, many researchers claimed that two aspects of information should be considered in the context of social media ads . The first one, product information, focuses on informativeness, relevance, and intrusiveness of information . The second one, personal interaction, refers to social interaction, which reflects the impact of social identity and group norms on community users’ group intention of accepting advertising . Windels et al. examined the differences between native advertisements and friend referrals on social networking sites. They found that social relationships did not always work . In this paper, we consider these related studies as well as the features of short-video ads to design antecedents toward the perceived usefulness of users and the attitude to short-video ads. As a result, four antecedents including informativeness, relevance, intrusiveness, and social interaction are designed in our study.
The Technology Acceptance Model (TAM) was developed to describe users’ behavior to accept or reject the use of new technologies . The TAM model defines two variables, namely perceived usefulness and perceived ease of use, to quantify user attitude to information technology, which in turn can be used to measure user acceptance of information technologies. Although the TAM model is initially designed to explain and predict the behavior of individuals on the use of information systems, it has been used in many studies . Previous studies have demonstrated the applicability of the TAM model to online advertising. For example, Demangeot and Broderick examined a modified technology acceptance model (TAM) that was developed to test the intention to use SMS advertising . Based on the ability of TAM and short video including a wide variety of online media services and SNS, it is a reasonable choice to consider the TAM model to model and quantify user acceptance of short-video ads. The TAM model has been widely adopted in many existing studies. In this paper, we also adopt the TAM model as the basic research model. There are two reasons. First, many studies in social media have applied the TAM model to analyze the acceptance factors of online advertising and social network advertising. The research scope of this paper, i.e., short video, has the characteristics of both online media and social networks; thus, it is a reasonable choice to select the TAM model. Second, the basic factors in the TAM model, namely perceived usefulness (PU) and perceptual ease of use (PEOU), are suitable to distinguish the usefulness of live advertising and the impact of live-video technologies. On the other hand, differing from the traditional TAM model, we integrate social interaction, intrusiveness, informativeness, and relevance with TAM and propose to use perceived usefulness as the mediate factor, forming an augmented TAM model that is more suitable for studying the intact path of short-video ads. Even though research integrating these external variables with the TAM is limited, available empirical findings generally support the influence of these factors on perceptions about users’ ad attributes and adoption intentions. In addition to the TAM model, another widely used model for predicting user acceptance of short-video ads is the Unified Theory of Acceptance and Use of Technology (UTAUT) model , which was proposed to incorporate various models of human behavior theory. The UTAUT model was constructed by extracting three variables that affect users’ behavioral intentions, one variable that influences action, and four moderators that mediate the effects of the process. Some of the variables had similar concepts with variables that construct the TAM model. One major difference between UTAUT and TAM was that UTAUT proposed four control variables (i.e., gender, age, experience, and voluntariness) to further enhance the predictive power of the model. Based on UTAUT, Venkatesh et al. incorporated three other constructs into UTAUT, namely hedonic motivation, price value, and habit, extending UTAUT into UTAUT 2 . Compared to UTAUT, the extensions proposed in UTAUT2 produced a substantial improvement in the variance explained in behavioral intention. As a result, both UTAUT and UTAUT 2 have been actively used in predicting users’ purchase behavior in many areas like healthcare systems and social-network-based advertising . However, as suggested by Venkatesh et al. , in order to increase the applicability of the original UTAUT as well as the UTAUT 2 model, other relevant factors are usually needed to be extended to meet the specific requirements of different applications, technologies, countries, etc. In summary, both TAM and UTAUT are actively utilized in the research of user adoption of new technologies. Whether or not to use TAM or UTAUT mainly depends on the particular type of the application. In this paper, we select TAM rather than UTAUT as the basic model for analyzing user acceptance of health-related short-video ads for the following reasons. First, UTAUT is a behavioral model aiming to explain the behavior of people in the use of information systems , but in this study, the content of short-videos, as measured by the antecedents named informativeness, relevance, and intrusiveness in our model, is also an important factor. Second, the UTAUT model is more complex than TAM; thus, extending TAM is easier than extending UTAUT. Nevertheless, as Venkatesh et al. and Dwivedi et al. reported, most studies only employed a subset of the UTAUT model, and the moderators were typically removed. To this end, the TAM model can have similar predictive power as the UTAUT model without moderators. However, it is still worth investigating the extension of the UTAUT or UTAUT 2 model to predict the user adoption of health-related short-video ads in the future.
3.1. Research Model In this paper, we first selected the main factors in the TAM model as basic independent variables, i.e., perceived usefulness (PU), perceived ease of use (PE), and ad attitudes (AT). Further, we introduced social interaction (SI), informativeness (IR), intrusiveness (IN), and relevance (RE) as new antecedents of perceived usefulness. The selection of the four factors (i.e., SI, IR, IN, RE) was mostly based on two aspects. First, previous studies in social media have shown that these factors are critical to the usefulness of social media advertising . Second, these factors are suitable for short-video ads, as they reflect the information features of live video ads. Thus, it is reasonable to introduce them to the research model. As a result, the perceived usefulness (PU) in our research model became a mediator variable. The purchase intention (PI) was set to be the dependent variable. In the study of behavior intention, attitude has been commonly recognized as a variable that affects the willingness of behavior. Thus, we chose attitude as another mediator variable. On this basis, this paper put forward the research model of user responses to health-related short-video advertisements. shows the components of the proposed research model. 3.2. Research Hypothesis According to the research model, there are three independent variables, namely perceived usefulness (PU), perceived ease of use (PE), and ad attitude (AT), and four antecedent variables, namely social interaction (SI), informativeness (IR), intrusiveness (IN), and relevance (RE). In addition, there are two mediator variables called perceived usefulness (PU) and ad attitude (AT). These variables are supposed to impact the dependent variable named purchase intention (PI). To reveal the relationships among these factors, we propose hypotheses to the research questions presented earlier, as listed in . The right column in shows the corresponding hypotheses that are proposed to answer the research question. In the following subsections, we will explain each hypothesis. Sociability has been defined as ‘the extent to which the computer-mediated communication environment facilitates the emergence of social space by allowing social affordance . Social interaction can mitigate users’ perceptions of ad avoidance and enhance the effectiveness of ads . Interactive online media are an increasingly preferred format for users and advertisers. Particularly, health-related short-video ads are more social than traditional social media advertising, whereby audiences can, in a timely manner, view, share, and post barrages on these ads with anchors and peer audiences. Given the massively real-time interactivity distinguishing live video from traditional social media, real-time interactivity may enhance the influence of social interactions or depress viewers’ experience because of barrages filled with mobile screens. Therefore, we make the following hypothesis: Hypothesis 1 (H1) . Social interaction of health-related short-video ads positively affects users’ perceived usefulness (PU) of ads. Intrusiveness describes the extent to which the content is messy and irritating to users . Intrusiveness in social media advertising will be negatively related to the perceived usefulness of ads . When an inconsistent ad is posted with live videos, audiences may spend efforts on non-major tasks and cannot effectively deal with advertising information, resulting in the ineffectiveness of advertising. In addition, the small screen of a mobile phone may also make intrusiveness be a negative factor in live video ads. Moreover, intrusive ads may bring the negative emotion to users . Hence, we make Hypothesis 2: Hypothesis 2a (H2a) . The intrusiveness of health-related short-video ads negatively affects users’ perceived usefulness (PU) of ads. Hypothesis 2b (H2b) . The intrusiveness of health-related short-video ads negatively affects users’ perceived ease of use of ads. Informativeness represents the information that is helpful and resourceful . It can inform users about product alternatives. Consumers tend to gain information more through unconditional, interpersonal information exchange . Studies have found informativeness to be important in the formation of consumer attitudes to electronic commerce websites as well as to SNS advertising and to social media advertising . The informative content of a message positively influences the perceived value of online advertising . Hence, we make Hypothesis 3: Hypothesis 3 (H3) . Informativeness of health-related short-video ads positively affects users’ perceived usefulness (PU) of ads. Relevance is used to capture users’ general perception of similarity between short-video ads and live video content. According to the limited capacity model of attention , the total capacity allocated to process activities can be divided into the primary capacity for the most important task and the spare capacity for less important tasks. When an inconsistent ad is placed in the primary task field, audiences may reduce the likelihood of primary capability being allocated to processing the ad. Advertising relevance decreases ad skepticism and ad avoidance in e-mails, direct mails, telemarketing, and text messages . Hence, we make Hypothesis 4: Hypothesis 4 (H4) . The relevance of health-related short-video ads positively affects users’ perceived usefulness (PU) of ads. Perceived usefulness (PU) refers to the degree to which individuals perceive the use of health-related short-video ads. If users think that short-video ads are useful, they are much likely to have a positive attitude on using them. Attracting and motivating short-video ads can also lead to purchase intention . Hence, we make Hypotheses 5 and 6 as follows: Hypothesis 5 (H5) . Users’ perceived usefulness of health-related short-video ads positively affects their attitudes toward ads. Hypothesis 6 (H6) . Users’ perceived usefulness of health-related short-video ads positively affects their purchase intentions . Perceived ease of use (PEOU) is defined as the mobile phone users’ expectations about the effort required to use live video advertising messages. Short-video advertising requires some cognitive effort from users such as maneuvering through screens, reading the advertising, and making a quick evaluation of whether it is worthy (in terms of time or effort) to take further action. Therefore, the easier they perceive using short-video advertisements to be in general, the more likely they are to later engage in the action implied by the ad. Hence, we make Hypotheses 7 and 8: Hypothesis 7 (H7) . Users’ perceived ease of use of health-related short-video ads positively affects their perceived usefulness. Hypothesis 8 (H8) . Users’ perceived ease of use of health-related short-video ads positively affects their attitudes toward ads. Attitude towards advertising is regarded as an important factor for advertising management because consumers’ attitude can influence purchase intention . Previous research has indicated that purchase intention is a critical indicator of advertising effectiveness and may be affected by indicators such as attitude towards ads . For example, Lin and Kim found that the attitude towards ads affected brand awareness and purchase intentions . Tran found that purchase intention was positively affected by ad attitude on Facebook . Whether consumers prefer a product or an advertisement depends on the informativeness of the advertisement, i.e., whether the advertisement can provide the opportunity and convenient ways for purchase. Therefore, the informational nature of advertisement affects the consumer’s purchase intention. Based on these observations, we make the following hypothesis: Hypothesis 9 (H9) . Users’ attitudes of health-related short-video ads positively affect their purchase intentions. Note that we only set up the Hypotheses H1–H4 to measure the impact of the four constructs on PU but not on PEOU. This is mainly because the constructs are all about the information offered by advertisements, but not about the operations or behavior of advertising. On the other hand, the perceived ease of use (PEOU) refers to the ease of operations that users perceive, i.e., PEOU is operation related. Thus, in this paper, we only focus on the impact of the constructs on PU.
In this paper, we first selected the main factors in the TAM model as basic independent variables, i.e., perceived usefulness (PU), perceived ease of use (PE), and ad attitudes (AT). Further, we introduced social interaction (SI), informativeness (IR), intrusiveness (IN), and relevance (RE) as new antecedents of perceived usefulness. The selection of the four factors (i.e., SI, IR, IN, RE) was mostly based on two aspects. First, previous studies in social media have shown that these factors are critical to the usefulness of social media advertising . Second, these factors are suitable for short-video ads, as they reflect the information features of live video ads. Thus, it is reasonable to introduce them to the research model. As a result, the perceived usefulness (PU) in our research model became a mediator variable. The purchase intention (PI) was set to be the dependent variable. In the study of behavior intention, attitude has been commonly recognized as a variable that affects the willingness of behavior. Thus, we chose attitude as another mediator variable. On this basis, this paper put forward the research model of user responses to health-related short-video advertisements. shows the components of the proposed research model.
According to the research model, there are three independent variables, namely perceived usefulness (PU), perceived ease of use (PE), and ad attitude (AT), and four antecedent variables, namely social interaction (SI), informativeness (IR), intrusiveness (IN), and relevance (RE). In addition, there are two mediator variables called perceived usefulness (PU) and ad attitude (AT). These variables are supposed to impact the dependent variable named purchase intention (PI). To reveal the relationships among these factors, we propose hypotheses to the research questions presented earlier, as listed in . The right column in shows the corresponding hypotheses that are proposed to answer the research question. In the following subsections, we will explain each hypothesis. Sociability has been defined as ‘the extent to which the computer-mediated communication environment facilitates the emergence of social space by allowing social affordance . Social interaction can mitigate users’ perceptions of ad avoidance and enhance the effectiveness of ads . Interactive online media are an increasingly preferred format for users and advertisers. Particularly, health-related short-video ads are more social than traditional social media advertising, whereby audiences can, in a timely manner, view, share, and post barrages on these ads with anchors and peer audiences. Given the massively real-time interactivity distinguishing live video from traditional social media, real-time interactivity may enhance the influence of social interactions or depress viewers’ experience because of barrages filled with mobile screens. Therefore, we make the following hypothesis: Hypothesis 1 (H1) . Social interaction of health-related short-video ads positively affects users’ perceived usefulness (PU) of ads. Intrusiveness describes the extent to which the content is messy and irritating to users . Intrusiveness in social media advertising will be negatively related to the perceived usefulness of ads . When an inconsistent ad is posted with live videos, audiences may spend efforts on non-major tasks and cannot effectively deal with advertising information, resulting in the ineffectiveness of advertising. In addition, the small screen of a mobile phone may also make intrusiveness be a negative factor in live video ads. Moreover, intrusive ads may bring the negative emotion to users . Hence, we make Hypothesis 2: Hypothesis 2a (H2a) . The intrusiveness of health-related short-video ads negatively affects users’ perceived usefulness (PU) of ads. Hypothesis 2b (H2b) . The intrusiveness of health-related short-video ads negatively affects users’ perceived ease of use of ads. Informativeness represents the information that is helpful and resourceful . It can inform users about product alternatives. Consumers tend to gain information more through unconditional, interpersonal information exchange . Studies have found informativeness to be important in the formation of consumer attitudes to electronic commerce websites as well as to SNS advertising and to social media advertising . The informative content of a message positively influences the perceived value of online advertising . Hence, we make Hypothesis 3: Hypothesis 3 (H3) . Informativeness of health-related short-video ads positively affects users’ perceived usefulness (PU) of ads. Relevance is used to capture users’ general perception of similarity between short-video ads and live video content. According to the limited capacity model of attention , the total capacity allocated to process activities can be divided into the primary capacity for the most important task and the spare capacity for less important tasks. When an inconsistent ad is placed in the primary task field, audiences may reduce the likelihood of primary capability being allocated to processing the ad. Advertising relevance decreases ad skepticism and ad avoidance in e-mails, direct mails, telemarketing, and text messages . Hence, we make Hypothesis 4: Hypothesis 4 (H4) . The relevance of health-related short-video ads positively affects users’ perceived usefulness (PU) of ads. Perceived usefulness (PU) refers to the degree to which individuals perceive the use of health-related short-video ads. If users think that short-video ads are useful, they are much likely to have a positive attitude on using them. Attracting and motivating short-video ads can also lead to purchase intention . Hence, we make Hypotheses 5 and 6 as follows: Hypothesis 5 (H5) . Users’ perceived usefulness of health-related short-video ads positively affects their attitudes toward ads. Hypothesis 6 (H6) . Users’ perceived usefulness of health-related short-video ads positively affects their purchase intentions . Perceived ease of use (PEOU) is defined as the mobile phone users’ expectations about the effort required to use live video advertising messages. Short-video advertising requires some cognitive effort from users such as maneuvering through screens, reading the advertising, and making a quick evaluation of whether it is worthy (in terms of time or effort) to take further action. Therefore, the easier they perceive using short-video advertisements to be in general, the more likely they are to later engage in the action implied by the ad. Hence, we make Hypotheses 7 and 8: Hypothesis 7 (H7) . Users’ perceived ease of use of health-related short-video ads positively affects their perceived usefulness. Hypothesis 8 (H8) . Users’ perceived ease of use of health-related short-video ads positively affects their attitudes toward ads. Attitude towards advertising is regarded as an important factor for advertising management because consumers’ attitude can influence purchase intention . Previous research has indicated that purchase intention is a critical indicator of advertising effectiveness and may be affected by indicators such as attitude towards ads . For example, Lin and Kim found that the attitude towards ads affected brand awareness and purchase intentions . Tran found that purchase intention was positively affected by ad attitude on Facebook . Whether consumers prefer a product or an advertisement depends on the informativeness of the advertisement, i.e., whether the advertisement can provide the opportunity and convenient ways for purchase. Therefore, the informational nature of advertisement affects the consumer’s purchase intention. Based on these observations, we make the following hypothesis: Hypothesis 9 (H9) . Users’ attitudes of health-related short-video ads positively affect their purchase intentions. Note that we only set up the Hypotheses H1–H4 to measure the impact of the four constructs on PU but not on PEOU. This is mainly because the constructs are all about the information offered by advertisements, but not about the operations or behavior of advertising. On the other hand, the perceived ease of use (PEOU) refers to the ease of operations that users perceive, i.e., PEOU is operation related. Thus, in this paper, we only focus on the impact of the constructs on PU.
4.1. Data Collection We mainly conducted questionnaires on users who have online shopping experiences due to mobile live ads, so that we could gain useful feedback from experienced users on short-video platforms. The data for this study were collected using an online survey in Douyu.com (a famous short-video platform in China) during January 2018, which lasted for three weeks. Three hundred and fifty questionnaires were sent to the viewers with anchors’ help, and 289 questionnaires were received. Thirty-three questionnaires that contained inconsistent answers or incomplete information filling were removed from the data set. Note that it is not possible to ask the Douyu platform to only present health-related short-video ads to the participants, because the platform does not offer the function of filtering health-related short-videos (actually, this could be a complicated task which needs effective algorithms). We offered the participants three weeks to return questionnaires, and all questions were restricted to health-related short-video ads (an example is given in ). Although we do not know what health-related videos the participants watched, we believe that the involved short-videos covers a wide range of health-related products. This is because the number of short videos on the Douyu platform is dramatically increasing day by day. In addition, different users have various interests, so the platform will recommend different short-video ads to each user. Specifically, we asked three Douyu anchors for help, namely Prisoner, Valila, and Liu shaji, to distribute the questionnaires to their fans. The time span of the questionnaires was from January 3, 2018 to January 24, 2018. Consequently, we collected 256 valid questionnaires, which met the analytical requirements of the structural equation model. In the data set, the proportions of males and females were 53.5% and 46.5%, respectively. There were 79.8% users that were under 30 years old. The duration of using live videos ranged from 0 to 2 years, and 58.6% of the users had experienced live videos for 1 to 2 years. Furthermore. 50.4% of the users were undergraduates and graduates (see ). The attributes of the surveyed data were similar to those in the IResearch report (IResearch, The research report on the China mobile live video market, http://wreport.iresearch.cn/uploadfiles/reports/636161408839324949.pdf ). 4.2. Measurement The survey was designed using a multi-item approach. All variables were carried out by a five-point Likert-scale, ranging from strongly disagree (1) to strongly agree (5). Items were borrowed from previous literature and modified for the context of this study. The questionnaire consisted of three parts: (1) Sample Selection: Users who have online shopping experiences due to mobile live ads are considered as samples in this study. (2) Sample Characteristics: This part mainly measures the sample statistical data of participants. (3) Variable Questionnaire: This study included eight latent variables, including Social interaction (SI), Intrusiveness (IR), Informativeness (IN), Relevance (RE), Perceived Usefulness (PU), Perceived ease of use (PE), Ad attitudes (AT), and Purchase Intention (PI). The whole items are shown in .
We mainly conducted questionnaires on users who have online shopping experiences due to mobile live ads, so that we could gain useful feedback from experienced users on short-video platforms. The data for this study were collected using an online survey in Douyu.com (a famous short-video platform in China) during January 2018, which lasted for three weeks. Three hundred and fifty questionnaires were sent to the viewers with anchors’ help, and 289 questionnaires were received. Thirty-three questionnaires that contained inconsistent answers or incomplete information filling were removed from the data set. Note that it is not possible to ask the Douyu platform to only present health-related short-video ads to the participants, because the platform does not offer the function of filtering health-related short-videos (actually, this could be a complicated task which needs effective algorithms). We offered the participants three weeks to return questionnaires, and all questions were restricted to health-related short-video ads (an example is given in ). Although we do not know what health-related videos the participants watched, we believe that the involved short-videos covers a wide range of health-related products. This is because the number of short videos on the Douyu platform is dramatically increasing day by day. In addition, different users have various interests, so the platform will recommend different short-video ads to each user. Specifically, we asked three Douyu anchors for help, namely Prisoner, Valila, and Liu shaji, to distribute the questionnaires to their fans. The time span of the questionnaires was from January 3, 2018 to January 24, 2018. Consequently, we collected 256 valid questionnaires, which met the analytical requirements of the structural equation model. In the data set, the proportions of males and females were 53.5% and 46.5%, respectively. There were 79.8% users that were under 30 years old. The duration of using live videos ranged from 0 to 2 years, and 58.6% of the users had experienced live videos for 1 to 2 years. Furthermore. 50.4% of the users were undergraduates and graduates (see ). The attributes of the surveyed data were similar to those in the IResearch report (IResearch, The research report on the China mobile live video market, http://wreport.iresearch.cn/uploadfiles/reports/636161408839324949.pdf ).
The survey was designed using a multi-item approach. All variables were carried out by a five-point Likert-scale, ranging from strongly disagree (1) to strongly agree (5). Items were borrowed from previous literature and modified for the context of this study. The questionnaire consisted of three parts: (1) Sample Selection: Users who have online shopping experiences due to mobile live ads are considered as samples in this study. (2) Sample Characteristics: This part mainly measures the sample statistical data of participants. (3) Variable Questionnaire: This study included eight latent variables, including Social interaction (SI), Intrusiveness (IR), Informativeness (IN), Relevance (RE), Perceived Usefulness (PU), Perceived ease of use (PE), Ad attitudes (AT), and Purchase Intention (PI). The whole items are shown in .
The research model was tested using AMOS 21, a structural modeling technique that is well suited for estimating parameters and theoretical models. In , we use confirmatory factor analysis (CFA) to validate the reliability and validity of the measurement model. In , we perform maximum likelihood estimation (MLE) and the bootstrapping method to examine the structural model. In , we measure the mediating effects of PU and AT in the research model. In , we summarize the conclusion of the research. Before measuring the model, we examined whether common method bias was a concern in this study with two tests of common method variance (CMV) . First, an exploratory factor analysis of all items extracted eight factors, which explained 73.72% of all the variance, with no single factor accounting for significant loadings (at the p < 0.10 level) for all items. Second, single method-factor approaches indicated that there was no significant difference among the method factors (ΔCMIN/DF = −0.032, ΔGFI = 0.009, ΔAGFI = 0.001, ΔIFI = 0.003, ΔRMSEA = −0.002). Thus, we concluded that CMV was not a concern in this data set. 5.1. Measurement Model Evaluation The measurement model evaluation mainly included three parts, namely reliability analysis, convergent validity, and discriminant validity. Reliability is usually examined by using internal consistency reliability and composite reliability. As shown in , the coefficient alpha for the eight variables scored in the range from 0.800 to 0.907 (more than 0.70 ). The composite reliability (CR) of all constructs was above 0.70 . The reliability of the model was achieved. Convergent validity is usually examined by using composite reliability (CR) and average variance extracted (AVE). As shown in , all items load significantly on their respective constructs and none of the loadings is below the cutoff value of 0.70. AVE of each variable was more than 0.50 . Thus, convergent validity was achieved. Discriminant validity is analyzed to examine whether a measurement is not a reflection of any other measurement or not. As shown in , the square root of AVE for each variable is greater than the other correlation coefficients. The discriminant validity of the model is achieved. 5.2. Structural Model Evaluation The research model was tested by using AMOS 23, and a bootstrapping resampling procedure (2000 samples) was used to ensure the solidity. The resulting indices indicated a good model fit (x2/df = 1.318; GFI = 0.903; RMSEA = 0.035; NFI = 0.909; CFI = 0.976; TLI = 0.972; RMR = 0.071; SRMR = 0.058). shows that social interaction (SI), informativeness (IN), and relevance (RE) of health-related short-video ads were significant and positive predictors of perceive usefulness of ads (β = 0.23, p < 0.01; β = 0.22, p < 0.01; β = 0.20, p < 0.01). These findings support H1, H3, and H4, respectively, and this supports previous studies . Similarly, intrusiveness (IR) of health-related short-video ads was a significant and negative predictor of the perceive usefulness of ads (β = −0.21, p < 0.01), and H2 is validated. This is consistent with previous research . These four factors (SI, IR, IN, and RE) explained 29% of the variance in the perceived usefulness of health-related short-video ads. Additionally, comparing these four coefficients, we found social interaction was more important to users’ perceived usefulness of ads. Users’ perceived usefulness of health-related short-video ads positively affected their attitudes toward ads (β = 0.59, p < 0.001) and purchase intention (β = 0.21, p < 0.05); these results validate H5 and H6. Users’ perceived ease of use of health-related short-video ads was a significant and positive predictor of perceived usefulness of ads (β = 0.27, p < 0.001), and H7 is validated. However, users’ perceived ease of use of health-related short-video ads could not significantly predict users’ attitudes toward ads. H8 is not validated and this is not consistent with Lin and Kim’ research, as they found users’ perceived ease of use of social media ads significantly predict users’ attitudes . This enlightens us that perceived ease of use may not be an important factor in health-related short-video ads. Therefore, only users’ perceived usefulness of ads explained 37% of the variance in attitudes toward ads. Finally, users’ attitudes of health-related short-video ads were found to positively predict their purchase intentions (β = 0.23, p < 0.05), and H9 is validated. Users’ perceived usefulness of ads and attitudes toward ads accounted for 33% of the variance in purchase intentions. 5.3. Mediating Effects Analysis Mediation analyses were tested using the Preacher and Hayes’ bootstrapping (2000 bootstrap samples) analysis with the PROCESS macro . We focused on two mediate variables (perceived usefulness and ad attitudes) to find the impact path of user responses to health-related short-video advertisements because users’ perceived ease of use of health-related short-video ads cannot significantly predict users’ attitudes toward ads. The results are shown in . The mediating path (Social Interaction→PU→AT) tested the specific indirect effect of social interaction of ads on ad attitudes through perceived usefulness of ads. As both the upper and lower limits of the confidence interval (CI) were above zero (0.014, 0.166), this indirect effect can be interpreted as significantly positive. Similarly, the direct effect of the perceived usefulness of ads can be interpreted as significantly positive. Hence, the perceived usefulness of ads served as a partial mediating variable between social interaction and ad attitudes. That means those who perceived the sociality of health-related short-video ads believed that the ad was more useful, and this increased usefulness enhances the viewers’ positive attitudes toward the ad. The mediating path (Intrusiveness→PU→AT) tested the specific indirect effect of intrusiveness of ads on ad attitudes through perceived usefulness of ads. As both the upper and lower limits of the confidence interval (CI) were above zero (−0.134, −0.018), this indirect effect can be interpreted as significantly positive. The perceived usefulness of ads served as a partial mediating variable between intrusiveness and ad attitudes. That means those who perceived intrusiveness of health-related short-video ads believed that the ad was less useful, and this decreased usefulness enhances the viewers’ negative attitudes toward the ad. The mediating path (Informativeness→PU→AT) tested the specific indirect effect of informativeness of ads on ad attitudes through perceived usefulness of ads. Perceived usefulness of ads served as a partial mediating variable between informativeness and ad attitudes (CI: 0.011, 0.164). That means those who perceived informativeness of health-related short-video ads believed that the ad was more useful, and this increased usefulness enhances the viewers’ positive attitudes toward the ad. The mediating path (PEOU→PU→AT) tested the specific indirect effect of perceived ease of use of ads on ad attitudes through perceived usefulness of ads. Perceived usefulness of ads served as a partial mediating variable between perceived ease of use and ad attitudes (CI: 0.024, 0.188). That means those who perceived ease of use of health-related short-video ads believed that the ad was more useful, and this increased usefulness enhances the viewers’ positive attitudes toward the ad. Similarly, the perceived usefulness of ads served as a partial mediating variable between perceived ease of use and purchase intentions (CI: 0.018, 0.195). That means those who perceived ease of use of health-related short-video ads believed that the ad was more useful, and this increased usefulness enhances the viewers’ purchase intentions. Finally, the mediating path (PU→AT→PI) tested the specific indirect effect of perceived usefulness of ads on purchase intention through ad attitudes. The indirect effect was significant (CI: 0.026, 0.368) while the direct effect was not significant (CI: −0.007, 0.511). Hence, ad attitudes served as a full mediating variable between perceived usefulness and purchase intention. That means users’ attitude toward health-related short-video ads was a key path to the effectiveness of ads. In summary, there were four impact paths of user responses to health-related short-video advertisements. First, social interaction had an indirect effect on purchase intentions through perceived usefulness and ad attitudes in serial (SI→PU→AT→PI). Second, intrusiveness had an indirect effect on purchase intentions through perceived usefulness and ad attitudes in serial (IR→PU→AT→PI). Third, informativeness had an indirect effect on purchase intentions through perceived usefulness and ad attitudes in serial (IN→PU→AT→PI). Fourth, perceived ease of use had an indirect effect on purchase intentions through perceived usefulness and ad attitudes in serial (PE→PU→AT→PI). 5.4. Summary of Hypothesis Validation By combing the results described in –5.3, we present the summary of the hypothesis validation in . The validation-result column in shows the final validation results of each hypothesis, from which we can see that H1-H4 are well established. These hypotheses correspond with the research questions RQ1 and RQ2 presented in . This implies that five qualities (social interaction, intrusiveness, informativeness, relevance, and perceived ease of use) of health-related short-video ads help enhance the perceived usefulness of ads. Among all the factors, according to different coefficients, social interaction of health-related short-video ads made a more valuable contribution to advertising usefulness. Thus, advertisers and marketers should not only focus on social interaction, intrusiveness, informativeness, relevance, and the perceived ease of use of health-related short-video ads, but also pay more attention to social interaction. Regarding hypotheses H5, H7, and H8, which aim to answer RQ3, only the perceived usefulness of health-related short-video ads helped produce positive user attitudes. H9 answers RQ4, finding that positive user attitudes significantly promotef purchase intentions. Combined with the analysis of the mediating effects in , we found four impact paths of user responses to health-related short-video advertisements (SI→PU→AT→PI; IR→PU→AT→PI; IN→PU→AT→PI; PE→PU→AT→PI). This answers RQ5. Thus, advertisers and marketers may maximize resource utilization through the impact paths.
The measurement model evaluation mainly included three parts, namely reliability analysis, convergent validity, and discriminant validity. Reliability is usually examined by using internal consistency reliability and composite reliability. As shown in , the coefficient alpha for the eight variables scored in the range from 0.800 to 0.907 (more than 0.70 ). The composite reliability (CR) of all constructs was above 0.70 . The reliability of the model was achieved. Convergent validity is usually examined by using composite reliability (CR) and average variance extracted (AVE). As shown in , all items load significantly on their respective constructs and none of the loadings is below the cutoff value of 0.70. AVE of each variable was more than 0.50 . Thus, convergent validity was achieved. Discriminant validity is analyzed to examine whether a measurement is not a reflection of any other measurement or not. As shown in , the square root of AVE for each variable is greater than the other correlation coefficients. The discriminant validity of the model is achieved.
The research model was tested by using AMOS 23, and a bootstrapping resampling procedure (2000 samples) was used to ensure the solidity. The resulting indices indicated a good model fit (x2/df = 1.318; GFI = 0.903; RMSEA = 0.035; NFI = 0.909; CFI = 0.976; TLI = 0.972; RMR = 0.071; SRMR = 0.058). shows that social interaction (SI), informativeness (IN), and relevance (RE) of health-related short-video ads were significant and positive predictors of perceive usefulness of ads (β = 0.23, p < 0.01; β = 0.22, p < 0.01; β = 0.20, p < 0.01). These findings support H1, H3, and H4, respectively, and this supports previous studies . Similarly, intrusiveness (IR) of health-related short-video ads was a significant and negative predictor of the perceive usefulness of ads (β = −0.21, p < 0.01), and H2 is validated. This is consistent with previous research . These four factors (SI, IR, IN, and RE) explained 29% of the variance in the perceived usefulness of health-related short-video ads. Additionally, comparing these four coefficients, we found social interaction was more important to users’ perceived usefulness of ads. Users’ perceived usefulness of health-related short-video ads positively affected their attitudes toward ads (β = 0.59, p < 0.001) and purchase intention (β = 0.21, p < 0.05); these results validate H5 and H6. Users’ perceived ease of use of health-related short-video ads was a significant and positive predictor of perceived usefulness of ads (β = 0.27, p < 0.001), and H7 is validated. However, users’ perceived ease of use of health-related short-video ads could not significantly predict users’ attitudes toward ads. H8 is not validated and this is not consistent with Lin and Kim’ research, as they found users’ perceived ease of use of social media ads significantly predict users’ attitudes . This enlightens us that perceived ease of use may not be an important factor in health-related short-video ads. Therefore, only users’ perceived usefulness of ads explained 37% of the variance in attitudes toward ads. Finally, users’ attitudes of health-related short-video ads were found to positively predict their purchase intentions (β = 0.23, p < 0.05), and H9 is validated. Users’ perceived usefulness of ads and attitudes toward ads accounted for 33% of the variance in purchase intentions.
Mediation analyses were tested using the Preacher and Hayes’ bootstrapping (2000 bootstrap samples) analysis with the PROCESS macro . We focused on two mediate variables (perceived usefulness and ad attitudes) to find the impact path of user responses to health-related short-video advertisements because users’ perceived ease of use of health-related short-video ads cannot significantly predict users’ attitudes toward ads. The results are shown in . The mediating path (Social Interaction→PU→AT) tested the specific indirect effect of social interaction of ads on ad attitudes through perceived usefulness of ads. As both the upper and lower limits of the confidence interval (CI) were above zero (0.014, 0.166), this indirect effect can be interpreted as significantly positive. Similarly, the direct effect of the perceived usefulness of ads can be interpreted as significantly positive. Hence, the perceived usefulness of ads served as a partial mediating variable between social interaction and ad attitudes. That means those who perceived the sociality of health-related short-video ads believed that the ad was more useful, and this increased usefulness enhances the viewers’ positive attitudes toward the ad. The mediating path (Intrusiveness→PU→AT) tested the specific indirect effect of intrusiveness of ads on ad attitudes through perceived usefulness of ads. As both the upper and lower limits of the confidence interval (CI) were above zero (−0.134, −0.018), this indirect effect can be interpreted as significantly positive. The perceived usefulness of ads served as a partial mediating variable between intrusiveness and ad attitudes. That means those who perceived intrusiveness of health-related short-video ads believed that the ad was less useful, and this decreased usefulness enhances the viewers’ negative attitudes toward the ad. The mediating path (Informativeness→PU→AT) tested the specific indirect effect of informativeness of ads on ad attitudes through perceived usefulness of ads. Perceived usefulness of ads served as a partial mediating variable between informativeness and ad attitudes (CI: 0.011, 0.164). That means those who perceived informativeness of health-related short-video ads believed that the ad was more useful, and this increased usefulness enhances the viewers’ positive attitudes toward the ad. The mediating path (PEOU→PU→AT) tested the specific indirect effect of perceived ease of use of ads on ad attitudes through perceived usefulness of ads. Perceived usefulness of ads served as a partial mediating variable between perceived ease of use and ad attitudes (CI: 0.024, 0.188). That means those who perceived ease of use of health-related short-video ads believed that the ad was more useful, and this increased usefulness enhances the viewers’ positive attitudes toward the ad. Similarly, the perceived usefulness of ads served as a partial mediating variable between perceived ease of use and purchase intentions (CI: 0.018, 0.195). That means those who perceived ease of use of health-related short-video ads believed that the ad was more useful, and this increased usefulness enhances the viewers’ purchase intentions. Finally, the mediating path (PU→AT→PI) tested the specific indirect effect of perceived usefulness of ads on purchase intention through ad attitudes. The indirect effect was significant (CI: 0.026, 0.368) while the direct effect was not significant (CI: −0.007, 0.511). Hence, ad attitudes served as a full mediating variable between perceived usefulness and purchase intention. That means users’ attitude toward health-related short-video ads was a key path to the effectiveness of ads. In summary, there were four impact paths of user responses to health-related short-video advertisements. First, social interaction had an indirect effect on purchase intentions through perceived usefulness and ad attitudes in serial (SI→PU→AT→PI). Second, intrusiveness had an indirect effect on purchase intentions through perceived usefulness and ad attitudes in serial (IR→PU→AT→PI). Third, informativeness had an indirect effect on purchase intentions through perceived usefulness and ad attitudes in serial (IN→PU→AT→PI). Fourth, perceived ease of use had an indirect effect on purchase intentions through perceived usefulness and ad attitudes in serial (PE→PU→AT→PI).
By combing the results described in –5.3, we present the summary of the hypothesis validation in . The validation-result column in shows the final validation results of each hypothesis, from which we can see that H1-H4 are well established. These hypotheses correspond with the research questions RQ1 and RQ2 presented in . This implies that five qualities (social interaction, intrusiveness, informativeness, relevance, and perceived ease of use) of health-related short-video ads help enhance the perceived usefulness of ads. Among all the factors, according to different coefficients, social interaction of health-related short-video ads made a more valuable contribution to advertising usefulness. Thus, advertisers and marketers should not only focus on social interaction, intrusiveness, informativeness, relevance, and the perceived ease of use of health-related short-video ads, but also pay more attention to social interaction. Regarding hypotheses H5, H7, and H8, which aim to answer RQ3, only the perceived usefulness of health-related short-video ads helped produce positive user attitudes. H9 answers RQ4, finding that positive user attitudes significantly promotef purchase intentions. Combined with the analysis of the mediating effects in , we found four impact paths of user responses to health-related short-video advertisements (SI→PU→AT→PI; IR→PU→AT→PI; IN→PU→AT→PI; PE→PU→AT→PI). This answers RQ5. Thus, advertisers and marketers may maximize resource utilization through the impact paths.
6.1. Research Implications (1) The study of this paper is based on the background of online ads in the health-related short video, which has become an important field for the expansion of social media and a part of people’s daily life. Due to the value of short-video ads, it is necessary to explore user responses to short-video advertisements. We propose a research model based on the integration of the TAM model, social interaction, intrusiveness, informativeness, and relevance. This model augments the application of the widely used TAM model and offers referential values for other related researches. In addition, we present empirical results on user acceptance of participating in health-related short-video ads. These results can provide new research insights for advancing health-related short-video advertisements, e.g., a socially interactive mechanism for mobile ads. (2) This paper studies users’ purchase intention towards health-related short-video ads and is valuable for advertisers and marketers to realize the importance of developing health-related short-video advertisements. With the rapid development of technologies and social entertainment, users’ attitudes toward online ads may have changed a lot. Short video has been a promising tool for seeking business opportunities and establishing brand expression, and advertisers need to keep reforming their advertising strategy to meet user needs. (3) According to the empirical study conducted in this paper, the five factors (social interaction, intrusiveness, informativeness, relevance, and perceived ease of use) defined in the research model are helpful to enhance the perceived usefulness of health-related short-video ads, which can in turn affect user acceptance of health-related short-video ads. The results indicated that social interaction, informativeness, and relevance of advertisements are all positive factors, while intrusiveness is a negative factor, meaning that the increasing of intrusiveness will lower the user acceptance of health-related short-video ads. These results are consistent to what were revealed by previous studies . Furthermore, compared with other factors, social interaction showed a higher impact to the perceived usefulness of short-video ads. This is a new finding of the study. While advertisers should be mindful of multiple aspects of ads, including intrusiveness, informativeness, relevance, and perceived ease of use, they should largely focus their efforts on the social interactions that stem from these ads. (4) We also find that users’ perceived ease of use of health-related short-video ads cannot significantly predict users’ attitudes toward short-video ads. This does not support Lin and Kim’ research conclusions , i.e., users’ perceived ease of use of social media ads significantly predict users’ attitudes toward ads. To this end, the perceived ease of use may not be an important factor in health-related short-video ads. (5) We find four impact paths of user responses to health-related short-video advertisements (SI→PU→AT→PI; IR→PU→AT→PI; IN→PU→AT→PI; PE→PU→AT→PI) by mediating effects analysis. Thus, advertisers and marketers can maximize resource utilization through the impact paths and promote the effectiveness of health-related short-video ads. 6.2. Suggestions The empirical analysis of this study can offer some useful hints for advertisers to advance short-video ads. Below, we present some suggestions for advertisers and marketers to better develop health-related short-video ads: (1) Consider various factors when promoting short-video ads. The results of this study show that the five factors (social interaction, intrusiveness, informativeness, relevance, and perceived ease of use) defined in the model can all positively affect the perceived usefulness of short-video ads. Thus, we suggest that advertisers should consider all five factors rather than focusing on one or some specific factors. For example, if advertisers neglect the ‘relevance’ factor, they might issue short-video ads in which the video content is irrelevant to the topic of the ads. (2) Emphasize the social interactions of short-video ads. Our study shows that social interaction is more influential to the perceived usefulness of short-video ads compared with other factors. Thus, we suggest advertisers to pay more attention to the social interactions of short-video ads, which is a shortcoming of current short-video ads. As shown in , current short-video ads provide few ways for social interaction. They are much like an introduction of a product, which neglect the use of social interaction. However, the results of this study show that social interaction is a key construct for enhance user acceptance of short-video ads. Therefore, we strongly suggest advertisers to design social-interaction tools in short-video ads. For example, they can embed a voting button in a video by using new technologies like HTML5 to let video viewers submit their choices. (3) Focus on users’ perceived usefulness of short-video ads. Through the mediating factor analysis, we found that users’ perceived usefulness of short-video ads plays an important role in the impact path from the influential factors to users’ purchase intention. Therefore, enhancing users’ perceived usefulness of short-video ads is the key point of improving the effectiveness of short-video ads. On the one hand, advertisers should improve the quality of short-video ads, e.g., by increasing the informativeness and relevance of ads, or by providing new kinds of social interactions to attract users’ attention. On the other hand, advertisers should avoid issuing any false advertisements, which are much harmful to consumers’ confidence on products. Once users find that the short-video ads issued by an advertiser are over-claimed or false, the fast spreading of word-of-mouth on social network platforms will do considerable harm to the reputation of the advertiser. (4) Advance ads on short-video social network platforms. Short videos provide a free, real-time, and easy-to-access online way for enterprises to deliver ads to potential users. Recently, the number of the users on short-video social network platforms has shown a dramatical increasing trend, because live videos provide more attractive presentation than conventional textual web pages or tweets. Thus, it is important for enterprises to develop their short-video-based advertising systems and departments to advance ads on short-video social network platforms. Accordingly, the live short video as a new style of advertising could be utilized as a new tool to enhance the market competitiveness of enterprises.
(1) The study of this paper is based on the background of online ads in the health-related short video, which has become an important field for the expansion of social media and a part of people’s daily life. Due to the value of short-video ads, it is necessary to explore user responses to short-video advertisements. We propose a research model based on the integration of the TAM model, social interaction, intrusiveness, informativeness, and relevance. This model augments the application of the widely used TAM model and offers referential values for other related researches. In addition, we present empirical results on user acceptance of participating in health-related short-video ads. These results can provide new research insights for advancing health-related short-video advertisements, e.g., a socially interactive mechanism for mobile ads. (2) This paper studies users’ purchase intention towards health-related short-video ads and is valuable for advertisers and marketers to realize the importance of developing health-related short-video advertisements. With the rapid development of technologies and social entertainment, users’ attitudes toward online ads may have changed a lot. Short video has been a promising tool for seeking business opportunities and establishing brand expression, and advertisers need to keep reforming their advertising strategy to meet user needs. (3) According to the empirical study conducted in this paper, the five factors (social interaction, intrusiveness, informativeness, relevance, and perceived ease of use) defined in the research model are helpful to enhance the perceived usefulness of health-related short-video ads, which can in turn affect user acceptance of health-related short-video ads. The results indicated that social interaction, informativeness, and relevance of advertisements are all positive factors, while intrusiveness is a negative factor, meaning that the increasing of intrusiveness will lower the user acceptance of health-related short-video ads. These results are consistent to what were revealed by previous studies . Furthermore, compared with other factors, social interaction showed a higher impact to the perceived usefulness of short-video ads. This is a new finding of the study. While advertisers should be mindful of multiple aspects of ads, including intrusiveness, informativeness, relevance, and perceived ease of use, they should largely focus their efforts on the social interactions that stem from these ads. (4) We also find that users’ perceived ease of use of health-related short-video ads cannot significantly predict users’ attitudes toward short-video ads. This does not support Lin and Kim’ research conclusions , i.e., users’ perceived ease of use of social media ads significantly predict users’ attitudes toward ads. To this end, the perceived ease of use may not be an important factor in health-related short-video ads. (5) We find four impact paths of user responses to health-related short-video advertisements (SI→PU→AT→PI; IR→PU→AT→PI; IN→PU→AT→PI; PE→PU→AT→PI) by mediating effects analysis. Thus, advertisers and marketers can maximize resource utilization through the impact paths and promote the effectiveness of health-related short-video ads.
The empirical analysis of this study can offer some useful hints for advertisers to advance short-video ads. Below, we present some suggestions for advertisers and marketers to better develop health-related short-video ads: (1) Consider various factors when promoting short-video ads. The results of this study show that the five factors (social interaction, intrusiveness, informativeness, relevance, and perceived ease of use) defined in the model can all positively affect the perceived usefulness of short-video ads. Thus, we suggest that advertisers should consider all five factors rather than focusing on one or some specific factors. For example, if advertisers neglect the ‘relevance’ factor, they might issue short-video ads in which the video content is irrelevant to the topic of the ads. (2) Emphasize the social interactions of short-video ads. Our study shows that social interaction is more influential to the perceived usefulness of short-video ads compared with other factors. Thus, we suggest advertisers to pay more attention to the social interactions of short-video ads, which is a shortcoming of current short-video ads. As shown in , current short-video ads provide few ways for social interaction. They are much like an introduction of a product, which neglect the use of social interaction. However, the results of this study show that social interaction is a key construct for enhance user acceptance of short-video ads. Therefore, we strongly suggest advertisers to design social-interaction tools in short-video ads. For example, they can embed a voting button in a video by using new technologies like HTML5 to let video viewers submit their choices. (3) Focus on users’ perceived usefulness of short-video ads. Through the mediating factor analysis, we found that users’ perceived usefulness of short-video ads plays an important role in the impact path from the influential factors to users’ purchase intention. Therefore, enhancing users’ perceived usefulness of short-video ads is the key point of improving the effectiveness of short-video ads. On the one hand, advertisers should improve the quality of short-video ads, e.g., by increasing the informativeness and relevance of ads, or by providing new kinds of social interactions to attract users’ attention. On the other hand, advertisers should avoid issuing any false advertisements, which are much harmful to consumers’ confidence on products. Once users find that the short-video ads issued by an advertiser are over-claimed or false, the fast spreading of word-of-mouth on social network platforms will do considerable harm to the reputation of the advertiser. (4) Advance ads on short-video social network platforms. Short videos provide a free, real-time, and easy-to-access online way for enterprises to deliver ads to potential users. Recently, the number of the users on short-video social network platforms has shown a dramatical increasing trend, because live videos provide more attractive presentation than conventional textual web pages or tweets. Thus, it is important for enterprises to develop their short-video-based advertising systems and departments to advance ads on short-video social network platforms. Accordingly, the live short video as a new style of advertising could be utilized as a new tool to enhance the market competitiveness of enterprises.
In this paper, we analyze the major factors that influence user responses to health-related short-video advertisements. Particularly, we made three contributions. First, we extended the Technology Acceptance Model (TAM) with new antecedents including social interaction (SI), intrusiveness (IR), informativeness (IN), and relevance (RE) to analyze the users’ response to health-related short-video ads. We also introduced two mediate factors, i.e., perceived usefulness (PU) and ad attitude (AT), to reflect the influence of independent variables on the dependent variable named purchase intention (PI). To the best of our knowledge, this is the first study that extends the TAM model to analyze user acceptance of health-related short-video ads. Second, we conducted a survey on the Internet and short-video social network platforms and performed systematical data analysis over the surveyed data. The data analysis consisted of many aspects, including measurement model evaluation, structural model evaluation, and mediating effect analysis. The results showed that social interaction, intrusiveness, informativeness, relevance, and the TAM factors (perceived usefulness and perceived ease of use) had a significant influence on user acceptance of health-related short-video ads. Further, we found that users’ perceived ease of use on health-related short-video ads could not significantly predict users’ attitudes toward ads. This is a new finding that is contrary to previous studies in social media-oriented ads. Third, we presented reasonable suggestions for advertisers and marketers to better develop health-related short-video ads. This study is beneficial for advertisers to realize the importance of understanding the effectiveness of health-related short-video ads. Base on the empirical results of this study, we gained some new findings, some of which are contrary to existing research. These findings are helpful to reveal the major factors that influence users’ ad attitudes and intentions to purchase and to quantify the impacts of these factors on them. The suggestions made based on the empirical results of this study can provide some management ideas for advertisers and academia to develop health-related short-video ads. Some limitations of this study can be summarized as follows. First, although hundreds of valid questionnaires are theoretically enough to conduct data analysis, in this big data era, this number is relatively small to draw reliable and robust conclusions. Second, the results of this study have shown that the four antecedents, as well as the perceived ease of use, have significant influences on perceived usefulness, attitude, and purchase intention towards mobile live ads. However, the inherent theoretical basis has not been revealed yet. Third, the survey in this study has some limitations. Currently, we are only able to survey people on a Chinese short-video platform. The model, as well as the results, may not extend to people in other countries or on other platforms. In addition, as the respondents in the survey mainly cover young people (79.8% of them were under 30 years old), the results may not suit those advertisements that target old people. Thus, in the future, there are some research issues that are worth further investigating. First, a further study on secondary data collected from some crowdsourcing platforms like Amazon Mechanical Turk could be better to analyze user acceptance of advertising and marketing on short-video platforms. Second, because users’ decision-making behavior could be impacted by other factors, future work can be focused on other possible factors, such as users’ educational background and online experiences. Finally, in addition to the TAM model, it is also worth studying other research models within the big data and online community context .
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Proteomic Changes Associated With Endogenous | 1f586ae5-031c-499b-a245-bd11fc268c07 | 11909011 | Biochemistry[mh] | Introduction Endometrial cancer (EC) is currently the fifth leading cause of cancer death among women in the United States (US) , and by 2040 it is expected to become the 4th leading cause of cancer death for US women . Endometrioid ECs (EECs) are the most frequently diagnosed histological subtype, representing approximately 80% of new diagnoses and causing approximately 50% of EC deaths. The majority of non‐endometrioid tumors are serous, clear cell, or metaplastic ECs (uterine carcinosarcomas). Endometrioid and serous ECs segregate across four prognostically distinct molecular subgroups in The Cancer Genome Atlas (TCGA): copy number high/serous‐like, microsatellite instability (MSI)/hypermutated, copy number low/microsatellite stable (MSS), and POLE/ultramutated (; reviewed in ). The POLE/ultramutated subgroup had the best prognosis, and the copy number high/serous‐like subgroup had the worst prognosis . The FBXW7 tumor suppressor protein is a subunit of a ubiquitin ligase complex that regulates proteolytic and non‐proteolytic ubiquitination of substrate proteins . The functional domains of FBXW7 include a C‐terminal WD repeat region that mediates FBXW7‐substrate binding. FBXW7 is one of the most frequently mutated genes in EC (reviewed in ). Somatic mutations in FBXW7 occur in 10%–12% of endometrioid, 15%–29% of serous, 10%–39% of clear cell, and 13%–25% of metaplastic ECs (; and reviewed in ). The R465, R479, and R505 residues within the WD repeats mediate binding of FBXW7 to proteins targeted for ubiquitination (reviewed in ), and are mutation hotspots in EC and in some other tumor types . Studies in other cancer cell types have shown that FBXW7 hotspot mutants at residues R465 and R479 are dominant‐negative mutants that fail to bind substrate proteins, resulting in elevated substrate levels . Despite the frequent occurrence of FBXW7 mutations in EC and the pathogenicity of Fbxw7 deletion in a genetically engineered mouse model of uterine cancer , little is known regarding the functional consequences of FBXW7 mutations in the context of human EC cells. Because many FBXW7 substrates are oncoproteins that regulate gene transcription or signal transduction, we hypothesized that FBXW7 mutations in EC will result in altered levels of a myriad of proteins and post‐translationally modified proteins, including phosphoproteins. We recently reported the proteomic and phosphoproteomic effects of knocking in the FBXW7‐R465, ‐R479, or ‐R505 mutations into serous and high‐grade EC cells . Here, we aimed to determine the proteomic and phosphoproteomic effects of endogenous FBXW7 mutations in EC cells. Using liquid chromatography–tandem mass spectrometry (LC–MS/MS), we profiled the proteomes and phosphoproteomes of two moderately differentiated EC cell lines (HEC‐1‐B FBXW7−R367X and JHUEM‐1 FBXW7 − R505C ) and isogenic mutation‐corrected cell lines that we previously generated by CRISPR‐Cas9 editing . We identified 98 total proteins at significantly ( p < 0.05) different levels in both HEC‐1‐B FBXW7−R367X and JHUEM‐1 FBXW7 − R505C cells as compared with the isogenic mutation‐corrected cells. Intriguingly, we found that both of the endogenously mutated EC cell lines had significantly increased levels of TROP2 (Trophoblast Cell Surface Antigen 2), a druggable protein targeted by the antibody‐drug conjugate Sacituzumab Govitecan (IMMU‐132), which is currently being evaluated in a phase II clinical trial for the treatment of persistent or recurrent EC ( https://clinicaltrials.gov/study/NCT04251416 ) (See interim results at https://ascopubs.org/doi/abs/10.1200/JCO.2020.38.15_suppl.6081 ).
Results A comparison of mass spectrometry‐detected protein levels in HEC‐1‐B FBXW7−R367X parental cells to those in isogenic mutation‐corrected cells revealed significantly different ( p < 0.05) levels of 171 total proteins and 717 phosphorylated peptides (corresponding to 475 unique phosphorylated proteins), all of which were reproducibly altered in triplicate lysate preparations (Figure , Tables and ). Among total proteins with significantly different levels in HEC‐1‐B FBXW7−R367X parental cells compared to isogenic mutation‐corrected cells, 17% (29/171) also exhibited significantly different levels of phosphoproteins; considering total and phosphorylated proteins combined, fold change > 5 was observed in 11% (67/617) of proteins (Figure ). The significantly altered proteins and phosphoproteins observed between the HEC‐1‐B cell lines included 18% (17/94) of known and predicted FBXW7 substrates (Figure ), 7% (8/114) of proteins encoded by genes in an expression signature that predicts FBXW7 mutation status in human cancer (Figure ), 5% (22/456) of proteins encoded by orthologs of murine genes differentially expressed following re‐introduction of Fbxw7 into uterine carcinosarcoma cells from Fbxw7 / Pten deficient animals (Figure ), 3% (30/929) of proteins encoded by ubiquitin‐related genes and 4% (4/95) of proteins encoded by deubiquitinating genes (Figure ). Comparing JHUEM‐1 FBXW7−R505C cells to the isogenic mutation‐corrected cells, we found 2193 total proteins (including 2 isoforms for 9 proteins) and 3770 phosphorylated peptides (corresponding to 1926 unique phosphorylated proteins) that exhibited significantly different levels, all of which were reproducibly altered in triplicate lysate preparations (Figure , and Tables and ). Among the total proteins with significantly different levels in JHUEM‐1 FBXW7−R505C cells compared to the isogenic mutation‐corrected cells, 23% (513/2184) also exhibited significantly different levels of phosphorylation; among these proteins, 25.7% (132/513) have a fold change > 5 for the total protein level (Figure ). Proteins and/or phosphoproteins present at significantly different levels between the JHUEM‐1 cell lines included 57% (54/94) of known and predicted FBXW7 substrates (Figure ), and proteins corresponding to 40% (46/114) of genes in an expression signature that predicts FBXW7 mutation status (Figure ), 13% (61/456) of proteins encoded by orthologues of murine genes differentially expressed following re‐introduction of Fbxw7 into uterine carcinosarcoma cells from Fbxw7 / Pten deficient animals (Figure ), 25% (230/929) of ubiquitin‐related genes and 24% (23/95) of deubiquitinating genes (Figure ). We identified 397 phosphorylated and/or total proteins that exhibited significantly different levels in the FBXW7 ‐mutant parental cell line compared to the isogenic mutation‐corrected cell line for both HEC‐1‐B and JHUEM‐1 (Figure ). Ninety‐eight proteins exhibited significantly different total protein levels in both HEC‐1‐B FBXW7−R367X and JHUEM‐1 FBXW7 − R505C , 40 of which exhibited higher levels in the FBXW7 ‐mutant parental lines compared to the isogenic mutation‐corrected lines (Figure ); 40 of the remaining 58 proteins exhibited lower levels in the FBXW7 ‐mutant parental lines compared to the isogenic CRISPR‐edited FBXW7 non‐mutant derivative lines. A majority (22/40) of the proteins exhibiting higher levels in FBXW7 ‐mutant cells annotated to the gene ontology (GO) term vesicle (GO:0031982); the most significantly associated gene ontology terms were extracellular exosome (GO:0070062), extracellular vesicle (GO:1903561) and extracellular organelle (GO:0043230), which were all comprised of the same set of 18 proteins (ASS1, BCAM, BLVRA, CA2, CLN5, DNASE2, GLB1, HEXB, galectin‐3 ( LGALS3 ), LGALS3BP, MYH14, PADI2, PLCG2, PODXL, PPL, SCEL, SLPI, TROP2 ( TACSTD2 )) with significantly different levels in FBXW7 ‐mutant HEC‐1‐B FBXW7−R367X and JHUEM‐1 FBXW7−R505C cells compared to the isogenic mutation‐corrected cell lines (Table ). The gene ontology terms most significantly associated with proteins that exhibited lower levels in FBXW7 ‐mutant cells were cytoskeletal protein binding (GO:0008092), protein localization to secretory granule (GO:0033366), transport vesicle (GO:0030133), and transport vesicle membrane (GO:0030658) (Table ). GO terms that were most significantly associated with the 98 total proteins present at significantly different levels in both FBXW 7‐mutant cell lines were identical protein binding (GO:0042802), gamma‐aminobutyric acid metabolic process (GO:0009448), neurotransmitter catabolic process (GO:0042135), and gamma‐aminobutyric acid catabolic process (GO:0009450) (Table ). Ten significantly altered total proteins that increased in both HEC‐1‐B FBXW7−R367X and JHUEM‐1 FBXW7−R505C mutant lines, compared to the isogenic mutation‐corrected cells, exhibited a normalized fold change of ≥ 30‐fold in JHUEM‐1 FBXW7−R505C cells (Figure ). We used Western blotting to orthogonally assess levels of six of these proteins (TROP2 ( TACSTD2 ), galectin‐3 ( LGALS3 ), ASS1, PLCG2, AGR2, NaPi2b ( SLC34A2 )) for which antibodies were available from Cell Signaling Technology. Possibly due to the small fold change observed for AGR2 and NaPi2b ( SLC34A2 ) in HEC‐1‐B FBXW7‐R367X (Figure ), and the differential sensitivities of LC–MS/MS and Western blotting, we did not observe increased levels of AGR2 (Anterior gradient protein 2 homolog) or NaPi2b (Sodium‐dependent phosphate transport protein 2B) in HEC‐1‐B FBXW7‐R367X cells, compared to isogenic mutation‐corrected cells (Figure ). We confirmed increased levels of the remaining four proteins, namely TROP2 (Trophoblast cell surface antigen 2), galectin‐3, ASS1 (Argininosuccinate synthase 1) and PLCG2 (Phospholipase C gamma 2), in HEC‐1‐B FBXW7−R367X cells and of all six proteins in JHUEM‐1 FBXW7‐R505C cells, compared to isogenic mutation‐corrected lines (Figure , Figures and ).
Materials and Methods 3.1 Ethics Statement The research conducted in this study was excluded from IRB Review per the Common Rule 45 CFR 46 and NIH policy for the use of specimens/data. 3.2 Cell Culture HEC‐1‐B was originally established from a moderately differentiated papillary adenocarcinoma . JHUEM‐1 was established from a grade‐2 EEC (URL: https://cellbank.brc.riken.jp/cell_bank/CellInfo/?cellNo=RCB1548 ). HEC‐1‐B EC cells were acquired from ATCC (HTB‐113) and maintained in EMEM + 10% FBS at 37°C in a humidified atmosphere with 5% CO 2 . JHUEM‐1 EEC cells were purchased from Riken Bioresource Research Center and maintained in DMEM/HamF12 + 15% FBS at 37°C in a humidified atmosphere with 5% CO 2 . Cells were authenticated by Laragen Inc. (Culver City, CA) using short tandem repeat profiling prior to shipment to the Washington University Genome Engineering and iPSC Center (GEIC; St. Louis, MO). All cell lines were verified mycoplasma free by the Washington University GEIC and re‐authenticated by Laragen Inc. (Culver City, CA) at the time frozen stocks were established. Both cell lines were previously inferred to harbor microsatellite instability ; HEC‐1‐B was reported to have an FBXW7 deep deletion, while JHUEM‐1 had no detectable FBXW7 copy number aberration . 3.3 Generation of CRISPR ‐Edited FBXW7 Non‐Mutated Cell Lines HEC‐1‐B and JHUEM‐1 cells were CRISPR‐edited by the GEIC at Washington University, St. Louis, to correct the endogenous FBXW7 c.C1099T (p.R367X) and c.C1513T (p.R505C) mutations, respectively, following methods published previously . 3.4 DNA Extraction, Polymerase Chain Reaction ( PCR ) Amplification and Sanger Sequencing DNA was extracted using the Gentra Puregene Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. Verification of single mutations and wildtype status of all remaining coding exons of FBXW7 in JHUEM‐1 and HEC‐1‐B parental cells and wildtype status of all FBXW7 coding exons of CRISPR‐edited derivative lines (Figure ) was verified using FBXW7 primers , PCR conditions, purification, and Sanger sequencing exactly as previously reported . 3.5 Lysate Preparation and Liquid Chromatography Tandem Mass Spectrometry ( LC – MS / MS ) Analysis HEC‐1‐B FBXW7−R367X (passage (P)5), HEC‐1‐B mutation corrected (P5), JHUEM‐1 FBXW7−R505C (P3), and JHUEM‐1 mutation corrected (P5) cells were counted with a Countess Cell Counter (Thermo Fisher Scientific, Waltham, MA), plated into four 150 mm dishes at a density of 4 × 10 6 cells per dish, and scraped into a total of 4 mL urea lysis buffer (Cell Signaling Technology (CST), Danvers, MA) the following day. Triplicate lysates were prepared for each cell line from the same passage number to minimize proteomic variation due to cell age and were immediately frozen on dry ice before shipment to CST. Sonication, centrifugation, reduction, digestion, and purification of cellular extracts and LC–MS/MS analysis were performed exactly as previously described . Also, as previously described , Immobilized Metal Affinity Chromatography (IMAC) enrichment with Fe‐NTA magnetic beads (CST) was used for phosphorylated protein analysis, and labeling with TMT10plex reagent (Thermo Fisher Scientific) was used for total protein analysis. LC–MS/MS analysis was performed using an Orbitrap‐Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) with replicate injections of each sample for the IMAC analysis. An HCD‐MS2 acquisition method was used for phosphopeptide analysis, and an MS3 acquisition method was used for total proteome analysis. Details of acquisition methods have been previously provided . 3.6 Peptide and Protein Identification Mass spectra evaluations and peptide and protein identification were performed by CST exactly as previously described . 3.7 Filtering to Proteins With Significantly Different Levels in FBXW7 ‐Mutated Parental Cells Proteins exhibiting significantly different levels in FBXW7 non‐mutant CRISPR‐edited HEC‐1‐B and JHUEM‐1 cells compared to isogenic FBXW7 ‐mutant parental cells were determined using a previously published filtering scheme . Briefly, total proteins with ≥ ± 2.0 average fold change were filtered to those with significantly ( p < 0.05) different levels in FBXW7 non‐mutant CRISPR‐edited cells compared to isogenic FBXW7 ‐mutant parental cells. p ‐values were based on a two‐tailed t ‐test using the signal:noise for each CRISPR‐edited derivative line versus isogenic parental cells across three replicates. Phosphorylated peptides were filtered using these same criteria with additional filters of maximum abundance > 1,000,000 and percent coefficient of variance (%CV) ≤ 50% for peptides with ≤ 10 average fold change or ≤ 80% for peptides with > 10 average fold change. Unsupervised hierarchical clustering was performed using Partek Genomics Suite. 3.8 Functional Annotation Proteins were functionally annotated using g:Profiler ( RRID :S CR_008653 ) and Ingenuity Pathway Analysis (IPA) (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway‐analysis ). 3.9 Western Blot Analysis For HEC‐1‐B FBXW7−R367X (P8), HEC‐1‐B mutation corrected (P7 or P8), JHUEM‐1 FBXW7−R505C (P11), and JHUEM‐1 mutation corrected (P11) cell lines, protein isolation, quantification, gel electrophoresis, and transfer were performed as previously described , from cells collected 1 day after plating 1.6 × 10 6 cells per 100 mm dish, in RIPA buffer (Thermo Fisher Scientific) containing 1× Protease/Phosphatase inhibitor cocktail (#5872; Cell Signaling Technology). Triplicate lysates were prepared for each cell line from the same passage number to minimize proteomic variation due to cell age. Proteins were subjected to electrophoresis into 10% Mini‐PROTEAN‐TGX gels or 4%–15% gradient Mini‐PROTEAN‐TGX gels (Bio‐Rad Laboratories) and wet transferred to PVDF membranes (Bio‐Rad Laboratories, Hercules, CA). Blots were blocked in tris‐buffered saline containing 0.1% Tween 20 (TBST) + 5% milk and incubated in the following antibodies from Cell Signaling Technology and R&D systems according to their suggested protocols at dilutions of 1:1000: AGR2 (#13062), LGALS3 (#87985), TACSTD2/TROP2 (#47866), PLCG2 (#3872), NaPi2b/SLC34A2 (#66445), ASS1 (#70720). As a loading control, blots were incubated in β‐Actin antibody (A2228; Sigma‐Aldrich, St. Louis MO) overnight at 4°C at a dilution of 1:10,000 in TBST + 5% BSA. Secondary antibodies were Cell Signaling Technology # 7074 and #7076 used at 1:2000 in TBST + 5% milk and incubated for 1–2 h. Proteins were detected using Clarity and Clarity Max Western ECL Substrate (Bio‐Rad Laboratories) prior to film exposure (Carestream Health Inc., Rochester, NY or Agfa Healthcare NV, Mortsel, Belgium) and development using a film processor (Konica Minolta, Ramsey, NJ). To enable probing with multiple antibodies, blots were incubated in Restore Western Blot Stripping Buffer (Thermo Fisher Scientific). All Western blots are representative of a minimum of three lysate replicates.
Ethics Statement The research conducted in this study was excluded from IRB Review per the Common Rule 45 CFR 46 and NIH policy for the use of specimens/data.
Cell Culture HEC‐1‐B was originally established from a moderately differentiated papillary adenocarcinoma . JHUEM‐1 was established from a grade‐2 EEC (URL: https://cellbank.brc.riken.jp/cell_bank/CellInfo/?cellNo=RCB1548 ). HEC‐1‐B EC cells were acquired from ATCC (HTB‐113) and maintained in EMEM + 10% FBS at 37°C in a humidified atmosphere with 5% CO 2 . JHUEM‐1 EEC cells were purchased from Riken Bioresource Research Center and maintained in DMEM/HamF12 + 15% FBS at 37°C in a humidified atmosphere with 5% CO 2 . Cells were authenticated by Laragen Inc. (Culver City, CA) using short tandem repeat profiling prior to shipment to the Washington University Genome Engineering and iPSC Center (GEIC; St. Louis, MO). All cell lines were verified mycoplasma free by the Washington University GEIC and re‐authenticated by Laragen Inc. (Culver City, CA) at the time frozen stocks were established. Both cell lines were previously inferred to harbor microsatellite instability ; HEC‐1‐B was reported to have an FBXW7 deep deletion, while JHUEM‐1 had no detectable FBXW7 copy number aberration .
Generation of CRISPR ‐Edited FBXW7 Non‐Mutated Cell Lines HEC‐1‐B and JHUEM‐1 cells were CRISPR‐edited by the GEIC at Washington University, St. Louis, to correct the endogenous FBXW7 c.C1099T (p.R367X) and c.C1513T (p.R505C) mutations, respectively, following methods published previously .
DNA Extraction, Polymerase Chain Reaction ( PCR ) Amplification and Sanger Sequencing DNA was extracted using the Gentra Puregene Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. Verification of single mutations and wildtype status of all remaining coding exons of FBXW7 in JHUEM‐1 and HEC‐1‐B parental cells and wildtype status of all FBXW7 coding exons of CRISPR‐edited derivative lines (Figure ) was verified using FBXW7 primers , PCR conditions, purification, and Sanger sequencing exactly as previously reported .
Lysate Preparation and Liquid Chromatography Tandem Mass Spectrometry ( LC – MS / MS ) Analysis HEC‐1‐B FBXW7−R367X (passage (P)5), HEC‐1‐B mutation corrected (P5), JHUEM‐1 FBXW7−R505C (P3), and JHUEM‐1 mutation corrected (P5) cells were counted with a Countess Cell Counter (Thermo Fisher Scientific, Waltham, MA), plated into four 150 mm dishes at a density of 4 × 10 6 cells per dish, and scraped into a total of 4 mL urea lysis buffer (Cell Signaling Technology (CST), Danvers, MA) the following day. Triplicate lysates were prepared for each cell line from the same passage number to minimize proteomic variation due to cell age and were immediately frozen on dry ice before shipment to CST. Sonication, centrifugation, reduction, digestion, and purification of cellular extracts and LC–MS/MS analysis were performed exactly as previously described . Also, as previously described , Immobilized Metal Affinity Chromatography (IMAC) enrichment with Fe‐NTA magnetic beads (CST) was used for phosphorylated protein analysis, and labeling with TMT10plex reagent (Thermo Fisher Scientific) was used for total protein analysis. LC–MS/MS analysis was performed using an Orbitrap‐Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) with replicate injections of each sample for the IMAC analysis. An HCD‐MS2 acquisition method was used for phosphopeptide analysis, and an MS3 acquisition method was used for total proteome analysis. Details of acquisition methods have been previously provided .
Peptide and Protein Identification Mass spectra evaluations and peptide and protein identification were performed by CST exactly as previously described .
Filtering to Proteins With Significantly Different Levels in FBXW7 ‐Mutated Parental Cells Proteins exhibiting significantly different levels in FBXW7 non‐mutant CRISPR‐edited HEC‐1‐B and JHUEM‐1 cells compared to isogenic FBXW7 ‐mutant parental cells were determined using a previously published filtering scheme . Briefly, total proteins with ≥ ± 2.0 average fold change were filtered to those with significantly ( p < 0.05) different levels in FBXW7 non‐mutant CRISPR‐edited cells compared to isogenic FBXW7 ‐mutant parental cells. p ‐values were based on a two‐tailed t ‐test using the signal:noise for each CRISPR‐edited derivative line versus isogenic parental cells across three replicates. Phosphorylated peptides were filtered using these same criteria with additional filters of maximum abundance > 1,000,000 and percent coefficient of variance (%CV) ≤ 50% for peptides with ≤ 10 average fold change or ≤ 80% for peptides with > 10 average fold change. Unsupervised hierarchical clustering was performed using Partek Genomics Suite.
Functional Annotation Proteins were functionally annotated using g:Profiler ( RRID :S CR_008653 ) and Ingenuity Pathway Analysis (IPA) (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway‐analysis ).
Western Blot Analysis For HEC‐1‐B FBXW7−R367X (P8), HEC‐1‐B mutation corrected (P7 or P8), JHUEM‐1 FBXW7−R505C (P11), and JHUEM‐1 mutation corrected (P11) cell lines, protein isolation, quantification, gel electrophoresis, and transfer were performed as previously described , from cells collected 1 day after plating 1.6 × 10 6 cells per 100 mm dish, in RIPA buffer (Thermo Fisher Scientific) containing 1× Protease/Phosphatase inhibitor cocktail (#5872; Cell Signaling Technology). Triplicate lysates were prepared for each cell line from the same passage number to minimize proteomic variation due to cell age. Proteins were subjected to electrophoresis into 10% Mini‐PROTEAN‐TGX gels or 4%–15% gradient Mini‐PROTEAN‐TGX gels (Bio‐Rad Laboratories) and wet transferred to PVDF membranes (Bio‐Rad Laboratories, Hercules, CA). Blots were blocked in tris‐buffered saline containing 0.1% Tween 20 (TBST) + 5% milk and incubated in the following antibodies from Cell Signaling Technology and R&D systems according to their suggested protocols at dilutions of 1:1000: AGR2 (#13062), LGALS3 (#87985), TACSTD2/TROP2 (#47866), PLCG2 (#3872), NaPi2b/SLC34A2 (#66445), ASS1 (#70720). As a loading control, blots were incubated in β‐Actin antibody (A2228; Sigma‐Aldrich, St. Louis MO) overnight at 4°C at a dilution of 1:10,000 in TBST + 5% BSA. Secondary antibodies were Cell Signaling Technology # 7074 and #7076 used at 1:2000 in TBST + 5% milk and incubated for 1–2 h. Proteins were detected using Clarity and Clarity Max Western ECL Substrate (Bio‐Rad Laboratories) prior to film exposure (Carestream Health Inc., Rochester, NY or Agfa Healthcare NV, Mortsel, Belgium) and development using a film processor (Konica Minolta, Ramsey, NJ). To enable probing with multiple antibodies, blots were incubated in Restore Western Blot Stripping Buffer (Thermo Fisher Scientific). All Western blots are representative of a minimum of three lysate replicates.
Discussion In the present study, we searched for proteomic and phosphoproteomic changes associated with endogenous FBXW7 mutations in the moderately differentiated EC cell lines, HEC‐1‐B FBXW7 − R367X and JHUEM‐1 FBXW7 − R505C . To more accurately reflect tumor cells in vivo, we utilized unsynchronized cell lines for these experiments. It is conceivable that synchronized cell populations might yield different results for proteins that are present at variable levels throughout the cell cycle. In our experimental conditions, we identified 3809 proteins and/or phosphoproteins that exhibited increased or decreased levels in at least one of the mutated cell lines compared to isogenic mutation‐corrected cells; the levels of 98 total proteins and 270 phosphoproteins were altered in both mutant cell lines. The 98 total proteins included TROP2 ( TACSTD2 ), a protein being clinically evaluated as a druggable target for EC, and galectin‐3 ( LGALS3 ), which is a potentially druggable target. TROP2 is a transmembrane glycoprotein that regulates cell proliferation, migration, and intracellular signal transduction pathways (reviewed in ). Upregulation of this protein has been detected by IHC in 60% of SECs and 84% of EECs . Among endometrioid tumors, strong expression of TROP2 was associated with higher tumor grade and cervical involvement, which are adverse prognosticators . The mechanistic basis for increased TROP2 protein levels in FBXW7 ‐mutated EC cells remains to be determined. One possible mechanism is that the FBXW7‐SKP1‐CUL1 ubiquitin ligase complex regulates TROP2 via ubiquitin‐mediated degradation, and therefore mutations disrupting the FBXW7 WD repeats result in the loss of FBXW7/TROP2 binding and elevated TROP2 levels. Alternatively, FBXW7 substrates, or their downstream effectors, might regulate TROP2 levels either transcriptionally or post‐translationally. In this regard, it is interesting to speculate that galectin‐3 might mediate the effect of FBXW7 on TROP2 levels because galectin‐3 positively regulates TROP2 ( TACSTD2 ) gene expression in colorectal cancer cells and both galectin‐3 and TROP2 exhibited increased levels in the two FBXW7 ‐mutated EC cell lines in our study. Galectin‐3 is a β‐galactosidase binding protein involved in the regulation of apoptosis, cell cycle, cell growth, metastasis, and other cellular processes . Our study is the first to report that increased galectin‐3 protein levels are associated with FBXW7 mutations in EC cells. Dysregulation of this protein has previously been reported in EC, but results were conflicting. Whereas some studies observed galectin‐3 upregulation in EC as compared with control tissues , others observed downregulation or no change in expression . It has been suggested that these contradictory findings might be due to methodological variability across studies . It is also possible that the analysis of unsynchronized cell populations may contribute to the interstudy variability regarding galectin‐3 levels in EC cells. Our results raise the additional possibility that interstudy differences in the FBXW7 mutation status of endometrial tumors may be an additional confounding factor contributing to these contradictory findings. Interestingly, several of the orthogonally validated proteins that exhibited increased levels in one or both cell lines with endogenous FBXW7 mutations in our study are druggable or potentially druggable , including galectin‐3 (; reviewed in ) and TROP2 . A recent preclinical study using serous EC cell lines, xenografts, and patient‐derived organoids showed that galectin‐3 knockout or treatment with a galectin‐3 inhibitor resulted in the reduction of several tumorigenic features, including reduced tumor growth in vivo . Regarding druggability, TROP2 is particularly interesting because sacituzumab govitecan (SG) (an antibody‐drug conjugate that targets TROP2 and which has SN‐38, an active metabolite of irinotecan, as the payload) is currently being evaluated for the treatment of EC. Preclinical studies in EC cells have shown that increased TROP2 levels are a biomarker of sensitivity to killing by SG. Moreover, SG exhibited antitumor activity in TROP2‐positive EC cell line‐derived xenografts . Clinical trials evaluating SG for the treatment of EC have thus far yielded encouraging results ( ClinicalTrials.gov Identifier: NCT04251416) (URL: https://classic.clinicaltrials.gov/ct2/show/NCT04251416 ) . Given our finding that endogenous FBXW7 mutations are associated with increased levels of TROP2 in EC cells, it would be interesting, in future studies, to determine whether FBXW7 mutation status, either alone or in combination with TROP2 protein status, is a molecular correlate of clinical responsiveness of EC patients to SG. The clinical management of ECs is continuing to evolve with increased knowledge of the molecular landscapes of these tumors. Surrogate tests for the four TCGA prognostic subgroups, based on determining POLE , MSI, and p53 status, have been developed and validated (; and reviewed in ). The FIGO staging system for EC was recently revised to include consideration of additional features including molecular subtype based on the results of surrogate testing , although this has been somewhat controversial . It should be noted, however, that although advances have been made in disease risk stratification over the past decade, there are still some challenges to incorporating molecular subtyping of EC into routine clinical practice (reviewed in ). Despite this, a better understanding of the molecular and proteomic landscapes of ECs provides momentum for useful clinical developments that could benefit EC patients. For example, we previously reported that FBXW7‐mutant EC cells exhibit significantly altered levels of L1CAM as compared to their isogenic FBXW7‐wildtype counterparts . L1CAM is being studied for its value as a prognostic biomarker, and overexpression has recently been associated with adverse outcomes . In conclusion, our study provides novel insights into the proteomic and phosphoproteomic effects of endogenous FBXW7 mutations in EC cells, including thousands of significantly altered protein levels as well as increased levels of galectin‐3 and the druggable target TROP2. Future studies to (1) elucidate the mechanisms underlying increased levels of these proteins in FBXW7 ‐mutant cell lines, (2) determine whether the increased levels of TROP2 and galectin‐3 associated with FBXW7 mutations are detectable on EC tissue specimens by immunohistochemistry and whether the combined results are prognostic and/or predictive, and (3) determine whether FBXW7 mutation status is a biomarker of clinical responsiveness to SG are warranted.
Mary Ellen Urick: conceptualization (equal); project administration (equal); investigation (lead); formal analysis (lead); writing‐original draft (equal); writing – review and editing (equal); visualization (lead). Suresh Kumar Chalapareddy: investigation (equal); writing – review and editing (equal); visualization (equal). Eun‐Jeong Yu: investigation (equal); writing – review and editing (equal); visualization (equal). Daphne W. Bell: conceptualization (equal); funding acquisition (lead); project administration (equal); supervision (lead); writing – original draft (equal); writing – review and editing (equal); visualization (equal).
The research conducted in this study was excluded from IRB Review per the Common Rule 45 CFR 46 and NIH policy for the use of specimens/data.
Dr. Mary Ellen Urick, Dr. Suresh Kumar Chalapareddy, and Dr. Eun‐Jeong Yu have no conflicts of interest. Dr. Daphne W. Bell is an inventor on US patent no. 7,294,468, which has been licensed and provides royalty income for work performed outside the current study.
Figure S1. Table S1.
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Metabolic dysfunction-associated fatty liver disease and implications for cardiovascular risk and disease prevention | 0323e1a3-eee0-4de3-810d-f024aca9ff9e | 9719631 | Internal Medicine[mh] | Many Cardiologists are not aware of the increased risk of cardiovascular disease (CVD) among patients with non-alcoholic fatty liver disease (NAFLD) . Whilst Cardiologists pay close attention to traditional CVD risk factors, there is currently little awareness that fatty liver per se may contribute to CVD risk. To date, however, it remains debatable whether screening for fatty liver disease should be given the same priority as other established cardiometabolic risk factors. Although NAFLD is associated with increased CVD risk, routine screening is not recommended in current cardiovascular guidelines. It is reasonable to assume that the lack of clear recommendations for NAFLD screening likely relates to the lack of any effective pharmacotherapies other than lifestyle modification. The lack of awareness of the existing link between NAFLD and increased CVD risk further exacerbates clinical inertia amongst Cardiologists, Primary-care practitioners and non-liver clinician specialists . In 2020, metabolic dysfunction-associated fatty liver disease (MAFLD) was proposed as a more appropriate term than NAFLD, because this nomenclature better defines the pathophysiology of this liver disease and its associated metabolic abnormalities . The proposed change is more than a name change because it affects how clinicians perceive the association of this common liver disease with CVD and metabolic risk. NAFLD is defined as a group of heterogeneous conditions in which there is liver fat accumulation in the absence of secondary causes of hepatic steatosis, such as excessive alcohol consumption, viral hepatitis and other known causes of hepatic steatosis . These “negative” (by exclusion) diagnostic criteria are not appropriate, meaning that NAFLD is only present when all other causes of fatty liver are excluded. In addition, fatty liver disease may coexist with viral hepatitis, excessive alcohol intake or other liver diseases. This renders it difficult for clinicians to make a definitive diagnosis of NAFLD in the face of other potential causes of hepatic steatosis. The term “non-alcoholic” may also confuse patients in terms of the real cause of their disease, which is not conducive to a good therapeutic relationship. Significantly different from NAFLD, MAFLD is defined as a condition characterized by liver fat accumulation in the presence of at least one of the following three metabolic conditions: overweight/obesity, T2DM, or at least two of seven metabolic risk abnormalities in those subjects who do not have T2DM and are lean by ethnic-specific body mass index (BMI) criteria (Fig. ) . The “positive” diagnostic criteria for MAFLD are based on the coexistence of hepatic steatosis and metabolic dysfunction and hence MAFLD may also coexist with other liver diseases. This is not possible when using the NAFLD definition, which requires the exclusion of all other causes of hepatic steatosis as a prerequisite for diagnosis. To date, the newly proposed definition of MAFLD has been accepted by many experts in the field, and by some pan-national societies; although debate is ongoing and there is not uniform agreement . For some experts the change in terminology/definition from NAFLD to MAFLD seems premature and they suggest that such a change could also lead to confusion . In addition, there is not consensus on what constitutes “metabolic health”. That said, taken together, the MAFLD definition better emphasizes the pathogenic role of metabolic dysregulation in the development and progression of this common and burdensome liver disease. Additionally, the inclusion of recognized cardiovascular risk factors within the definition, highlights the need for treatment of these specific coexisting cardiometabolic risk factors. To date, there are few consensus statements about NAFLD or MAFLD published by national or international cardiovascular societies (Fig. ). The first position paper was published by the Indian College of Cardiology in 2015 and raised questions as to whether NAFLD itself may predispose to CVD risk, independent of other common CVD risk factors . In 2022, the American Heart Association (AHA) issued the first scientific statement on NAFLD and CVD risk . This AHA statement highlighted the strong and independent association between NAFLD and increased risk of CVD and sounded the alarm to increase awareness among clinicians, particularly Cardiologists. We are now at the stage where it is germane to consider and understand the emerging relationship between MAFLD and CVD risk from a Cardiologist’s perspective (Table ). This Perspectives article discusses issues related to NAFLD and MAFLD that are of concern for Cardiologists, divided into the following five sections: is the estimated risk of CVD similar when using the NAFLD or MAFLD definitions? Why is MAFLD associated with an increased risk of CVD? What is the role of MAFLD in CVD; is it a bystander or a mediator of CVD? Is routine screening for MAFLD necessary for CVD risk assessment? What is the effect of treatment interventions for MAFLD on the risk of CVD?
Because the overlap between the NAFLD and MAFLD definitions in the general population is reported to be around 70–90%, it is expected that patients with MAFLD have essentially similar CVD risk to those with NAFLD . However, emerging evidence suggests a greater risk of CVD events in patients with MAFLD than in those with NAFLD. Using the National Health and Nutrition Examination Survey (NHANES 1999–2016) database, Zhang et al. reported that patients with MAFLD had a significantly higher 10-year CVD risk (as assessed by the Framingham risk score) compared to those with NAFLD. These data provided the first hint that the CVD risk burden may be greater for MAFLD. Kim et al. analyzed data in 2144 subjects without a prior history of CVD and showed that individuals with MAFLD had a remarkably higher risk of intermediate to high 10-year CVD risk compared to those with NAFLD only (defined as presence of NAFLD but not MAFLD), with an odds ratio (OR) of 8.17 (95% CI 2.40–36.1) in adjusted regression analyses. It is known that the Suita score is a CVD risk prediction tool that may improve CVD risk prediction relative to the Framingham risk score in Japanese individuals . Tsutsumi et al. reported that MAFLD better identified patients at high CVD risk (as estimated by Suita and Framingham risk scores) compared with NAFLD. In a community-based cohort of 6232 participants followed for a median of 4.3 years, Liu et al. reported that MAFLD was associated with a greater risk of developing subclinical atherosclerosis, defined as increased carotid intima-media thickness and plaque, elevated brachial ankle pulse wave velocity, or microalbuminuria. Liu H et al. reported that MAFLD was associated with an increased CVD risk in a cohort of 3306 patients with chronic coronary syndrome. Finally, in a prospective study of nearly 500 hospitalized patients with acute coronary syndromes (ACS) and hepatic steatosis, Noda et al. showed that the coexistence of MAFLD and impaired physical function tests independently predicted the risk of adverse CVD outcomes. Collectively, therefore, accumulating evidence indicates that MAFLD may increase the risk of developing adverse CVD outcomes. A recent large meta-analysis of 17 observational studies (including more than 12 million individuals) also reported that MAFLD is significantly associated with higher risk of overall mortality (hazard ratio (HR) 1.24, 95% confidence interval [CI] 1.13–1.34), CVD mortality (HR 1.28, 95% CI 1.03–1.53), nonfatal CVD events (HR 1.49, 95% CI 1.34–1.64) and stroke (HR: 1.55, 95% CI 1.37–1.73) . Moreover, a matched cohort study, using electronic primary healthcare databases from four European countries, reported that NAFLD appears not to be significantly associated with risk of acute myocardial infarction or stroke after adjustment for common CVD risk factors, (although it should be noted that in this large registry-based study it was not possible to prove that control subjects did not have NAFLD, giving rise to the potential for misclassification bias attenuating the strength of any association between NAFLD and CVD, towards the null) . Additionally, although there are important limitations of Mendelian randomization studies, a recent study did not find evidence supporting the existence of causal associations of NAFLD itself with acute myocardial infarction and any stroke subtypes . In contrast to the criteria necessary for diagnosing NAFLD, MAFLD by definition, is closely associated with T2DM, obesity and atherogenic dyslipidaemia, which are established risk factors for CVD . Recent cohort studies that compared MAFLD-only and NAFLD-only patient populations suggest that the MAFLD-only status is more strongly associated with risk of overall mortality, CVD mortality and nonfatal CVD events, compared with the NAFLD-only status (Fig. ) . In particular, as shown in Fig. A, the MAFLD-only status seems to be more closely associated with a higher risk of nonfatal CVD events. In a retrospective cohort study of 2985 participants followed for 7 years, Niriella et al. reported that the NAFLD-only status was not associated with CVD events compared with control individuals (HR = 1.90, 95% CI = 0.25–14.8) (although it should be noted the CIs were wide and the study may be underpowered), whilst the MAFLD-only status was associated with a greater risk of CVD events compared with control individuals (HR = 7.2, 95% CI = 2.4–21.5). In another study of ~ 9.5 million South Korean subjects from a health screening population, Lee et al. reported that individuals with MAFLD only were at higher risk of CVD events compared with those without MAFLD or NAFLD (HR = 1.43, 95% CI = 1.41–1.45), whereas the association between the NAFLD-only status and risk of CVD events was modest (HR = 1.09, 95% CI = 1.03–1.15). As shown in Fig. B, in the study by Lee et al. , patients with MAFLD were also at higher risk of CVD mortality compared with individuals without MAFLD or NAFLD (HR = 1.46, 95% CI = 1.41–1.52), whereas NAFLD patients were not (HR = 1.12, 95% CI = 0.96–1.30). For all-cause mortality (Fig. C), the difference in CVD risk associated with MAFLD or NAFLD was even more apparent. Kim et al. analyzed data from 7,761 participants in the NHANES-III database and showed that MAFLD was associated with a higher risk of all-cause mortality compared to those without MAFLD or NAFLD (HR = 1.66, 95% CI = 1.19–2.32), whereas NAFLD was not (HR = 0.94, 95% CI = 0.60–1.46). Similarly, Nguyen et al. reported that the MAFLD-only status identified a group of patients with higher all-cause mortality compared with individuals without MAFLD or NAFLD (HR = 2.4, 95% CI = 1.2–4.6), whereas there was no increased risk for all-cause mortality with the NAFLD-only status (HR = 1.5, 95% CI = 0.8–2.8). We recently performed a meta-analysis of seven observational cohort studies (mostly from Asian countries) that examined the comparative effects of NAFLD and MAFLD definitions on risk of CVD events . This meta-analysis showed that each of the two definitions were significantly associated with a higher risk of incident CVD events (pooled random-effects HR 1.50, 95% CI 1.30–1.72 for MAFLD vs. no-MAFLD; and pooled random-effects HR 1.27, 95% CI 1.12–1.45 for NAFLD vs. no-NAFLD, respectively). Although MAFLD identified a numerically greater number of CVD events than NAFLD, the risk for incident CVD events associated with either definition was not significantly different . Collectively, since the MAFLD definition better captures underlying metabolic dysfunction, it is perhaps not surprising that MAFLD definition might also increase CVD risk more strongly than NAFLD definition. However, further cohort studies from different countries are certainly needed to elucidate whether MAFLD may better predict the risk of developing incident CVD events than NAFLD.
There are at least two possible explanations for the increased CVD risk observed in individuals with MAFLD. First, the MAFLD definition has as an obligate requirement for the presence of overweight/obesity, T2DM or other features of the metabolic syndrome, all of which are associated with increased CVD risk. In MAFLD, the presence of T2DM marks the most severe form of metabolic dysfunction and hence has the worst prognosis . Indeed, recent studies have shown that MAFLD patients with T2DM have a worse clinical outcome than their counterparts without T2DM (i.e. MAFLD patients with overweight/obesity, or nondiabetic MAFLD patients with other metabolic risk abnormalities) . Several pathophysiological pathways may link MAFLD and T2DM to an increased CVD risk, including a proatherogenic lipid phenotype, as well as an increase in prothrombotic factors, insulin resistance, low-grade inflammation, and intestinal dysbiosis . Second, the impact of MAFLD on CVD risk may also be affected by other coexisting liver diseases, such as viral hepatitis or moderate alcohol consumption. Whereas it is necessary to always exclude these coexisting liver diseases to establish a diagnosis of NAFLD, this is not necessary for a diagnosis of MAFLD. Indeed, some studies showed that patients with MAFLD and concomitant viral hepatitis or moderate alcohol consumption have a higher 10-year calculated CVD risk compared to those with MAFLD only . That said, MAFLD itself may increase risk of CVD possibly via multiple pathophysiological mechanisms associated with metabolic dysfunction; these include increased oxidative stress, systemic/hepatic insulin resistance, low-grade inflammation and endothelial dysfunction (Fig. ) . Patients with MAFLD exhibit excessive reactive oxygen species (ROS), and ROS overproduction leads to hepatic inflammation and fibrosis, mostly through activation of hepatic stellate cells . ROS overproduction also leads to low-density lipoprotein (LDL)-cholesterol oxidation, which may promote transformation of macrophages into foam cells, which is a key step in the formation of atherosclerotic lesions and atherosclerosis progression. The latter occurs through a variety of pathways, including endothelial cell dysfunction and vascular smooth muscle cell proliferation . Insulin resistance is considered one of the core pathophysiological changes in MAFLD . Insulin resistance promotes hepatic de novo lipogenesis and may affect microvascular and macrovascular homeostasis in a variety of ways to promote atherosclerosis . In addition, previous studies confirmed that chronic hyperglycemia damages vascular endothelial cells, stimulates proliferation of smooth muscle cells, improves platelet activity, and induces ROS overproduction, thus promoting accelerated atherogenesis . Low-grade inflammation also aggravates endothelial dysfunction, changes vascular tone, and promotes vascular plaque formation . All these mechanisms promote the development and progression of CVD including vascular inflammation, lipid deposition, vascular remodeling, endothelial injury and hypercoagulability. Given that MAFLD is defined by the presence of hepatic steatosis plus at least one of its diagnostic cardiometabolic criteria , it is reasonable to hypothesize that there will be a strong mechanistic association between MAFLD and adverse CVD outcomes .
To date, most Cardiologists are not aware that NAFLD (or MAFLD) is a CVD risk factor . In 2015, a position paper published by the Indian College of Cardiology identified the increased CVD risk in patients with NAFLD, but raised some doubts as to whether NAFLD per se may predispose to CVD development. However, since the publication of that position paper, further new data has provided yet more evidence that MAFLD is a CVD risk factor . Interestingly, there is a discrepancy for the risk of CVD outcomes in MAFLD and NAFLD after adjustment for coexisting cardiometabolic risk factors (Fig. ). As shown in Fig. A, in a cohort study of ~ 6.8 million Japanese individuals, Yoneda et al. reported that the risk of CVD events was almost the same (adjusted HR 1.02, 95% CI 0.92–1.14) in the NAFLD and non-NAFLD groups after adjusting for cardiometabolic risk factors. In contrast, after adjusting for the same cardiometabolic risk factors, the risk of CVD was higher in the MAFLD group compared with the non-MAFLD group (adjusted HR 1.89, 95% CI 1.78–2.01). However, there are conflicting data (Fig. B) . In a smaller prospective study, Kim et al. reported a significant association between MAFLD and CVD mortality (HR 2.14, 95% CI 1.71–2.70), but this risk was attenuated after adjusting for cardiometabolic risk factors. Similarly, these authors did not find any association between NAFLD and CVD mortality in adjusted regression analyses. Using the NHANES III database, Huang et al. reported that MAFLD was associated with a greater risk of CVD mortality compared with NAFLD (HR 2.01, 95% CI 1.66–2.44 vs. HR 1.53, 95% CI 1.26–1.86, respectively). However, the increased risk of CVD mortality was attenuated after adjustment for cardiometabolic risk factors. Previous meta-analyses reported that NAFLD was associated with a higher risk of nonfatal CVD events but not CVD mortality . However, the largest updated meta-analysis to date by Mantovani et al. has clearly shown that NAFLD was associated with a higher risk of both nonfatal CVD events (pooled random-effects HR1.40, 95% CI 1.20–1.64) and CVD mortality (pooled random-effects HR 1.30, 95% CI 1.08–1.56), and that this risk was further increased with the severity of NAFLD (especially with higher fibrosis stage). A nationwide Swedish cohort study by Simon et al. provided further evidence of a strong association between the presence and severity of biopsy-proven NAFLD and the risk of CVD mortality. MAFLD is associated with a higher risk of all-cause mortality but this association is attenuated after adjustment for cardiometabolic risk factors (Fig. C) . For example, Kim et al. reported that the association with higher all-cause mortality in MAFLD became non-significant, after adjustment for cardiometabolic risk factors. Huang et al. showed that MAFLD was associated with higher all-cause mortality compared with NAFLD and control subjects, but the associations lost significance after adjustment for cardiometabolic risk factors, in both MAFLD and NAFLD. On the other hand, in a community-based cohort study of 8919 subjects Moon et al. reported that MAFLD significantly predicted the risk of all-cause mortality even after adjustment for cardiometabolic risk factors (HR 1.36, 95% CI 1.08–1.73), whereas NAFLD did not (HR 1.20, 95% CI 0.94–1.53). Although the recent AHA scientific statement identified NAFLD as an independent risk factor for CVD, the question as to whether MAFLD is a simple bystander or an active mediator in the pathogenesis of CVD remains . Based on the available evidence , the shared cardiometabolic risk factors play an important role but likely do not account for the entire relationship between MAFLD and the risk of CVD events. Apart from shared cardiometabolic risk factors, the precise mechanism(s) underlying the association between MAFLD and CVD risk is (are) not clear, but some potential mechanisms (such as, for example, activation of the renin–angiotensin–aldosterone system, some NAFLD-related genetic polymorphisms and intestinal dysbiosis) may also play a role in both MAFLD and CVD , but further research is needed.
Based on current evidence, whether a diagnosis of MAFLD improves CVD risk prediction remains uncertain . Currently, in high-risk patient populations with obesity, T2DM or MetS, screening for MAFLD has been recommended by many scientific guidelines . Conversely, routine screening for MAFLD has not been recommended by scientific guidelines from cardiovascular societies . Before MAFLD screening can be recommended, it is necessary to demonstrate that routine screening may improve both liver-related and cardiovascular outcomes in a cost-effective manner . Wong et al. performed a study of 612 patients referred for coronary angiography with 3679 patient-years of follow-up to test the utility of MAFLD for CVD risk prediction. These authors found that whilst the presence of MAFLD was associated with significant coronary artery disease and need for coronary revascularization procedures, the rates of mortality and CVD events were the same among the MAFLD and non-MAFLD patient cohorts. In fatty liver disease, it is often overlooked that the severity of liver fibrosis is strongly associated with an increased risk of fatal and nonfatal CVD events . Non-invasive tests for diagnosing liver fibrosis may reduce the number for unnecessary liver biopsies and identify patients at higher risk of CVD. As proof, in a population-based cohort study of 3512 individuals, Tamaki et al. examined the associations between non-invasive biomarkers of liver fibrosis [including Fibrosis-4 (FIB-4) index, non-alcoholic fatty liver disease fibrosis score (NFS), and Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA + -M2BP)] and risk of CVD events. The authors showed that advanced fibrosis (defined as FIB-4 ≥ 2.67, NFS ≥ 0.675, or WFA + -M2BP ≥ 1.0) was associated with higher CVD risk (using the Framingham risk score), independent of traditional CVD risk factors. In another prospective study of nearly 900 outpatients with metabolic syndrome followed for a median period of 41 months, Baratta et al. showed that subjects with NAFLD and FIB-4 ≥ 2.67 had a fourfold increase in fatal and nonfatal CVD events (HR 4.02, 95% CI 1.06–5.74). Although further prospective studies are needed, these findings are proof of concept for the use of non-invasive tools for a better CVD risk stratification in MAFLD.
Safe, effective and acceptable pharmacotherapies for MAFLD must halt or delay the progression from simple steatosis to cirrhosis, end-stage liver disease and/or hepatocellular carcinoma. The efficacy and safety of potential treatments for MAFLD that reduce the risk of CVD are summarized in Fig. . Interventions with benefit in both CVD and MAFLD Lifestyle intervention continues to play a key role in the primary and secondary prevention of CVD, as also recommended in several guidelines for management of MAFLD . Adopting a diet rich in vegetables, fruits, legumes, nuts, whole grains and fish is recommended in order to reduce CVD risk and to improve hepatic steatosis and inflammation . A Mediterranean-type diet may reduce hepatic steatosis, improve insulin resistance , and is also effective in primary and secondary prevention of CVD . Weight loss is an essential treatment component for reducing CVD risk. Weight loss of 5% to 10% has been shown to be an achievable goal in most lifestyle interventions and results in significant improvements of hepatic histology features (steatosis, inflammation and fibrosis) and CVD risk reduction . With regard to physical activity, at least 150 min per week of accumulated moderate-intensity aerobic physical activity or 75 min per week of vigorous-intensity aerobic physical activity, can improve hepatic steatosis and reduce CVD risk . There may be no lower limit to the amount of moderate to vigorous physical activity at which the benefits of CVD risk reduction begins . Therefore, for adults who cannot meet the minimum level of physical activity, engaging in some moderate or vigorous physical activity may help to reduce risk of CVD . Sleep is an emerging risk factor for cardiometabolic disease with strong relationships between obstructive sleep apnea and fatty liver disease, possibly mediated (at least in part) by recurrent nocturnal hypoxemia . Importantly, in light of the increasing prevalence of inadequate sleep worldwide, sleep deprivation has been causally implicated in increased visceral fat deposition even in young and healthy subjects . Indeed, the AHA recently included sleep in its list of “Life’s Essential 8”, as a behavioral strategy for improving cardiovascular and metabolic population health (see https://www.heart.org/en/healthy-living/healthy-lifestyle/lifes-essential-8 ). T2DM is often present in MAFLD and diabetic cardiomyopathy is a risk factor for CVD . Recent data also suggests that some newer glucose-lowering agents may not only improve the histological features of NAFLD, but also significantly reduce CVD outcomes because these agents induce weight loss and improve glycemic control . Glucagon-like peptide 1 receptor agonists (GLP-1RAs) and sodium-glucose cotransporter 2 inhibitors (SGLT-2i) are two newer classes of glucose-lowering agents that are highly effective for both T2DM treatment and risk reduction of CVD and kidney outcomes . A meta-analysis of phase-2 randomized controlled trials demonstrated that treatment with GLP-1RAs (especially subcutaneous liraglutide and semaglutide) significantly reduce body weight and improve liver histology in NAFLD . Tirzepatide, a novel, dual GLP-1RAs and glucose-dependent insulinotropic polypeptide (GIP) may also exert beneficial effects on liver fat content and the volume of visceral and abdominal subcutaneous adipose tissues. Importantly, tirzepatide did not increase the risk of major CVD events in patients with T2DM . Some phase-2 randomized controlled trials have reported that SGLT2i treatment may also improve hepatic fat content and fibrosis . Nevertheless, the beneficial effects of these newer glucose-lowering agents on hepatic fibrosis beyond weight loss require further study. Pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, is another glucose-lowering drug that also improves hepatic histology features in patients with biopsy-proven non-alcoholic steatohepatitis, irrespective of the coexistence of T2DM . The benefits of pioglitazone on CVD outcomes in patients with and without T2DM are also well-known . However, safety concerns and moderate weight gain have severely impacted the long-term use of this drug in clinical practice . Therapies with benefit in MAFLD but with cardiovascular safety concerns Acetyl-CoA carboxylase (ACC) is a key enzyme in fatty acid synthesis that has been explored as a therapeutic target for metabolic steatohepatitis . ACC inhibitors may improve hepatic steatosis, inflammation and fibrosis . Unfortunately, in a randomized controlled trial, ACC inhibitors reduced liver fat content but increased plasma triglyceride levels, raising concerns about their CVD safety . To date, Mendelian randomization studies have not provided sufficient evidence to support the conclusion that hepatic fat accumulation is causally associated with CVD . Conversely, some studies reported that MAFLD susceptibility genotypes (e.g., genetic variants in patatin-like phospholipase domain containing 3 ( PNPLA3 ) and trans-membrane 6 superfamily member 2 ( TM6SF2 )) are associated with higher risk of fatty liver and steatohepatitis, but with a less atherogenic lipid profile and lower risk of CVD . Farnesoid X receptor (FXR) agonists have therapeutic potential for MAFLD by correcting abnormalities in intermediary metabolism and lipid accumulation, inhibiting p53 activation induced by metabolic stress, inhibiting the progression of fibrosis, and reducing hepatic inflammation . However, obeticholic acid as the first FXR agonist to be submitted for approval for treatment of nonalcoholic steatohepatitis was rejected by the U.S. Food and Drug Administration in 2020 citing uncertainty over the expected benefits based on alternative histopathological endpoints and after consideration that the treatment benefits did not outweigh the potential risks of increasing plasma LDL-C concentrations. Saroglitazar, a peroxisome proliferator-activated receptor (PPAR) α/γ dual agonist is the first drug to be approved for non-cirrhotic non-alcoholic steatohepatitis (NASH). A randomized, double-blind, placebo-controlled trial demonstrated that high dose saroglitazar (4 mg daily) for 16 weeks reduced liver fat content and improved insulin resistance, serum triglyceride, and transaminase levels in obese patients with NAFLD or NASH . Saroglitazar was approved in India in 2020, but regulatory approval outside of India has not occurred. Lanifibranor is a pan-PPAR agonist that activates PPAR, α, γ and δ receptors. In the phase 2B placebo-controlled NATIVE trial , the histological SAF-A (activity of liver steatosis, activity, and fibrosis) score was reduced in obese patients with biopsy-confirmed nonalcoholic steatohepatitis. Additionally, multiple secondary endpoints were achieved with satisfactory resolution of steatohepatitis without worsening of fibrosis, and improvement in fibrosis stage of at least one stage without worsening of NASH. However, there is little evidence of its impact on CVD risk. Vitamin E effectively improves hepatic histology in adult patients with biopsy-proven NASH . Combined low-dose spironolactone plus vitamin E also decreased NAFLD liver fat score . However, studies evaluating vitamin E for histological benefit have generally been negative or have produced inconsistent results in small groups of patients . The results of some randomized placebo-controlled clinical trials also indicate that vitamin E supplementation not only failed to prevent major CVD events, but in fact may increase the risk of developing heart failure . Therapies with benefit in CVD but unknown or uncertain effects in MAFLD Statins are the first-line treatment to prevent atherosclerotic CVD in patients with hypercholesterolemia . Statins reduce the risk of CVD in MAFLD patients with dyslipidemia, even without any beneficial effect on liver histology . Statins are known to be safe in NAFLD and statin use is not associated with abnormal serum liver enzyme levels, even in patients with hepatic steatosis . An unexpected concern is that statin treatment might be suboptimal for subjects with MAFLD , however further research is needed to test this further. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors represent an alternative pharmacological approach to reducing plasma LDL-C concentrations. While some studies reported a possible beneficial effect on hepatic pathology, it is premature to recommend this agent for specifically treating MAFLD. Daily aspirin use has been associated with fewer severe histologic features of MAFLD and a lower risk of progressing to advanced fibrosis in a recent observational study . Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARBs) are also thought to exert a moderate anti-fibrotic effect on the liver in experimental and clinical studies . Given the current evidence, more and larger controlled clinical trials are needed before a recommendation for use of these anti-hypertensive agents can be recommended for specifically treating MAFLD.
Lifestyle intervention continues to play a key role in the primary and secondary prevention of CVD, as also recommended in several guidelines for management of MAFLD . Adopting a diet rich in vegetables, fruits, legumes, nuts, whole grains and fish is recommended in order to reduce CVD risk and to improve hepatic steatosis and inflammation . A Mediterranean-type diet may reduce hepatic steatosis, improve insulin resistance , and is also effective in primary and secondary prevention of CVD . Weight loss is an essential treatment component for reducing CVD risk. Weight loss of 5% to 10% has been shown to be an achievable goal in most lifestyle interventions and results in significant improvements of hepatic histology features (steatosis, inflammation and fibrosis) and CVD risk reduction . With regard to physical activity, at least 150 min per week of accumulated moderate-intensity aerobic physical activity or 75 min per week of vigorous-intensity aerobic physical activity, can improve hepatic steatosis and reduce CVD risk . There may be no lower limit to the amount of moderate to vigorous physical activity at which the benefits of CVD risk reduction begins . Therefore, for adults who cannot meet the minimum level of physical activity, engaging in some moderate or vigorous physical activity may help to reduce risk of CVD . Sleep is an emerging risk factor for cardiometabolic disease with strong relationships between obstructive sleep apnea and fatty liver disease, possibly mediated (at least in part) by recurrent nocturnal hypoxemia . Importantly, in light of the increasing prevalence of inadequate sleep worldwide, sleep deprivation has been causally implicated in increased visceral fat deposition even in young and healthy subjects . Indeed, the AHA recently included sleep in its list of “Life’s Essential 8”, as a behavioral strategy for improving cardiovascular and metabolic population health (see https://www.heart.org/en/healthy-living/healthy-lifestyle/lifes-essential-8 ). T2DM is often present in MAFLD and diabetic cardiomyopathy is a risk factor for CVD . Recent data also suggests that some newer glucose-lowering agents may not only improve the histological features of NAFLD, but also significantly reduce CVD outcomes because these agents induce weight loss and improve glycemic control . Glucagon-like peptide 1 receptor agonists (GLP-1RAs) and sodium-glucose cotransporter 2 inhibitors (SGLT-2i) are two newer classes of glucose-lowering agents that are highly effective for both T2DM treatment and risk reduction of CVD and kidney outcomes . A meta-analysis of phase-2 randomized controlled trials demonstrated that treatment with GLP-1RAs (especially subcutaneous liraglutide and semaglutide) significantly reduce body weight and improve liver histology in NAFLD . Tirzepatide, a novel, dual GLP-1RAs and glucose-dependent insulinotropic polypeptide (GIP) may also exert beneficial effects on liver fat content and the volume of visceral and abdominal subcutaneous adipose tissues. Importantly, tirzepatide did not increase the risk of major CVD events in patients with T2DM . Some phase-2 randomized controlled trials have reported that SGLT2i treatment may also improve hepatic fat content and fibrosis . Nevertheless, the beneficial effects of these newer glucose-lowering agents on hepatic fibrosis beyond weight loss require further study. Pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, is another glucose-lowering drug that also improves hepatic histology features in patients with biopsy-proven non-alcoholic steatohepatitis, irrespective of the coexistence of T2DM . The benefits of pioglitazone on CVD outcomes in patients with and without T2DM are also well-known . However, safety concerns and moderate weight gain have severely impacted the long-term use of this drug in clinical practice .
Acetyl-CoA carboxylase (ACC) is a key enzyme in fatty acid synthesis that has been explored as a therapeutic target for metabolic steatohepatitis . ACC inhibitors may improve hepatic steatosis, inflammation and fibrosis . Unfortunately, in a randomized controlled trial, ACC inhibitors reduced liver fat content but increased plasma triglyceride levels, raising concerns about their CVD safety . To date, Mendelian randomization studies have not provided sufficient evidence to support the conclusion that hepatic fat accumulation is causally associated with CVD . Conversely, some studies reported that MAFLD susceptibility genotypes (e.g., genetic variants in patatin-like phospholipase domain containing 3 ( PNPLA3 ) and trans-membrane 6 superfamily member 2 ( TM6SF2 )) are associated with higher risk of fatty liver and steatohepatitis, but with a less atherogenic lipid profile and lower risk of CVD . Farnesoid X receptor (FXR) agonists have therapeutic potential for MAFLD by correcting abnormalities in intermediary metabolism and lipid accumulation, inhibiting p53 activation induced by metabolic stress, inhibiting the progression of fibrosis, and reducing hepatic inflammation . However, obeticholic acid as the first FXR agonist to be submitted for approval for treatment of nonalcoholic steatohepatitis was rejected by the U.S. Food and Drug Administration in 2020 citing uncertainty over the expected benefits based on alternative histopathological endpoints and after consideration that the treatment benefits did not outweigh the potential risks of increasing plasma LDL-C concentrations. Saroglitazar, a peroxisome proliferator-activated receptor (PPAR) α/γ dual agonist is the first drug to be approved for non-cirrhotic non-alcoholic steatohepatitis (NASH). A randomized, double-blind, placebo-controlled trial demonstrated that high dose saroglitazar (4 mg daily) for 16 weeks reduced liver fat content and improved insulin resistance, serum triglyceride, and transaminase levels in obese patients with NAFLD or NASH . Saroglitazar was approved in India in 2020, but regulatory approval outside of India has not occurred. Lanifibranor is a pan-PPAR agonist that activates PPAR, α, γ and δ receptors. In the phase 2B placebo-controlled NATIVE trial , the histological SAF-A (activity of liver steatosis, activity, and fibrosis) score was reduced in obese patients with biopsy-confirmed nonalcoholic steatohepatitis. Additionally, multiple secondary endpoints were achieved with satisfactory resolution of steatohepatitis without worsening of fibrosis, and improvement in fibrosis stage of at least one stage without worsening of NASH. However, there is little evidence of its impact on CVD risk. Vitamin E effectively improves hepatic histology in adult patients with biopsy-proven NASH . Combined low-dose spironolactone plus vitamin E also decreased NAFLD liver fat score . However, studies evaluating vitamin E for histological benefit have generally been negative or have produced inconsistent results in small groups of patients . The results of some randomized placebo-controlled clinical trials also indicate that vitamin E supplementation not only failed to prevent major CVD events, but in fact may increase the risk of developing heart failure .
Statins are the first-line treatment to prevent atherosclerotic CVD in patients with hypercholesterolemia . Statins reduce the risk of CVD in MAFLD patients with dyslipidemia, even without any beneficial effect on liver histology . Statins are known to be safe in NAFLD and statin use is not associated with abnormal serum liver enzyme levels, even in patients with hepatic steatosis . An unexpected concern is that statin treatment might be suboptimal for subjects with MAFLD , however further research is needed to test this further. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors represent an alternative pharmacological approach to reducing plasma LDL-C concentrations. While some studies reported a possible beneficial effect on hepatic pathology, it is premature to recommend this agent for specifically treating MAFLD. Daily aspirin use has been associated with fewer severe histologic features of MAFLD and a lower risk of progressing to advanced fibrosis in a recent observational study . Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARBs) are also thought to exert a moderate anti-fibrotic effect on the liver in experimental and clinical studies . Given the current evidence, more and larger controlled clinical trials are needed before a recommendation for use of these anti-hypertensive agents can be recommended for specifically treating MAFLD.
The AHA statement in 2022 identifies liver fat accumulation (NAFLD) as an independent risk factor for CVD. However, routine screening for MAFLD in patients with pre-existing CVD is not currently recommended. There is increasing scientific and clinical interest in the link between MAFLD and CVD risk, not least because newer glucose-lowering drugs, such as GLP-1RAs and SGLT2i may exert benefit on both hepatic fat content and CVD outcomes. That said, when safe and effective pharmacological treatments for MAFLD are licensed, management will involve close liaison between Cardiologists and other physicians treating this multisystem disease.
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Genomics and Epigenomics in Parathyroid Neoplasia: from Bench to Surgical Pathology Practice | 12b13f86-7e8a-43a2-8f6f-c2d28d4d4607 | 7960610 | Pathology[mh] | General Background In the clinical setting, the tumor responsible for primary hyperparathyroidism (PHPT) is usually a parathyroid adenoma. The vast majority of parathyroid adenomas are functioning due to an altered set point in terms of calcium sensing mechanisms, and the ensuing parathyroid hormone (PTH) secretion leads to hypercalcemia that may cause diverse symptoms in the afflicted patient . However, the majority of parathyroid adenomas are identified through serum calcium screening. The peak incidence is among 50–60-year-old individuals, and the female to male ratio increases with increased age at diagnosis, reaching 5:1 among patients > 75 years of age . The treatment is surgical, and cure rates at tertiary centers are usually high . Most cases are preoperatively localized using combinations of various imaging techniques, such as neck ultrasound, single-photon emission computed tomography (SPECT/CT), and/or technetium (99mTc) sestamibi scintigraphy, and the endocrine surgeon can thus plan for a focused parathyroidectomy . Although the bulk of PHPT cases are sporadic tumors arising through the somatic acquisition of genetic aberrancies in driver genes, approximately 5% of cases are associated with familial disease. If a familial syndrome is suspected, a four-gland exploration with subtotal parathyroidectomy or total parathyroidectomy with intramuscular reimplantation is often the preferred strategy, as these patients may develop multiglandular, metachronous disease—and some also carry a risk of developing parathyroid carcinoma . From a histopathological perspective, parathyroid adenomas are usually well-circumscribed tumors composed of chief cells arranged in micro-acinar or palisading formations (Fig. a) . Subsets of cells can exhibit hyperchromatic nuclei with nuclear atypia, and multinucleated tumor cells are sometimes observed. Mitotic figures can be seen in parathyroid adenoma and parathyroid carcinoma. The stromal fat content is reduced compared to normal gland histology, and a rim of normal appearing (although suppressed) parathyroid tissue can usually be seen, particularly in smaller parathyroid adenomas and less commonly seen in larger adenomas (Fig. a). What appears to be a rim of normal-appearing parathyroid tissue cannot be used to differentiate parathyroid adenoma from multiglandular disease, as up to 10% of parathyroid “hyperplasia” (multiglandular disease) may have what appear to be rims of normal parathyroid. Moreover, as intraoperative frozen section analyses not always are effective in distinguishing single parathyroid adenomas from multiglandular involvement, intraoperative PTH assays are probably more reliable in this context . In order to diagnose a parathyroid adenoma, there must be no signs of malignant behavior, such as vascular or perineural invasion. Regarding their immunophenotype, most parathyroid adenomas are positive for chromogranin A, PTH, and GATA3. Numerous histological variants have been described, of which oncocytic parathyroid adenomas are the most common, followed by unusual variants such as parathyroid lipoadenomas and water-clear cell adenomas . From a clinical perspective, oncocytic features associate to larger tumor size. Oncocytic parathyroid tumors are functional but may not have as elevated serum calcium or parathyroid hormone levels as comparable conventional parathyroid adenomas. Lipoadenomas might possibly correlate to the presence of arterial hypertension—suggesting that these subclassifications might disclose clinical considerations of importance . Subsets of parathyroid tumors exhibit atypical features usually observed in carcinomas, but yet lacking unequivocal criteria of malignancy such as invasive growth into periparathyroidal tissues (thyroid gland or soft tissues), lymphovascular or perineural invasion, regional and/or distant metastases. These features may include a trabecular growth pattern, fibrotic bands, tumor cells within the capsule, increased mitotic/proliferative activity, and nuclear atypia with macronucleoli (Fig. b, c) . Parathyroid lesions with these features are termed “atypical parathyroid tumors” and could be considered tumors with unknown malignant potential—even though the majority of these cases will behave benign in a clinical context, with few recurrences following parathyroidectomy . Due to the fact that small subsets of atypical tumors will recur as metastatic parathyroid carcinomas, numerous studies have tried to highlight the malignant potential of this tumor subgroup using various combinations of histology and molecular markers, which are discussed in detail below. Parathyroid carcinoma is the malignant form of PHPT. Quite rare, accounting for < 1% of PHPT, parathyroid carcinoma is an entity which from an endocrine pathology perspective always is feared, often discussed, but rarely diagnosed (personal observations) . Unlike parathyroid adenomas, which are more common in women than in men, parathyroid carcinomas affect woman and men equally. Clues to a parathyroid carcinoma diagnosis can be obtained preoperatively, as these tumors often are larger than adenomas, may be clinically palpable, and usually are associated with high serum calcium levels (often > 13.5 mg/dl) with patients exhibiting various symptoms related to hypercalcemia, such as nephrolithiasis and bone disease . Perioperative findings of the tumor being adherent to adjacent structures can also be information of value when assessing these rare lesions, and for this reason, surgeons usually opt in for an en bloc resection of the afflicted ipsilateral thyroid lobe when suspecting parathyroid carcinoma (Fig. d) . However, one must be cautious in evaluating a parathyroid that may be adhesed to adjacent structures such as the thyroid gland, as parathyroid adenomas may also be firmly adhesed to this organ. The difference of course is that parathyroid adenomas are not invasive of the thyroid while parathyroid carcinomas may be invasive of the adjacent thyroid. Parathyroid carcinomas exhibit local invasive behavior and may spread locally to adjacent structures and later on to distant sites (Fig. e, f) . Chemotherapy and/or radiotherapy are largely ineffective, and the 10-year survival rate is around 50–70% , with death often due to hypercalcemia. Morbidity due to complications following repetitive neck surgery (hypoparathyroidism, recurrent nerve palsy) is also high . The differentiation of parathyroid carcinoma from other carcinomas in the neck is usually straightforward. Parathyroid carcinomas are neuroendocrine tumors and generally positive for chromogranin A. Other neuroendocrine tumors in the neck such as medullary thyroid carcinoma will also shows staining for chromogranin A, but medullary thyroid carcinomas are usually positive for thyroid transcription factor 1 while negative for parathyroid hormone. Calcitonin is usually positive in medullary thyroid carcinoma and usually negative in parathyroid carcinoma; however, unusual staining patterns can be seen such an exceptional case of parathyroid carcinoma positive for calcitonin and calcitonin gene related peptide . Moreover, practicing pathologist should also be aware that medullary thyroid carcinomas can express PAX8 in a clone-dependent manner, in which absence of immunoreactivity is noted when monoclonal PAX8 antibodies are applied, as opposed to positive staining using polyclonal antibodies . As parathyroid tissue might stain positive for polyclonal PAX8 antibodies, we recommend a distinguishing panel of PTH and TTF1 when assessing these differentials . Although a plethora of epigenetic and genetic aberrancies have been identified in parathyroid tumors, few markers have paved their way into clinical routine practice. In this review, we focus on two main clinical predicaments concerning parathyroid tumors, namely, (1) the identification of tumors associated to various underlying syndromes of importance for genetic counseling, and (2) the distinction between benign and malignant tumors to triage each patient to correct follow-up and treatment algorithms. In the following sections, we highlight molecular markers of importance that facilitate these diagnostic quandaries, and discuss their potential as discriminative clinical markers of value to the surgical pathologist. Familial PHPT: Underlying Causes Much of what we today know about mutational driver events in parathyroid tumorigenesis stems from earlier work in kindreds with familial PHPT, long before the appearance of next-generation sequencing techniques. By genetic linkage analyses of family members with autosomal dominant PHTP, candidate gene loci segregating with disease-afflicted individuals were identified, followed by identification of mutational events by cumbersome Sanger sequencing of a large number of candidate genes within these regions. By this methodology, germline mutations of the MEN1 ( multiple endocrine neoplasia type 1 ), RET ( rearranged during transfection ), and CDC73 ( cell division cycle protein 73 ) (originally entitled hyperparathyroidism type 2 , HRPT2 ) genes as events underlying the development of multiple endocrine neoplasia type 1 (MEN1), multiple endocrine neoplasia type 2A (MEN2A), and hyperparathyroidism-jaw tumor (HPT-JT) syndromes, respectively (Table ) . These three conditions exhibit high prevalence of PHPT, occurring in approximately 90% of MEN1 kindred, 20–30% of MEN2A kindred, and in 80% of HTP-JT kindred . In MEN1 patients, PHPT is the most common disease manifestation, followed by pituitary tumors (30–40% of patients), duodenal and pancreatic neuroendocrine tumors (most often gastrinoma, insulinoma and/or glucagonoma, 40% of patients), and adrenocortical lesions (20–45% of patients) . In MEN2A, most patients (> 90%) develop medullary thyroid carcinoma and pheochromocytoma (50%), whereas PHPT is more uncommon, occurring in 15–30% of patients . For HPT-JT kindred, 70–80% develop PHPT, whereas approximately 10% of patients also develop ossifying fibromas of the mandible or maxilla . PHPT in MEN1 usually presents as multiglandular disease that may develop in synchronous or metachronous settings and may be asymmetric. For the MEN2A syndrome, the PHPT may present as multiglandular or single gland disease. There are exceedingly few reports of unequivocal parathyroid carcinomas arising in MEN1 or MEN2A kindred . The hyperparathyroidism in HPT-JT syndrome is usually associated with a parathyroid adenoma; however, 15–40% of patients carrying CDC73 mutations or gene deletions will develop parathyroid carcinoma . Apart from these three syndromes, hyperparathyroidism is also seen as the sole feature in familial isolated hyperparathyroidism (FIHP). These families often, but not always, present with germline mutations in either MEN1 , CDC73 , or the calcium sensing receptor ( CaSR ) gene, of which the latter is also associated to the development of familial hypocalciuric hypercalcemia type 1 (FHH1) . The reason why some patients with germline mutations develop full-blown MEN1, HPT-JT, and FHH1 syndromes, while others develop FIHP, is not clearly understood—as there is no apparent genotype to phenotype correlation in terms of mutation types and exon localization. Recently, germline activating glial cells missing transcription factor 2 ( GCM2 ) gene mutations were also coupled to FIHP, adding yet another candidate to the growing palette of genes underlying the development of familial hyperparathyroidism . Moreover, germline inactivating mutations in cyclin-dependent kinase inhibitor (CDKI) genes have been found in rare families with MEN1-like syndromes (with mutations in either CDKN1A , CDKN2B , or CDKN2C ) or the MEN4 syndrome, a phenotypic MEN1 syndrome characterized by mutations in CDKN1B . Somatic Genetics in Parathyroid Adenomas Given the identification of the MEN1 , RET , and CDC73 gene aberrancies as main responsible for the development of familial PHPT, numerous studies followed in which the involvement of these genes were assessed in sporadic parathyroid tumors. Approximately 25–40% of all sporadic parathyroid adenomas harbor LOH of the MEN1 gene locus at 11q13, and half of these cases also exhibit an inactivating MEN1 mutation of the remaining allele (Table , Fig. ) . Interestingly, an association between somatic MEN1 mutations and mild PHPT symptoms has been observed, possibly arguing for early-stage events in parathyroid tumorigenesis . Moreover, while somatic CDC73 gene mutations have been reported in small subsets of sporadic parathyroid adenomas, no reports on somatic RET gene mutations in parathyroid adenoma have been noted . Moreover, mutational and/or epigenetic silencing of other genes predisposing for FIHP and MEN1-like syndromes have also been detected in small subsets of apparent sporadic parathyroid adenomas, including CDKN1A , CDKN1B , CDKN2A , CDKN2B , CDKN2C , and GCM2 . Of particular interest, CDKN1B encodes p27 and down-regulation of p27 has been described in PAs, both on the RNA- and protein level . Moreover, CDKN1B mutations have also been functionally linked to the development of parathyroid tumors, thereby solidifying the role of aberrant cell cycle regulation in the development of parathyroid adenomas . Continuing on the cell cycle topic, a recurrent chromosomal inversion involving the peri-centromeric portion of chromosome 11 has been observed in exceedingly small subsets of sporadic parathyroid adenomas. This rearrangement causes the juxtaposition of the PTH 5′ regulatory region to the CCND1 oncogene coding region, causing a constitutively expression of the CCND1 corresponding protein cyclin D1. Although rare in sporadic parathyroid adenoma, overexpression of cyclin D1 is a common event, and therefore, other mechanisms apart from rare chromosomal inversions involving CCND1 or mutations in cyclin D1-regulating CDKIs are expected to play a role. Instead, promoter hypermethylation and down-regulation of various CDKIs could probably explain the commonly observed cyclin D1 upregulation in parathyroid adenomas (Fig. ) . Apart from the discovery of somatic alterations in established parathyroid adenoma susceptibility genes discussed above, the advent of next-generation sequencing techniques has led to the discovery of additional gene mutations of possible importance to parathyroid adenoma development. By whole-exome sequencing of parathyroid adenomas, an activating missense mutation in the methyltransferase gene enhancer of zeste homolog 2 ( EZH2 ) was detected in one out of 8 adenomas interrogated, and additional targeted sequencing of 185 adenomas revealed one additional case with the same mutation . The EZH2 gene is an epigenetic regulator of chromatin accessibility with an association to tumorigenesis in general, thereby providing additional validity of this gene constituting a possible contributor of parathyroid adenoma development—which was also verified using functional experiments in a parathyroid cell line . Since then, additional whole-exome sequencing studies have corroborated low frequencies of EZH2 mutations in sporadic adenomas . Additional mutational events occurring in low frequencies of parathyroid adenomas include activating CTNNB1 mutations, of which some have been reported as homozygous in small numbers , although not reproduced by others . CTNNB1 encodes beta-catenin, a central onco-protein regulating the Wingless type (Wnt) pathway, and data suggests that nuclear accumulation of beta-catenin could be an important player in the development of parathyroid adenomas—either through activating mutations or through aberrantly expressed Wnt co-receptors (Fig. ) . On the epigenetic level, apart from hypermethylation of CDKIs mentioned above, aberrant methylation has also been reported for numerous tumor-related genes, including WT1 , SFRP1 , SFRP2 , SFRP4 , RIZ1 , APC , and RASSF1A . Most notably, RASSF1A hypermethylation was strongly associated to down-regulation on the mRNA level in virtually all parathyroid adenomas, thereby constituting one of the most commonly known genetic aberrancies in this disease (Table ) . Moreover, on a global level, adenomas seem to exhibit similar levels of methylation as non-tumorous parathyroid tissues, suggesting that epigenetic dysregulations are driven by gene-specific events and not due to a general hypo- or hypermethylation pattern . Regarding specific parathyroid adenoma subtypes, there is also an established correlation between oncocytic parathyroid adenomas and somatic mutations in genes encoded by mitochondrial DNA (mtDNA), especially mitochondrial respiratory chain complex genes NADH dehydrogenase 1 , 4 , and 5 ( ND1 , ND4 , and ND5 ) (Table , Fig. ). As oncocytic tumors in general exhibit prominent amounts of mitochondria, the association is intriguing . However, as no recurrent mutations were observed, these findings mandate functional verification before a true driver status could be assigned to any of these alterations. Somatic Genetics in Parathyroid Carcinomas Somatic CDC73 gene mutations are the most frequent somatic alteration in parathyroid carcinoma (Table , Fig. ) . These mutations are in general disruptive due to premature truncations or frameshift alterations, alternatively the mutations are of missense nature in conserved regions encoding the nuclear localization signals (NLSs) or the human polymerase–associated factor 1 (hPAF1) complex of the corresponding protein product, termed parafibromin. Apart from mutations, LOH encompassing the CDC73 gene locus and aberrant CDC73 promoter methylation have also been reported as somatic events in parathyroid carcinoma . Parafibromin is a member of the hPAF regulatory complex, a key transcriptional unit that interacts with RNA polymerase II and facilitates transcriptional activity due to histone-modifying and chromatin remodeling processes . Parafibromin is associated with tumor-suppressive properties, as (a) the majority of CDC73 germline mutations in HPT-JT and FIHP kindred as well as the bulk of somatic mutations in sporadic parathyroid carcinomas are disruptive , (b) the majority of tumors with CDC73 mutations exhibit loss of parafibromin expression , and (c) functional experiments with CDC73 plasmids support an anti-proliferative effect of the wild-type protein . Indeed, parafibromin has been found to regulate cyclin D1 levels, exhibit pro-apoptotic effects, regulate the Wnt pathway through interactions with beta-catenin, as well as the c-Myc oncogene through direct binding to the promoter region of this gene . Intriguingly, parafibromin also seems to exhibit oncogenic features in the presence of certain molecular partners, suggesting a yin-yang modus operandi of this protein . In contrast to parathyroid adenomas, somatic MEN1 mutations are very infrequent in parathyroid carcinomas, but nonetheless reported . Given the exceedingly low rate of malignant PHPT in MEN1 kindred, other genetic events apart from this aberrancy are expected to drive the invasive behavior in parathyroid carcinoma. TP53 gene mutations are among the most common genetic aberrations in malignant epithelial tumors, although these genetic events seem to be unusual in parathyroid carcinomas . Even so, LOH of one TP53 allele seems to be more common . Similarly, loss of the retinoblastoma 1 ( RB1 ) gene, encoding pRB, is observed in the majority of parathyroid carcinomas, however inactivating mutations have not been reported . The P53 and pRB proteins are regulators of cell cycle progression and two bona fide tumor suppressors usually required to be silenced on both alleles in order to promote neoplasia, and given the fact that parathyroid carcinomas recurrently exhibit absent pRB expression, additional inactivating mechanisms apart from mutations are most likely operational . Next-generation sequencing studies on parathyroid carcinomas are rare, not surprising given the low prevalence of this disease in general. In a recently published whole-genome sequencing study of 23 parathyroid carcinomas, the authors conclude that CDC73 gene mutations were the most common sequence aberration, occurring in almost 40% of cases. Increased copy number variants were seen in parathyroid carcinomas with CDC73 mutations, and these cases also carried an increased tumor mutational burden and poorer patient outcome. In unrelated, exome-based studies, recurrent mutations in AarF domain containing kinase 1 ( ADCK1 ) and prune homolog 2 with BCH domain ( PRUNE2 ) have been reported in parathyroid carcinomas; however, their functional roles has not been elucidated . Moreover, a general overrepresentation of mutations in genes associated with DNA repair and cell cycle regulation seems evident , and rare mutations in established cancer-associated genes have also been identified, for example succinate dehydrogenase complex flavoprotein subunit A ( SDHA ) and DICER1 . From a therapeutic perspective, the majority of parathyroid carcinomas might carry alterations suitable for molecular targeted therapies, thus highlighting the potential role for next-generation sequencing as a tool to identify cases with potential for targeted therapeutic interventions . Promoter mutations of the telomerase reverse transcriptase ( TERT ) gene are heavily implicated in human cancers, as they convey increased TERT expression, which in turn promotes immortalization. However, these mutations seem fairly rare in parathyroid carcinomas, although these tumors in general express TERT protein . As of this, alterative mechanisms leading to increased TERT expression in parathyroid carcinomas are suspected. Aberrant epigenetic mechanisms are also at play in parathyroid carcinomas. Via global methylome analyses, parathyroid carcinomas and adenomas seem to exhibit hypermethylation of CDKN2B , CDKN2A , WT1 , SFRP1 , SFRP2 , and SFRP4 . Moreover, beside the recurrent CDC73 promoter hypermethylation discussed above, altered methylation levels of the adenomatous polyposis coli (APC) promoter 1A region is recurrently seen in parathyroid tumors, although the APC mRNA expression seem to be retained by an unmethylated 1B promoter region . In parathyroid carcinomas specifically, loss of APC protein expression is a frequent event, and this could in part be due to aberrant methylation patterns rather than gene mutations . The APC protein is a tumor suppressor that regulates the Wnt pathway, and loss of APC expression is therefore thought to stimulate proliferation in parathyroid cells. In more recent years, the regulation of epigenetic de-methylation has been highlighted in cancer, especially the discovery of the TET1/TET2 enzymatic activity catalyzing oxidation of 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC). In parathyroid carcinomas, 5hmC levels and TET1 expression have been found extensively reduced, suggesting a general reduction in de-methylation events across the genome . As this phenomenon is tightly linked to the presence of TERT promoter mutations in unrelated cancer types, the general absence of these mutations in parathyroid carcinoma would suggest that other molecular mechanisms influence the observed lack of global de-methylation—a subject worthy of follow-up studies . In the clinical setting, the tumor responsible for primary hyperparathyroidism (PHPT) is usually a parathyroid adenoma. The vast majority of parathyroid adenomas are functioning due to an altered set point in terms of calcium sensing mechanisms, and the ensuing parathyroid hormone (PTH) secretion leads to hypercalcemia that may cause diverse symptoms in the afflicted patient . However, the majority of parathyroid adenomas are identified through serum calcium screening. The peak incidence is among 50–60-year-old individuals, and the female to male ratio increases with increased age at diagnosis, reaching 5:1 among patients > 75 years of age . The treatment is surgical, and cure rates at tertiary centers are usually high . Most cases are preoperatively localized using combinations of various imaging techniques, such as neck ultrasound, single-photon emission computed tomography (SPECT/CT), and/or technetium (99mTc) sestamibi scintigraphy, and the endocrine surgeon can thus plan for a focused parathyroidectomy . Although the bulk of PHPT cases are sporadic tumors arising through the somatic acquisition of genetic aberrancies in driver genes, approximately 5% of cases are associated with familial disease. If a familial syndrome is suspected, a four-gland exploration with subtotal parathyroidectomy or total parathyroidectomy with intramuscular reimplantation is often the preferred strategy, as these patients may develop multiglandular, metachronous disease—and some also carry a risk of developing parathyroid carcinoma . From a histopathological perspective, parathyroid adenomas are usually well-circumscribed tumors composed of chief cells arranged in micro-acinar or palisading formations (Fig. a) . Subsets of cells can exhibit hyperchromatic nuclei with nuclear atypia, and multinucleated tumor cells are sometimes observed. Mitotic figures can be seen in parathyroid adenoma and parathyroid carcinoma. The stromal fat content is reduced compared to normal gland histology, and a rim of normal appearing (although suppressed) parathyroid tissue can usually be seen, particularly in smaller parathyroid adenomas and less commonly seen in larger adenomas (Fig. a). What appears to be a rim of normal-appearing parathyroid tissue cannot be used to differentiate parathyroid adenoma from multiglandular disease, as up to 10% of parathyroid “hyperplasia” (multiglandular disease) may have what appear to be rims of normal parathyroid. Moreover, as intraoperative frozen section analyses not always are effective in distinguishing single parathyroid adenomas from multiglandular involvement, intraoperative PTH assays are probably more reliable in this context . In order to diagnose a parathyroid adenoma, there must be no signs of malignant behavior, such as vascular or perineural invasion. Regarding their immunophenotype, most parathyroid adenomas are positive for chromogranin A, PTH, and GATA3. Numerous histological variants have been described, of which oncocytic parathyroid adenomas are the most common, followed by unusual variants such as parathyroid lipoadenomas and water-clear cell adenomas . From a clinical perspective, oncocytic features associate to larger tumor size. Oncocytic parathyroid tumors are functional but may not have as elevated serum calcium or parathyroid hormone levels as comparable conventional parathyroid adenomas. Lipoadenomas might possibly correlate to the presence of arterial hypertension—suggesting that these subclassifications might disclose clinical considerations of importance . Subsets of parathyroid tumors exhibit atypical features usually observed in carcinomas, but yet lacking unequivocal criteria of malignancy such as invasive growth into periparathyroidal tissues (thyroid gland or soft tissues), lymphovascular or perineural invasion, regional and/or distant metastases. These features may include a trabecular growth pattern, fibrotic bands, tumor cells within the capsule, increased mitotic/proliferative activity, and nuclear atypia with macronucleoli (Fig. b, c) . Parathyroid lesions with these features are termed “atypical parathyroid tumors” and could be considered tumors with unknown malignant potential—even though the majority of these cases will behave benign in a clinical context, with few recurrences following parathyroidectomy . Due to the fact that small subsets of atypical tumors will recur as metastatic parathyroid carcinomas, numerous studies have tried to highlight the malignant potential of this tumor subgroup using various combinations of histology and molecular markers, which are discussed in detail below. Parathyroid carcinoma is the malignant form of PHPT. Quite rare, accounting for < 1% of PHPT, parathyroid carcinoma is an entity which from an endocrine pathology perspective always is feared, often discussed, but rarely diagnosed (personal observations) . Unlike parathyroid adenomas, which are more common in women than in men, parathyroid carcinomas affect woman and men equally. Clues to a parathyroid carcinoma diagnosis can be obtained preoperatively, as these tumors often are larger than adenomas, may be clinically palpable, and usually are associated with high serum calcium levels (often > 13.5 mg/dl) with patients exhibiting various symptoms related to hypercalcemia, such as nephrolithiasis and bone disease . Perioperative findings of the tumor being adherent to adjacent structures can also be information of value when assessing these rare lesions, and for this reason, surgeons usually opt in for an en bloc resection of the afflicted ipsilateral thyroid lobe when suspecting parathyroid carcinoma (Fig. d) . However, one must be cautious in evaluating a parathyroid that may be adhesed to adjacent structures such as the thyroid gland, as parathyroid adenomas may also be firmly adhesed to this organ. The difference of course is that parathyroid adenomas are not invasive of the thyroid while parathyroid carcinomas may be invasive of the adjacent thyroid. Parathyroid carcinomas exhibit local invasive behavior and may spread locally to adjacent structures and later on to distant sites (Fig. e, f) . Chemotherapy and/or radiotherapy are largely ineffective, and the 10-year survival rate is around 50–70% , with death often due to hypercalcemia. Morbidity due to complications following repetitive neck surgery (hypoparathyroidism, recurrent nerve palsy) is also high . The differentiation of parathyroid carcinoma from other carcinomas in the neck is usually straightforward. Parathyroid carcinomas are neuroendocrine tumors and generally positive for chromogranin A. Other neuroendocrine tumors in the neck such as medullary thyroid carcinoma will also shows staining for chromogranin A, but medullary thyroid carcinomas are usually positive for thyroid transcription factor 1 while negative for parathyroid hormone. Calcitonin is usually positive in medullary thyroid carcinoma and usually negative in parathyroid carcinoma; however, unusual staining patterns can be seen such an exceptional case of parathyroid carcinoma positive for calcitonin and calcitonin gene related peptide . Moreover, practicing pathologist should also be aware that medullary thyroid carcinomas can express PAX8 in a clone-dependent manner, in which absence of immunoreactivity is noted when monoclonal PAX8 antibodies are applied, as opposed to positive staining using polyclonal antibodies . As parathyroid tissue might stain positive for polyclonal PAX8 antibodies, we recommend a distinguishing panel of PTH and TTF1 when assessing these differentials . Although a plethora of epigenetic and genetic aberrancies have been identified in parathyroid tumors, few markers have paved their way into clinical routine practice. In this review, we focus on two main clinical predicaments concerning parathyroid tumors, namely, (1) the identification of tumors associated to various underlying syndromes of importance for genetic counseling, and (2) the distinction between benign and malignant tumors to triage each patient to correct follow-up and treatment algorithms. In the following sections, we highlight molecular markers of importance that facilitate these diagnostic quandaries, and discuss their potential as discriminative clinical markers of value to the surgical pathologist. Much of what we today know about mutational driver events in parathyroid tumorigenesis stems from earlier work in kindreds with familial PHPT, long before the appearance of next-generation sequencing techniques. By genetic linkage analyses of family members with autosomal dominant PHTP, candidate gene loci segregating with disease-afflicted individuals were identified, followed by identification of mutational events by cumbersome Sanger sequencing of a large number of candidate genes within these regions. By this methodology, germline mutations of the MEN1 ( multiple endocrine neoplasia type 1 ), RET ( rearranged during transfection ), and CDC73 ( cell division cycle protein 73 ) (originally entitled hyperparathyroidism type 2 , HRPT2 ) genes as events underlying the development of multiple endocrine neoplasia type 1 (MEN1), multiple endocrine neoplasia type 2A (MEN2A), and hyperparathyroidism-jaw tumor (HPT-JT) syndromes, respectively (Table ) . These three conditions exhibit high prevalence of PHPT, occurring in approximately 90% of MEN1 kindred, 20–30% of MEN2A kindred, and in 80% of HTP-JT kindred . In MEN1 patients, PHPT is the most common disease manifestation, followed by pituitary tumors (30–40% of patients), duodenal and pancreatic neuroendocrine tumors (most often gastrinoma, insulinoma and/or glucagonoma, 40% of patients), and adrenocortical lesions (20–45% of patients) . In MEN2A, most patients (> 90%) develop medullary thyroid carcinoma and pheochromocytoma (50%), whereas PHPT is more uncommon, occurring in 15–30% of patients . For HPT-JT kindred, 70–80% develop PHPT, whereas approximately 10% of patients also develop ossifying fibromas of the mandible or maxilla . PHPT in MEN1 usually presents as multiglandular disease that may develop in synchronous or metachronous settings and may be asymmetric. For the MEN2A syndrome, the PHPT may present as multiglandular or single gland disease. There are exceedingly few reports of unequivocal parathyroid carcinomas arising in MEN1 or MEN2A kindred . The hyperparathyroidism in HPT-JT syndrome is usually associated with a parathyroid adenoma; however, 15–40% of patients carrying CDC73 mutations or gene deletions will develop parathyroid carcinoma . Apart from these three syndromes, hyperparathyroidism is also seen as the sole feature in familial isolated hyperparathyroidism (FIHP). These families often, but not always, present with germline mutations in either MEN1 , CDC73 , or the calcium sensing receptor ( CaSR ) gene, of which the latter is also associated to the development of familial hypocalciuric hypercalcemia type 1 (FHH1) . The reason why some patients with germline mutations develop full-blown MEN1, HPT-JT, and FHH1 syndromes, while others develop FIHP, is not clearly understood—as there is no apparent genotype to phenotype correlation in terms of mutation types and exon localization. Recently, germline activating glial cells missing transcription factor 2 ( GCM2 ) gene mutations were also coupled to FIHP, adding yet another candidate to the growing palette of genes underlying the development of familial hyperparathyroidism . Moreover, germline inactivating mutations in cyclin-dependent kinase inhibitor (CDKI) genes have been found in rare families with MEN1-like syndromes (with mutations in either CDKN1A , CDKN2B , or CDKN2C ) or the MEN4 syndrome, a phenotypic MEN1 syndrome characterized by mutations in CDKN1B . Given the identification of the MEN1 , RET , and CDC73 gene aberrancies as main responsible for the development of familial PHPT, numerous studies followed in which the involvement of these genes were assessed in sporadic parathyroid tumors. Approximately 25–40% of all sporadic parathyroid adenomas harbor LOH of the MEN1 gene locus at 11q13, and half of these cases also exhibit an inactivating MEN1 mutation of the remaining allele (Table , Fig. ) . Interestingly, an association between somatic MEN1 mutations and mild PHPT symptoms has been observed, possibly arguing for early-stage events in parathyroid tumorigenesis . Moreover, while somatic CDC73 gene mutations have been reported in small subsets of sporadic parathyroid adenomas, no reports on somatic RET gene mutations in parathyroid adenoma have been noted . Moreover, mutational and/or epigenetic silencing of other genes predisposing for FIHP and MEN1-like syndromes have also been detected in small subsets of apparent sporadic parathyroid adenomas, including CDKN1A , CDKN1B , CDKN2A , CDKN2B , CDKN2C , and GCM2 . Of particular interest, CDKN1B encodes p27 and down-regulation of p27 has been described in PAs, both on the RNA- and protein level . Moreover, CDKN1B mutations have also been functionally linked to the development of parathyroid tumors, thereby solidifying the role of aberrant cell cycle regulation in the development of parathyroid adenomas . Continuing on the cell cycle topic, a recurrent chromosomal inversion involving the peri-centromeric portion of chromosome 11 has been observed in exceedingly small subsets of sporadic parathyroid adenomas. This rearrangement causes the juxtaposition of the PTH 5′ regulatory region to the CCND1 oncogene coding region, causing a constitutively expression of the CCND1 corresponding protein cyclin D1. Although rare in sporadic parathyroid adenoma, overexpression of cyclin D1 is a common event, and therefore, other mechanisms apart from rare chromosomal inversions involving CCND1 or mutations in cyclin D1-regulating CDKIs are expected to play a role. Instead, promoter hypermethylation and down-regulation of various CDKIs could probably explain the commonly observed cyclin D1 upregulation in parathyroid adenomas (Fig. ) . Apart from the discovery of somatic alterations in established parathyroid adenoma susceptibility genes discussed above, the advent of next-generation sequencing techniques has led to the discovery of additional gene mutations of possible importance to parathyroid adenoma development. By whole-exome sequencing of parathyroid adenomas, an activating missense mutation in the methyltransferase gene enhancer of zeste homolog 2 ( EZH2 ) was detected in one out of 8 adenomas interrogated, and additional targeted sequencing of 185 adenomas revealed one additional case with the same mutation . The EZH2 gene is an epigenetic regulator of chromatin accessibility with an association to tumorigenesis in general, thereby providing additional validity of this gene constituting a possible contributor of parathyroid adenoma development—which was also verified using functional experiments in a parathyroid cell line . Since then, additional whole-exome sequencing studies have corroborated low frequencies of EZH2 mutations in sporadic adenomas . Additional mutational events occurring in low frequencies of parathyroid adenomas include activating CTNNB1 mutations, of which some have been reported as homozygous in small numbers , although not reproduced by others . CTNNB1 encodes beta-catenin, a central onco-protein regulating the Wingless type (Wnt) pathway, and data suggests that nuclear accumulation of beta-catenin could be an important player in the development of parathyroid adenomas—either through activating mutations or through aberrantly expressed Wnt co-receptors (Fig. ) . On the epigenetic level, apart from hypermethylation of CDKIs mentioned above, aberrant methylation has also been reported for numerous tumor-related genes, including WT1 , SFRP1 , SFRP2 , SFRP4 , RIZ1 , APC , and RASSF1A . Most notably, RASSF1A hypermethylation was strongly associated to down-regulation on the mRNA level in virtually all parathyroid adenomas, thereby constituting one of the most commonly known genetic aberrancies in this disease (Table ) . Moreover, on a global level, adenomas seem to exhibit similar levels of methylation as non-tumorous parathyroid tissues, suggesting that epigenetic dysregulations are driven by gene-specific events and not due to a general hypo- or hypermethylation pattern . Regarding specific parathyroid adenoma subtypes, there is also an established correlation between oncocytic parathyroid adenomas and somatic mutations in genes encoded by mitochondrial DNA (mtDNA), especially mitochondrial respiratory chain complex genes NADH dehydrogenase 1 , 4 , and 5 ( ND1 , ND4 , and ND5 ) (Table , Fig. ). As oncocytic tumors in general exhibit prominent amounts of mitochondria, the association is intriguing . However, as no recurrent mutations were observed, these findings mandate functional verification before a true driver status could be assigned to any of these alterations. Somatic CDC73 gene mutations are the most frequent somatic alteration in parathyroid carcinoma (Table , Fig. ) . These mutations are in general disruptive due to premature truncations or frameshift alterations, alternatively the mutations are of missense nature in conserved regions encoding the nuclear localization signals (NLSs) or the human polymerase–associated factor 1 (hPAF1) complex of the corresponding protein product, termed parafibromin. Apart from mutations, LOH encompassing the CDC73 gene locus and aberrant CDC73 promoter methylation have also been reported as somatic events in parathyroid carcinoma . Parafibromin is a member of the hPAF regulatory complex, a key transcriptional unit that interacts with RNA polymerase II and facilitates transcriptional activity due to histone-modifying and chromatin remodeling processes . Parafibromin is associated with tumor-suppressive properties, as (a) the majority of CDC73 germline mutations in HPT-JT and FIHP kindred as well as the bulk of somatic mutations in sporadic parathyroid carcinomas are disruptive , (b) the majority of tumors with CDC73 mutations exhibit loss of parafibromin expression , and (c) functional experiments with CDC73 plasmids support an anti-proliferative effect of the wild-type protein . Indeed, parafibromin has been found to regulate cyclin D1 levels, exhibit pro-apoptotic effects, regulate the Wnt pathway through interactions with beta-catenin, as well as the c-Myc oncogene through direct binding to the promoter region of this gene . Intriguingly, parafibromin also seems to exhibit oncogenic features in the presence of certain molecular partners, suggesting a yin-yang modus operandi of this protein . In contrast to parathyroid adenomas, somatic MEN1 mutations are very infrequent in parathyroid carcinomas, but nonetheless reported . Given the exceedingly low rate of malignant PHPT in MEN1 kindred, other genetic events apart from this aberrancy are expected to drive the invasive behavior in parathyroid carcinoma. TP53 gene mutations are among the most common genetic aberrations in malignant epithelial tumors, although these genetic events seem to be unusual in parathyroid carcinomas . Even so, LOH of one TP53 allele seems to be more common . Similarly, loss of the retinoblastoma 1 ( RB1 ) gene, encoding pRB, is observed in the majority of parathyroid carcinomas, however inactivating mutations have not been reported . The P53 and pRB proteins are regulators of cell cycle progression and two bona fide tumor suppressors usually required to be silenced on both alleles in order to promote neoplasia, and given the fact that parathyroid carcinomas recurrently exhibit absent pRB expression, additional inactivating mechanisms apart from mutations are most likely operational . Next-generation sequencing studies on parathyroid carcinomas are rare, not surprising given the low prevalence of this disease in general. In a recently published whole-genome sequencing study of 23 parathyroid carcinomas, the authors conclude that CDC73 gene mutations were the most common sequence aberration, occurring in almost 40% of cases. Increased copy number variants were seen in parathyroid carcinomas with CDC73 mutations, and these cases also carried an increased tumor mutational burden and poorer patient outcome. In unrelated, exome-based studies, recurrent mutations in AarF domain containing kinase 1 ( ADCK1 ) and prune homolog 2 with BCH domain ( PRUNE2 ) have been reported in parathyroid carcinomas; however, their functional roles has not been elucidated . Moreover, a general overrepresentation of mutations in genes associated with DNA repair and cell cycle regulation seems evident , and rare mutations in established cancer-associated genes have also been identified, for example succinate dehydrogenase complex flavoprotein subunit A ( SDHA ) and DICER1 . From a therapeutic perspective, the majority of parathyroid carcinomas might carry alterations suitable for molecular targeted therapies, thus highlighting the potential role for next-generation sequencing as a tool to identify cases with potential for targeted therapeutic interventions . Promoter mutations of the telomerase reverse transcriptase ( TERT ) gene are heavily implicated in human cancers, as they convey increased TERT expression, which in turn promotes immortalization. However, these mutations seem fairly rare in parathyroid carcinomas, although these tumors in general express TERT protein . As of this, alterative mechanisms leading to increased TERT expression in parathyroid carcinomas are suspected. Aberrant epigenetic mechanisms are also at play in parathyroid carcinomas. Via global methylome analyses, parathyroid carcinomas and adenomas seem to exhibit hypermethylation of CDKN2B , CDKN2A , WT1 , SFRP1 , SFRP2 , and SFRP4 . Moreover, beside the recurrent CDC73 promoter hypermethylation discussed above, altered methylation levels of the adenomatous polyposis coli (APC) promoter 1A region is recurrently seen in parathyroid tumors, although the APC mRNA expression seem to be retained by an unmethylated 1B promoter region . In parathyroid carcinomas specifically, loss of APC protein expression is a frequent event, and this could in part be due to aberrant methylation patterns rather than gene mutations . The APC protein is a tumor suppressor that regulates the Wnt pathway, and loss of APC expression is therefore thought to stimulate proliferation in parathyroid cells. In more recent years, the regulation of epigenetic de-methylation has been highlighted in cancer, especially the discovery of the TET1/TET2 enzymatic activity catalyzing oxidation of 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC). In parathyroid carcinomas, 5hmC levels and TET1 expression have been found extensively reduced, suggesting a general reduction in de-methylation events across the genome . As this phenomenon is tightly linked to the presence of TERT promoter mutations in unrelated cancer types, the general absence of these mutations in parathyroid carcinoma would suggest that other molecular mechanisms influence the observed lack of global de-methylation—a subject worthy of follow-up studies . MEN1-Related PHPT: Could the Pathologist be of any Help? The MEN1 syndrome is by far the most common among the hereditary conditions detailed in this review, with one case per 40,000 as opposed to MEN2A (1 case per 2 million) and the HPT-JT syndrome (unknown prevalence, but expected to be exceedingly low) . Therefore, surgical pathologists will most likely diagnose MEN1 related parathyroid adenomas to a much larger extent than MEN2A, HPT-JT, FIHP, or MEN1-like related cases. Although the MEN1 syndrome often is diagnosed long before the patient is subjected to parathyroidectomy, the non-total penetrance in younger years as well as the occurrence of de novo MEN1 mutations in subsets of patients with healthy parents allow for subsets of patients being misclassified as sporadic PHPT patients. As up to 10% of individuals with primary parathyroid “hyperplasia” (multiglandular disease, adenomatosis, multiple adenomas) have MEN1, genetic screening for familial disease has been considered for all patients with primary parathyroid “hyperplasia.” From a diagnostic pathology standpoint, is there a way for the attentive pathologist to aid in the detection of syndromic PHPT in cases where the syndromic association was not evident preoperatively? MEN1 was early on assigned a tumor-suppressor gene status, as the bulk of mutational events in MEN1 kindred reported represent inactivating events leading to premature truncations of the menin protein . Moreover, most parathyroid adenomas arising in MEN1 patients harbor loss of heterozygosity (LOH) of the MEN1 wild-type allele on the somatic level, thereby arguing in favor for the Knudson’s “two hit” theory in which bi-allelic inactivation of a tumor suppressor gene is needed in order for a tumor to develop . Therefore, the question arises if clinical screening using expressional analyses targeting menin could be a cheap and efficient way for the surgical pathologist to triage menin-negative PHPT cases for genetic screening—as loss of menin expression would be indicative of MEN1 gene aberrancies. Menin is a predominantly nuclear protein with a highly conserved sequence among species, and the protein is universally expressed in most human tissues . The protein harbors nuclear localization signals and leucine zipper motifs, both of which are features needed to regulate gene expression though direct interaction with DNA elements. For example, menin has been shown to interact with JunD , a proto-oncogene encoding a transcription factor regulating apoptosis and TP53 gene activation (Fig. ) . Moreover, menin has been proposed as an important player in the regulation of various signaling networks associated to tumor development, such as the TGF-beta, RAS, Wingless type (Wnt) pathways—as well as influencing the expression of the telomerase reverse transcriptase ( TERT ) gene . From a histological context, the MEN1-related parathyroid disease is often multiglandular, although not always synchronous in presentation . Unlike sporadic parathyroid adenomas, MEN1-related tumors are often devoid of a rim of normal parathyroid tissue, and they may be composed of chief cells arranged in compact, sheet-like formations . The tumors may exhibit fibrosis and have similar appearance as parathyroid hyperplastic lesions arising in the setting of chronic renal failure, and almost always lack invasive features. Overall, findings of multiglandular disease and absence of a normal parathyroid rim in a PHPT patient should raise the suspicion of the MEN1 syndrome; however, a “rim” can also be seen in up to 10% of multiglandular disease. In all, there is no histological feature that sets MEN1-related adenomas apart from sporadic ones. Expression studies regarding menin and associations to underlying MEN1 mutational status in parathyroid tumors have been promising, as menin immunohistochemistry seem to exhibit high sensitivity to detect underlying MEN1 mutations and/or gene deletions . Even so, the interpretation of menin immunohistochemistry is not straightforward, and the lack of comprehensive studies on the subject makes menin immunohistochemistry inappropriate for clinical routine screening purposes. Moreover, as the MEN1 gene is frequently deleted and mutated also on the somatic level in sporadic parathyroid adenomas, additional factors are needed to bring into consideration before suspecting MEN1 syndrome–related PHPT from expressional analyses in the pathology laboratory. For example, the penetrance of PHPT in MEN1 patients reaches 95% at 50 years of age, and therefore, the diagnosis of MEN1 in older patients with multiglandular disease should not be completely overlooked—although the bar for considering an MEN1 diagnosis should be even lower in adolescent patients . In all, a concerted teamwork between endocrine surgeons and pathologists covering the patient’s medical history, disease presentation, radiology, and histopathology is probably needed to properly identify all MEN1-related PHPT in the clinical setting. HPT-JT-Associated PHPT: Clues from the Histopathological Workup? As HPT-JT patients carry germline CDC73 gene mutations or deletions, it comes as no surprise that the prevalence of parathyroid carcinoma in this patient category is much higher (15–30%) than in unselected PHPT patient cohorts (< 1%). As parathyroid carcinoma is so uncommon, it has been suggested that individuals diagnosed with parathyroid carcinoma should be promptly evaluated for HPT-JT. Additionally, if the pathologist raises the suspicion that the patient is indeed an HPT-JT kindred, a raised awareness of the increased parathyroid carcinoma risk is mandated when diagnosing the tumor. Easily recognizable features such as a tumor being cystic may in itself raise the possibility of HPT-JT syndrome. In the most comprehensive study yet regarding morphological features of CDC73 mutated parathyroid tumors, the authors conclude that these lesions are characterized by sheet-like, compact growth rather than the usual acinar patterns visualized in the bulk of parathyroid tumors, as well as a typical eosinophilic cytoplasm distinct from the granular oxyphilic cell type often observed in areas of parathyroid adenomas . Many tumors also exhibited enlarged nuclei and a perinuclear cytoplasmic clearing. In all, these findings suggest that the occurrence of these histological parameters might suggest the occurrence of an underlying CDC73 gene mutation, and these cases should preferably be investigated more thoroughly with parafibromin immunohistochemistry and/or CDC73 gene sequencing. Pinpointing Parathyroid Carcinoma in the Diagnostic Setting Parathyroid carcinoma is the malignant form of PHPT. It constitutes only 0.5–3% of PHPT cases. As many parathyroid tumors present with various degrees of atypia, although not fulfilling the current WHO criteria for parathyroid carcinoma, there is a great need to identify malignant potential in these lesions by other means than histology alone. Moreover, any clinical marker of value in this context would need to exhibit very high specificity, in order to avoid falsely diagnosing benign tumors as carcinoma. The bulk of studies on this subject have attempted to address this dilemma via immunohistochemistry, which is a gold standard, inexpensive methodology used in most pathology laboratories and therefore suitable for rapid implementation in routine clinical practice . One of the earliest immunohistochemical markers proposed to identify parathyroid carcinoma was Ki-67, an extensively used proliferation marker used to identify cells in active (non-G0) phases of the cell cycle. Although there seem to be an overrepresentation of carcinomas among high-proliferative parathyroid tumors, the overlap between parathyroid adenomas and carcinomas is considerable . In a similar fashion, investigations of the cell cycle protein cyclin D1 was initially proven of value, as it was found upregulated in most parathyroid carcinoma compared to adenomas . However, subsequent studies have identified overlap in expression between benign and malignant groups . Detection of widespread LOH of the RB1 locus in parathyroid carcinoma with loss of its corresponding protein pRB prompted the investigation of pRB expression in parathyroid tumors—but with divergent results both concerning staining outcomes as well as LOH events . In short, while LOH of the RB1 locus seem to confer high sensitivity for the detection of parathyroid carcinoma, a significant amount of adenomas harbor the same genetic aberrancy, and the reduced specificity renders this marker of less value when screening for rare carcinomas in PHPT. Moreover, a positive pRb staining seems to be variable in terms of the proportion tumor cells stained, making the interpretation somewhat challenging . Following the advent of tissue microarrays, a study concluded that the combined positivity of p27, B-cell lymphoma 2 (bcl-2), and mouse double minute 2 homolog (mdm2) adjoined by a low Ki-67 proliferation index indicated a benign clinical behavior of any given parathyroid tumor, as this profile was not evident in any parathyroid carcinoma but found in the vast majority of adenomas . Both bcl-2 and mdm2 are members of the P53 pathway, thereby furthermore solidifying the relationship between aberrant P53 signaling and parathyroid carcinoma. However, follow-up studies have demonstrated overlaps between parathyroid adenomas and carcinomas, not least the finding of down-regulation of p27 in large subsets of parathyroid adenomas . P53 immunoreactivity shows variable staining in benign and malignant parathyroid tumors and does not appear to be useful as a single marker in separating these tumors . Other diagnostic nomograms have been evaluated to differentiate malignant from benign parathyroid neoplasms, including among others protein gene product 9.5 (PGP 9.5), Ki-67, galectin-3, E-cadherin, and pRb markers . More novel combinations of immunohistochemical and in situ hybridization approaches have also been evaluated, such as long noncoding RNA expression . The most well-studied and reproduced marker in the context of distinguishing parathyroid carcinoma from adenoma is parafibromin, the 531 amino acid protein product of the CDC73 main transcript . Given the increased parathyroid carcinoma risk in HPT-JT kindred as well as the association between somatic CDC73 mutations and sporadic parathyroid carcinoma, subsequent immunohistochemical analyses regarding parafibromin early-on proved of value to detect the majority of CDC73 mutated cases, including most parathyroid carcinomas . Moreover, the vast majority of parathyroid adenomas analyzed have been shown to retain parafibromin immunoreactivity, apart from subsets of sporadic cases with a predominant cystic growth pattern, HPT-JT-associated parathyroid adenomas, and atypical parathyroid tumors . Thus, with the exceptions of the previously mentioned exceptions, parafibromin is useful for screening purposes in the PHPT population and can be a highly useful marker of malignant potential in many cases. The value of this marker in the clinical setting has been assessed both by comprehensive meta-analyses and by recent reports from high-volume centers . As parafibromin is a predominant nuclear protein, loss of nuclear immunoreactivity is considered pathognomonic for an underlying inactivating CDC73 mutation. As most CDC73 mutations, be it germline or somatic, are either deleterious, located in early exons (causing premature termination) or missense alterations in important regulatory regions (such as the nuclear localization regions), mutated parafibromin is usually unable to reach its nuclear localization . Most studies therefore seem to agree that almost all parathyroid tumors with wild-type CDC73 sequence display a diffuse positive parafibromin stain (Fig. a), while reduced or absent nuclear parafibromin expression is an aberrant staining pattern and might signal the presence of a mutation (Fig. b, d, e). While diffuse loss of parafibromin expression is usually seen, reduced (but not complete) nuclear parafibromin expression can also be noted in CDC73 mutated cases, occurring in subsets of tumor nuclei in a chessboard type of pattern (Fig. b) . Additionally, rare cases with parafibromin-negative nucleolar compartments have also been reported, which could be of importance given the nucleolar roles of wild-type parafibromin . Specifically, subsets of parathyroid tumors with CDC73 mutations thought to disrupt the nucleolar localization signals might exhibit retained nuclear parafibromin immunoreactivity while evidently displaying negative nucleolar staining—and practicing pathologist should therefore be aware of this staining pattern as well (Fig. c) . As most studies focus on the 2H1 monoclonal parafibromin antibody targeting the N-terminal, the usage of different antibodies should not explain these different staining patterns. Indeed, the “partial loss” pattern described above has been reported in cases assessed with four different parafibromin antibodies, suggesting that these observations are not biased by the selection of a specific target epitope . The interpretation of parafibromin immunohistochemistry is therefore not completely straightforward, and additionally, the laboratory processing (including different antigen retrieval techniques and primary antibody incubation time) might affect the overall staining outcome . Moreover, parafibromin expression is often found stronger in the tumor periphery than in central aspects of the lesion, and therefore internal controls (such as endothelial cells) should be assessed and noted as positive before a negative parafibromin stain is called out (Fig. d, e) . Most parathyroid tumors will be assessed for parafibromin immunoreactivity if they are atypical, i.e., lack undisputable evidence of malignancy but display worrisome features (be it clinical or histological) often seen in parathyroid carcinomas. The preponderance of these tumors stain positive, although large subsets can be parafibromin deficient . How should these cases be interpreted, and what should be reported to the surgeon? Consulting the literature, it seems evident the vast majority parafibromin-negative atypical tumors will behave benign, even after long-term follow-up . Even so, the recurrence rate is not entirely negligible, and the finding of an aberrant parafibromin stain should also highlight the need to exclude an underlying germline CDC73 gene mutation—especially if there is a positive family history or if the proband itself exhibits multiglandular disease (Fig. ) . Given the non-perfect parafibromin sensitivity and specificity, researchers have been looking for additional markers to complement parafibromin immunohistochemistry. Shortly after parafibromin was coupled to the Wnt pathway, investigations of Wnt regulators detected widespread loss of APC immunoreactivity in most parathyroid carcinomas—which has later been coupled to promoter hypermethylation in PCs (Fig. f) . As parathyroid adenomas are APC positive in general, the marker has gained ground as a clinical adjunct to parafibromin . However, as panels with two negative markers have its limitations in terms of interpretation associated to tissue fixation, researchers have looked for markers upregulated in parathyroid carcinomas. In this aspect, galectin-3 and PGP9.5 have both been shown to stain positive in the majority of parathyroid carcinomas while only expressed in few adenomas, and different panel combinations using galectin-3 and/or PGP9.5 with parafibromin have proven more reliable than using parafibromin alone . Overall, the parathyroid carcinoma diagnosis is reserved for cases exhibiting unequivocal histological signs of invasive behavior, and no single molecular marker has yet been able to safely predict the malignant potential in atypical cases—which is most likely influenced by the general abundance of atypia in benign tumors and the rarity of truly metastatic cases. Moreover, there is also a potential sampling bias, as malignant tumors are not primarily resected if advanced disease is present at the time of diagnosis, and the fact that tumors with a molecular potential for spread might be resected well before their dissemination. The MEN1 syndrome is by far the most common among the hereditary conditions detailed in this review, with one case per 40,000 as opposed to MEN2A (1 case per 2 million) and the HPT-JT syndrome (unknown prevalence, but expected to be exceedingly low) . Therefore, surgical pathologists will most likely diagnose MEN1 related parathyroid adenomas to a much larger extent than MEN2A, HPT-JT, FIHP, or MEN1-like related cases. Although the MEN1 syndrome often is diagnosed long before the patient is subjected to parathyroidectomy, the non-total penetrance in younger years as well as the occurrence of de novo MEN1 mutations in subsets of patients with healthy parents allow for subsets of patients being misclassified as sporadic PHPT patients. As up to 10% of individuals with primary parathyroid “hyperplasia” (multiglandular disease, adenomatosis, multiple adenomas) have MEN1, genetic screening for familial disease has been considered for all patients with primary parathyroid “hyperplasia.” From a diagnostic pathology standpoint, is there a way for the attentive pathologist to aid in the detection of syndromic PHPT in cases where the syndromic association was not evident preoperatively? MEN1 was early on assigned a tumor-suppressor gene status, as the bulk of mutational events in MEN1 kindred reported represent inactivating events leading to premature truncations of the menin protein . Moreover, most parathyroid adenomas arising in MEN1 patients harbor loss of heterozygosity (LOH) of the MEN1 wild-type allele on the somatic level, thereby arguing in favor for the Knudson’s “two hit” theory in which bi-allelic inactivation of a tumor suppressor gene is needed in order for a tumor to develop . Therefore, the question arises if clinical screening using expressional analyses targeting menin could be a cheap and efficient way for the surgical pathologist to triage menin-negative PHPT cases for genetic screening—as loss of menin expression would be indicative of MEN1 gene aberrancies. Menin is a predominantly nuclear protein with a highly conserved sequence among species, and the protein is universally expressed in most human tissues . The protein harbors nuclear localization signals and leucine zipper motifs, both of which are features needed to regulate gene expression though direct interaction with DNA elements. For example, menin has been shown to interact with JunD , a proto-oncogene encoding a transcription factor regulating apoptosis and TP53 gene activation (Fig. ) . Moreover, menin has been proposed as an important player in the regulation of various signaling networks associated to tumor development, such as the TGF-beta, RAS, Wingless type (Wnt) pathways—as well as influencing the expression of the telomerase reverse transcriptase ( TERT ) gene . From a histological context, the MEN1-related parathyroid disease is often multiglandular, although not always synchronous in presentation . Unlike sporadic parathyroid adenomas, MEN1-related tumors are often devoid of a rim of normal parathyroid tissue, and they may be composed of chief cells arranged in compact, sheet-like formations . The tumors may exhibit fibrosis and have similar appearance as parathyroid hyperplastic lesions arising in the setting of chronic renal failure, and almost always lack invasive features. Overall, findings of multiglandular disease and absence of a normal parathyroid rim in a PHPT patient should raise the suspicion of the MEN1 syndrome; however, a “rim” can also be seen in up to 10% of multiglandular disease. In all, there is no histological feature that sets MEN1-related adenomas apart from sporadic ones. Expression studies regarding menin and associations to underlying MEN1 mutational status in parathyroid tumors have been promising, as menin immunohistochemistry seem to exhibit high sensitivity to detect underlying MEN1 mutations and/or gene deletions . Even so, the interpretation of menin immunohistochemistry is not straightforward, and the lack of comprehensive studies on the subject makes menin immunohistochemistry inappropriate for clinical routine screening purposes. Moreover, as the MEN1 gene is frequently deleted and mutated also on the somatic level in sporadic parathyroid adenomas, additional factors are needed to bring into consideration before suspecting MEN1 syndrome–related PHPT from expressional analyses in the pathology laboratory. For example, the penetrance of PHPT in MEN1 patients reaches 95% at 50 years of age, and therefore, the diagnosis of MEN1 in older patients with multiglandular disease should not be completely overlooked—although the bar for considering an MEN1 diagnosis should be even lower in adolescent patients . In all, a concerted teamwork between endocrine surgeons and pathologists covering the patient’s medical history, disease presentation, radiology, and histopathology is probably needed to properly identify all MEN1-related PHPT in the clinical setting. As HPT-JT patients carry germline CDC73 gene mutations or deletions, it comes as no surprise that the prevalence of parathyroid carcinoma in this patient category is much higher (15–30%) than in unselected PHPT patient cohorts (< 1%). As parathyroid carcinoma is so uncommon, it has been suggested that individuals diagnosed with parathyroid carcinoma should be promptly evaluated for HPT-JT. Additionally, if the pathologist raises the suspicion that the patient is indeed an HPT-JT kindred, a raised awareness of the increased parathyroid carcinoma risk is mandated when diagnosing the tumor. Easily recognizable features such as a tumor being cystic may in itself raise the possibility of HPT-JT syndrome. In the most comprehensive study yet regarding morphological features of CDC73 mutated parathyroid tumors, the authors conclude that these lesions are characterized by sheet-like, compact growth rather than the usual acinar patterns visualized in the bulk of parathyroid tumors, as well as a typical eosinophilic cytoplasm distinct from the granular oxyphilic cell type often observed in areas of parathyroid adenomas . Many tumors also exhibited enlarged nuclei and a perinuclear cytoplasmic clearing. In all, these findings suggest that the occurrence of these histological parameters might suggest the occurrence of an underlying CDC73 gene mutation, and these cases should preferably be investigated more thoroughly with parafibromin immunohistochemistry and/or CDC73 gene sequencing. Parathyroid carcinoma is the malignant form of PHPT. It constitutes only 0.5–3% of PHPT cases. As many parathyroid tumors present with various degrees of atypia, although not fulfilling the current WHO criteria for parathyroid carcinoma, there is a great need to identify malignant potential in these lesions by other means than histology alone. Moreover, any clinical marker of value in this context would need to exhibit very high specificity, in order to avoid falsely diagnosing benign tumors as carcinoma. The bulk of studies on this subject have attempted to address this dilemma via immunohistochemistry, which is a gold standard, inexpensive methodology used in most pathology laboratories and therefore suitable for rapid implementation in routine clinical practice . One of the earliest immunohistochemical markers proposed to identify parathyroid carcinoma was Ki-67, an extensively used proliferation marker used to identify cells in active (non-G0) phases of the cell cycle. Although there seem to be an overrepresentation of carcinomas among high-proliferative parathyroid tumors, the overlap between parathyroid adenomas and carcinomas is considerable . In a similar fashion, investigations of the cell cycle protein cyclin D1 was initially proven of value, as it was found upregulated in most parathyroid carcinoma compared to adenomas . However, subsequent studies have identified overlap in expression between benign and malignant groups . Detection of widespread LOH of the RB1 locus in parathyroid carcinoma with loss of its corresponding protein pRB prompted the investigation of pRB expression in parathyroid tumors—but with divergent results both concerning staining outcomes as well as LOH events . In short, while LOH of the RB1 locus seem to confer high sensitivity for the detection of parathyroid carcinoma, a significant amount of adenomas harbor the same genetic aberrancy, and the reduced specificity renders this marker of less value when screening for rare carcinomas in PHPT. Moreover, a positive pRb staining seems to be variable in terms of the proportion tumor cells stained, making the interpretation somewhat challenging . Following the advent of tissue microarrays, a study concluded that the combined positivity of p27, B-cell lymphoma 2 (bcl-2), and mouse double minute 2 homolog (mdm2) adjoined by a low Ki-67 proliferation index indicated a benign clinical behavior of any given parathyroid tumor, as this profile was not evident in any parathyroid carcinoma but found in the vast majority of adenomas . Both bcl-2 and mdm2 are members of the P53 pathway, thereby furthermore solidifying the relationship between aberrant P53 signaling and parathyroid carcinoma. However, follow-up studies have demonstrated overlaps between parathyroid adenomas and carcinomas, not least the finding of down-regulation of p27 in large subsets of parathyroid adenomas . P53 immunoreactivity shows variable staining in benign and malignant parathyroid tumors and does not appear to be useful as a single marker in separating these tumors . Other diagnostic nomograms have been evaluated to differentiate malignant from benign parathyroid neoplasms, including among others protein gene product 9.5 (PGP 9.5), Ki-67, galectin-3, E-cadherin, and pRb markers . More novel combinations of immunohistochemical and in situ hybridization approaches have also been evaluated, such as long noncoding RNA expression . The most well-studied and reproduced marker in the context of distinguishing parathyroid carcinoma from adenoma is parafibromin, the 531 amino acid protein product of the CDC73 main transcript . Given the increased parathyroid carcinoma risk in HPT-JT kindred as well as the association between somatic CDC73 mutations and sporadic parathyroid carcinoma, subsequent immunohistochemical analyses regarding parafibromin early-on proved of value to detect the majority of CDC73 mutated cases, including most parathyroid carcinomas . Moreover, the vast majority of parathyroid adenomas analyzed have been shown to retain parafibromin immunoreactivity, apart from subsets of sporadic cases with a predominant cystic growth pattern, HPT-JT-associated parathyroid adenomas, and atypical parathyroid tumors . Thus, with the exceptions of the previously mentioned exceptions, parafibromin is useful for screening purposes in the PHPT population and can be a highly useful marker of malignant potential in many cases. The value of this marker in the clinical setting has been assessed both by comprehensive meta-analyses and by recent reports from high-volume centers . As parafibromin is a predominant nuclear protein, loss of nuclear immunoreactivity is considered pathognomonic for an underlying inactivating CDC73 mutation. As most CDC73 mutations, be it germline or somatic, are either deleterious, located in early exons (causing premature termination) or missense alterations in important regulatory regions (such as the nuclear localization regions), mutated parafibromin is usually unable to reach its nuclear localization . Most studies therefore seem to agree that almost all parathyroid tumors with wild-type CDC73 sequence display a diffuse positive parafibromin stain (Fig. a), while reduced or absent nuclear parafibromin expression is an aberrant staining pattern and might signal the presence of a mutation (Fig. b, d, e). While diffuse loss of parafibromin expression is usually seen, reduced (but not complete) nuclear parafibromin expression can also be noted in CDC73 mutated cases, occurring in subsets of tumor nuclei in a chessboard type of pattern (Fig. b) . Additionally, rare cases with parafibromin-negative nucleolar compartments have also been reported, which could be of importance given the nucleolar roles of wild-type parafibromin . Specifically, subsets of parathyroid tumors with CDC73 mutations thought to disrupt the nucleolar localization signals might exhibit retained nuclear parafibromin immunoreactivity while evidently displaying negative nucleolar staining—and practicing pathologist should therefore be aware of this staining pattern as well (Fig. c) . As most studies focus on the 2H1 monoclonal parafibromin antibody targeting the N-terminal, the usage of different antibodies should not explain these different staining patterns. Indeed, the “partial loss” pattern described above has been reported in cases assessed with four different parafibromin antibodies, suggesting that these observations are not biased by the selection of a specific target epitope . The interpretation of parafibromin immunohistochemistry is therefore not completely straightforward, and additionally, the laboratory processing (including different antigen retrieval techniques and primary antibody incubation time) might affect the overall staining outcome . Moreover, parafibromin expression is often found stronger in the tumor periphery than in central aspects of the lesion, and therefore internal controls (such as endothelial cells) should be assessed and noted as positive before a negative parafibromin stain is called out (Fig. d, e) . Most parathyroid tumors will be assessed for parafibromin immunoreactivity if they are atypical, i.e., lack undisputable evidence of malignancy but display worrisome features (be it clinical or histological) often seen in parathyroid carcinomas. The preponderance of these tumors stain positive, although large subsets can be parafibromin deficient . How should these cases be interpreted, and what should be reported to the surgeon? Consulting the literature, it seems evident the vast majority parafibromin-negative atypical tumors will behave benign, even after long-term follow-up . Even so, the recurrence rate is not entirely negligible, and the finding of an aberrant parafibromin stain should also highlight the need to exclude an underlying germline CDC73 gene mutation—especially if there is a positive family history or if the proband itself exhibits multiglandular disease (Fig. ) . Given the non-perfect parafibromin sensitivity and specificity, researchers have been looking for additional markers to complement parafibromin immunohistochemistry. Shortly after parafibromin was coupled to the Wnt pathway, investigations of Wnt regulators detected widespread loss of APC immunoreactivity in most parathyroid carcinomas—which has later been coupled to promoter hypermethylation in PCs (Fig. f) . As parathyroid adenomas are APC positive in general, the marker has gained ground as a clinical adjunct to parafibromin . However, as panels with two negative markers have its limitations in terms of interpretation associated to tissue fixation, researchers have looked for markers upregulated in parathyroid carcinomas. In this aspect, galectin-3 and PGP9.5 have both been shown to stain positive in the majority of parathyroid carcinomas while only expressed in few adenomas, and different panel combinations using galectin-3 and/or PGP9.5 with parafibromin have proven more reliable than using parafibromin alone . Overall, the parathyroid carcinoma diagnosis is reserved for cases exhibiting unequivocal histological signs of invasive behavior, and no single molecular marker has yet been able to safely predict the malignant potential in atypical cases—which is most likely influenced by the general abundance of atypia in benign tumors and the rarity of truly metastatic cases. Moreover, there is also a potential sampling bias, as malignant tumors are not primarily resected if advanced disease is present at the time of diagnosis, and the fact that tumors with a molecular potential for spread might be resected well before their dissemination. The molecular background of parathyroid neoplasia is well characterized from a driver gene perspective, not least due to the identification of mutations in genes responsible for the development of hereditary PHPT. These genes were subsequently found aberrant also in sporadic cases, and still—some 20 years after original identification—enjoy their position as the top recurrently mutated genes in parathyroid tumors. Modern molecular analyses have since then expanded our knowledge regarding common dysregulations in parathyroid tumors, and highlighted both genetic and epigenetic changes involved in the development of PHPT. Next-generation sequencing analyses of both parathyroid adenoma and carcinoma cohorts have helped identify additional genes of potential impact for both groups, although it is clearly demonstrated that the “low-hanging fruit” in terms of highly recurrent events already have been picked. From a clinical perspective, CDC73 mutations and loss of parafibromin expression have been firmly established as events coupled to parathyroid malignancy. Additionally, comprehensive sequencing of parathyroid carcinoma has identified mutations suitable for molecular targeted therapy as well as overall mutational burden as a prognostic tool of potential value, thereby highlighting the usage of modern genetic analyses where histology and immunohistochemistry might be insufficient. Given the rapid evolution of molecular techniques used for complimentary analyses in the routine clinical setting, the combination of histology, immunohistochemistry and next-generation sequencing might constitute standard work-up for parathyroid tumors displaying significant atypia in the near future. To summarize the field for the surgical pathologist, parathyroid adenoma is usually a straightforward diagnosis, but there are histological patterns coupled to underlying syndromes worth remembering—as they could help identify hereditary disease of importance for clinical follow-up. When present, histologically atypical features in a parathyroid lesion could signify malignant potential, but invasive growth is still the only accepted criteria to diagnose parathyroid carcinoma. A plethora of immunohistochemical markers could potentially aid in the identification of parathyroid carcinoma, but many display subpar specificity and should be interpreted with caution. Loss of parafibromin nuclear expression correlates with the presence of an underlying CDC7 3 gene mutation, which in turn is coupled to an increased risk of developing parathyroid carcinoma. The mutation might also be present in germline tissues and thus predispose to familial disease. Even though much of the genetic landscape of parathyroid tumors has been studied, two key queries remain to be deciphered, namely (1) the identification of additional driver genes responsible for the development of parathyroid adenomas, and (2) the quest for additional molecular events driving the malignant potential of parathyroid tumors besides parafibromin. As approximately half of parathyroid adenomas lack mutations in the most common driver genes, additional genetic events are expected to be found. Moreover, as the majority of HPT-JT kindred develop adenomas, other genetic aberrations apart from the inactivation of CDC73 are likely to be required to propel the invasive behavior in parathyroid carcinomas. Hopefully, these questions can be solved in the future by comprehensive, next-generation sequencing studies using multi-center tumor cohorts. |
Pharmacogenetics of | a5c5e4d3-2774-49cc-be78-b56c3f063892 | 10026301 | Pharmacology[mh] | INTRODUCTION Acute myeloid leukemia (AML) comprises a heterogenous group of diseases with dismal outcomes mainly in developing countries. Identification of genetic alterations is key for risk stratification, as it guides to appropriate treatment. Most patients with AML are classified as intermediate or unfavorable risk, usually leading to failure of induction therapy and early relapse after achieving complete remission. The identification of gene variants that participate in the processes of pharmacodynamics and pharmacokinetics (pharmacogenetics), have become relevant in the era of personalized medicine, in terms of the influence they exert on the response to treatment, , , and the incorporation of interindividual genetic differences for the design of more effective diagnostic and therapeutic strategies. , , , , Cytarabine (Cytosine arabinoside Ara‐c) and anthracyclines are routinely used in front‐line therapy for AML. , , , Resistance development to chemotherapy is a major obstacle in AML treatment and is responsible for relapses and increased toxicity in second‐line therapies. Ara‐C is a pyrimidine analog which is converted into ara‐CMP by deoxycytidine kinase ( DCK ), later on is converted to ara‐CDP and into ara‐CTP by cytidine/uridine monophosphate kinase 1 ( CMPK1 ) and nucleoside diphosphate kinase 1 (NME1), respectively. Ara‐CTP is a competitor of deoxycytidine 5′‐triphosphate that acts by inhibiting DNA synthesis. Cytidine deaminase ( CDA ) converts ara‐C to the inactive metabolite uracil arabinoside (ara‐U), which limits the amount of ara‐C to be converted to ara‐CTP. , , , ABCB1 , also known as multi‐drug resistance protein 1 (MDRP1), is 1 of 49 putative members in the superfamily of human adenosine triphosphate (ATP)‐binding cassette (ABC) transporters that encode transporter and channel proteins that function as efflux pumps, , codifies a P‐glycoprotein efflux transporter involved in mediating resistance to several drugs, multidrug resistance phenotype, in cancer , (Figure ). Different studies have shown that single nucleotide variants (SNV) in ABCB1, CDA, DCK, GSTT1 , and GSTM1 genes are related to drug toxicity in patients with AML. Two main SNV in the deoxycytidine kinase ( DCK ) gene (‐360C > T, rs377182313) and (‐201C > A, rs2306744), have been described associated to pharmacogenetic responses. Whether DCK mutations make AML cells resistant to cytarabine is controversial. Cytidine deaminase ( CDA ) irreversibly deaminates cytarabine, its overexpression results in Ara‐C resistance, while decreased expression is associated with toxicity. Two SNV in CDA gene have been found associated with pharmacogenetic responses, (79A > C, rs2072671) and (‐451C > T, rs532545). , For ABCB1 , three SNV have been associated to pharmacogenetic responses, ABCB1 (1236G > A, rs1128503), ABCB1 (2677C > A/T, rs2032582) and ABCB1 (3435G > A, rs1045642). , , Several antineoplastic drugs are metabolized by glutathione S‐transferase (GST) which catalyzes the conjugation of reduced glutathione to electrophilic centers of platinum drugs, anthracyclines, vinca alkaloids, cyclophosphamide, and epipodophylotoxins. The GSTT1 gene encodes the phase II metabolizing enzyme glutathione s‐transferase theta and GSTM1 encodes glutathione S‐transferase mu 1. The most studied variants of GSTT1 and GSTM1 are the null variant, which results from the complete or partial deletion of these genes. It has been suggested that individuals lacking GSTT1 and or GSTM1 have an impaired ability to detoxify environmental xenobiotics and are thus at elevated risk for cellular damage and resultant cancer. In the present study we analyzed 6 SNV in ABCB1 gene (3435C > T, rs1045642), (1236G > A, rs1128503), (2677G > T/A, rs2032582); CDA gene (79A > C, rs2072671), (‐451C > T, rs532545); DCK gene (‐201C > A, rs2306744), and the presence or absence of the GSTT1 and GSTM1 null alleles and their correlation with clinical outcomes and toxicity in a cohort of pediatric patients with AML from two pediatric cancer treatment centers in Colombia. MATERIALS AND METHODS 2.1 Patients Descriptive observational cohort study, 51 pediatric patients with a confirmed diagnosis of de novo AML (non‐promyelocitic) by convenience were included, with prior informed consent between March 2015 and June 2021, from HOMI Fundación Hospital Pediátrico La Misericordia and Clínica Infantil Colsubsidio Bogotá D.C., Colombia. Patients with Down syndrome or secondary AML were not included. This project was approved by the ethics committee of each institution, CEI 125‐18 and 243‐1, respectively, and the ethics committee of Universidad Nacional de Colombia (007‐091‐18). Patients received two cycles of induction chemotherapy including cytarabine 100 mg/m 2 /day for 7 days and daunorubycin 60 mg/m 2 /day for 3 days “7 × 3 cycle.” Consolidation chemotherapy was based on cytarabine high dose 3 mg/m 3 /day for 3 days, 2 or 3 cycles maximum. Minimal residual disease (MRD) evaluation was made based on multiparametric flow cytometry on bone marrow samples. Residual disease was detected using the leukemia‐associated immunophenotype at diagnosis and at follow‐up samples. A cutoff value of 0.1% was used as the threshold to distinguish MRD‐positive from MRD‐negative patients, acquiring around 2.5 million events (excluding all CD45‐negative cells and debris). 2.2 Pharmacogenetic testing We used a SNaPShot™ (Thermo Fisher Scientific) panel to simultaneously test for SNV (3435G > A, rs1045642), (1236C > T, rs1128503), (2677C > A/T, rs2032582) in ABCB1 gene; (79A > C, rs2072671), (‐451C > T, rs532545) CDA gene, and separately, (‐201C > A, rs2306744) in DCK gene. Primers and probes used for the SNaPshot™ assay are listed in Table . Primers were designed in order to perform a multiplex PCR reaction using Qiagen 2X PCR multiplex master mix (Qiagen). PCR amplification conditions were an initial denaturing step at 95°C for 15 min, followed by 32 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 90 s followed by extension at 72°C for 60 s, with a final extension step at 72°C for 10 min. For DCK rs2306744, PCR amplification as described previously. Amplified products were purified according to the SNaPshot™ protocol, following manufacturers' recommendations. An aliquot of the purified sample was hybridized with probes designed to align to the specified genetic variant with an additional tail of nonhuman DNA sequences to obtain better separation of each variant. The purified PCR products were analyzed in a ABI3500 Genetic Analyzer using Liz120 as sizing standard and analysis software GeneMapper 4.2 (Applied Biosystems, Thermo Fisher Scientific). The GSTT1 and GSTM1 deletions were analyzed by conventional PCR (Table ). Each reaction also contained a control gene ABCB1 (rs1045642) for amplification control. PCR amplification conditions were an initial denaturing step at 95°C for 3 min, followed by 32 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s followed by extension at 72°C for 60 s, with a final extension step at 72°C for 10 min. The PCR products were resolved by 2% Nusieve gel electrophoresis in 1X TBE buffer. 2.3 Genotype, allele frequencies, and Hardy Weinberg equilibrium Genotype and allele frequencies were determined by direct counting method. Hardy Weinberg equilibrium was calculated based on observed and expected genotype frequencies. Genotypes for each gene variant obtained for each sample tested are listed in Table . 2.4 Statistical analysis Quantitative variables were reported as means or medians with dispersion measures given in standard deviation and ranges, according to the nature and distribution of the variables, based on Shapiro Wilks normality test to establish the use of parametric tests or non‐parametric. We evaluated the association of genotypes from each gene variant, as well as the co‐occurrence of genotypic variants in the ABCB1, DCK , and CDA with clinical outcomes and toxicity using Chi‐square. Qualitative variables were analyzed with Pearson's Chi‐square test and Fisher's exact test. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 25.0. A p ‐value <.05 was considered significant. Logistic regression analysis was performed to analyze the relationship of the different variables with outcomes, MRD, relapse, event‐free survival, and overall survival. 2.5 Outcomes and definitions Organ toxicity was evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0, grading Scales 3–4 included: colitis, mucositis, cardiotoxicity, transaminitis, and aspergilosis (Table ). In addition, we also evaluated the efficacy of induction therapy, after 2 cycles 7 × 3, with complete remission (less than 5% of morphological blast count in bone marrow smear and hematological recovery in peripheral blood with platelet count >50 000/μl, >1000/μl leukocytes and absolute neutrophil count >500/μl) and induction failure (>5% of morphological blast count in bone marrow smear and without hematological recovery in peripheral blood counts), relapse and HSCT related toxicity. Other outcomes were overall survival, defined as the time between diagnosis and last contact alive or dead; event‐free survival was defined as the time between diagnosis and death, induction failure, relapse, abandonment, change of treatment institution, or last contact alive. Patients Descriptive observational cohort study, 51 pediatric patients with a confirmed diagnosis of de novo AML (non‐promyelocitic) by convenience were included, with prior informed consent between March 2015 and June 2021, from HOMI Fundación Hospital Pediátrico La Misericordia and Clínica Infantil Colsubsidio Bogotá D.C., Colombia. Patients with Down syndrome or secondary AML were not included. This project was approved by the ethics committee of each institution, CEI 125‐18 and 243‐1, respectively, and the ethics committee of Universidad Nacional de Colombia (007‐091‐18). Patients received two cycles of induction chemotherapy including cytarabine 100 mg/m 2 /day for 7 days and daunorubycin 60 mg/m 2 /day for 3 days “7 × 3 cycle.” Consolidation chemotherapy was based on cytarabine high dose 3 mg/m 3 /day for 3 days, 2 or 3 cycles maximum. Minimal residual disease (MRD) evaluation was made based on multiparametric flow cytometry on bone marrow samples. Residual disease was detected using the leukemia‐associated immunophenotype at diagnosis and at follow‐up samples. A cutoff value of 0.1% was used as the threshold to distinguish MRD‐positive from MRD‐negative patients, acquiring around 2.5 million events (excluding all CD45‐negative cells and debris). Pharmacogenetic testing We used a SNaPShot™ (Thermo Fisher Scientific) panel to simultaneously test for SNV (3435G > A, rs1045642), (1236C > T, rs1128503), (2677C > A/T, rs2032582) in ABCB1 gene; (79A > C, rs2072671), (‐451C > T, rs532545) CDA gene, and separately, (‐201C > A, rs2306744) in DCK gene. Primers and probes used for the SNaPshot™ assay are listed in Table . Primers were designed in order to perform a multiplex PCR reaction using Qiagen 2X PCR multiplex master mix (Qiagen). PCR amplification conditions were an initial denaturing step at 95°C for 15 min, followed by 32 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 90 s followed by extension at 72°C for 60 s, with a final extension step at 72°C for 10 min. For DCK rs2306744, PCR amplification as described previously. Amplified products were purified according to the SNaPshot™ protocol, following manufacturers' recommendations. An aliquot of the purified sample was hybridized with probes designed to align to the specified genetic variant with an additional tail of nonhuman DNA sequences to obtain better separation of each variant. The purified PCR products were analyzed in a ABI3500 Genetic Analyzer using Liz120 as sizing standard and analysis software GeneMapper 4.2 (Applied Biosystems, Thermo Fisher Scientific). The GSTT1 and GSTM1 deletions were analyzed by conventional PCR (Table ). Each reaction also contained a control gene ABCB1 (rs1045642) for amplification control. PCR amplification conditions were an initial denaturing step at 95°C for 3 min, followed by 32 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s followed by extension at 72°C for 60 s, with a final extension step at 72°C for 10 min. The PCR products were resolved by 2% Nusieve gel electrophoresis in 1X TBE buffer. Genotype, allele frequencies, and Hardy Weinberg equilibrium Genotype and allele frequencies were determined by direct counting method. Hardy Weinberg equilibrium was calculated based on observed and expected genotype frequencies. Genotypes for each gene variant obtained for each sample tested are listed in Table . Statistical analysis Quantitative variables were reported as means or medians with dispersion measures given in standard deviation and ranges, according to the nature and distribution of the variables, based on Shapiro Wilks normality test to establish the use of parametric tests or non‐parametric. We evaluated the association of genotypes from each gene variant, as well as the co‐occurrence of genotypic variants in the ABCB1, DCK , and CDA with clinical outcomes and toxicity using Chi‐square. Qualitative variables were analyzed with Pearson's Chi‐square test and Fisher's exact test. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 25.0. A p ‐value <.05 was considered significant. Logistic regression analysis was performed to analyze the relationship of the different variables with outcomes, MRD, relapse, event‐free survival, and overall survival. Outcomes and definitions Organ toxicity was evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0, grading Scales 3–4 included: colitis, mucositis, cardiotoxicity, transaminitis, and aspergilosis (Table ). In addition, we also evaluated the efficacy of induction therapy, after 2 cycles 7 × 3, with complete remission (less than 5% of morphological blast count in bone marrow smear and hematological recovery in peripheral blood with platelet count >50 000/μl, >1000/μl leukocytes and absolute neutrophil count >500/μl) and induction failure (>5% of morphological blast count in bone marrow smear and without hematological recovery in peripheral blood counts), relapse and HSCT related toxicity. Other outcomes were overall survival, defined as the time between diagnosis and last contact alive or dead; event‐free survival was defined as the time between diagnosis and death, induction failure, relapse, abandonment, change of treatment institution, or last contact alive. RESULTS Fifty‐one patients were included, demographic and clinical characteristics are shown in Table . Thirty patients (58%) were male, M:F ratio 1.4:1, the median age was 10 years (0.15–18 years), median leukocytes at diagnosis was 25 580 × 10 9 /L (1.190–1.896.000), IQR 87020, and CNS involvement in 16 patients (31%). One patient died before starting treatment. Thirty‐three (65%) patients required HSCT. Demographic and clinical data are shown in Table . Genotypes and allele frequencies for each of the gene variants analyzed for ABCB1, CDA, DCK, GSTT1 , and GSTM1 are shown in Table . All genotypes tested were found to be in Hardy–Weinberg equilibrium (data not shown). Several associations between genotypic variants and toxicity outcomes were found. First, we analyzed each genotypic variant independently and later on, genotypic associations within each gene or between genes. Odds ratios, CI 95%, and p ‐values are shown in Table . We found that patients carrying ABCB1 (1236C > T, rs1128503) GG genotype in had a 6.8 OR (CI 95% 1.08–42.73, p = .044) for cardiotoxicity at the end of induction, compared to patients carrying either AA or GA genotypes 0.14 OR (CI 95% 0.023–0.92, p = .044). Patients carrying ABCB1 (3435C > T, rs1045642) GG genotype had a 4.51 OR (CI 95% 1.15–17.75, p = .032) for transaminitis, as opposed to those carrying either AA or GA genotypes 0.22 OR (CI 95% 0.05–0.87, p = .032). Also, ABCB1 (3435C > T, rs1045642) AA genotype was identified as a protective factor for relapse 0.69 OR (CI 95% 0.56–0.85, p = .025), compared to those patients with either GG or GA genotype 1.44 OR (CI 95% 1.17–1.78, p = .025). For ABCB1 (1236G > A rs1128503/2677C > A/T rs2032582/3435G > A rs1045642) AA/AA/AA combined genotypes, a strong association was found with death after HSTC OR 13.73 (CI 95% 1.94–97.17, p = .009). In addition, these genotypes were protective factors against relapse 0.632 OR (CI 95% 0.495–0.805, p = .040). Measurable residual disease (MRD) >0.1% after first cycle of chemotherapy was associated with ABCB1 (1236G > A rs1128503/2677C > A/T rs2032582/3435G > A rs1045642) genotypes GG/CC/GG in addition to CDA (79A > C, rs2072671) CA genotype with 4.11 OR (CI 95% 2.32–725, p = .007), and CDA (−451G > A rs532545) CT genotype also was associated with 3.8 OR (CI 95% 2.23–6.47, p = .027). ABCB1 (1236G > A rs1128503/2677C > A/T rs2032582/3435G > A rs1045642) genotypes GG/CC/GG in addition to CDA (79A > C, rs2072671) CA genotype, showed a risk association with MRD >0.1% after first chemotherapy cycle 4.11 OR (CI 95% 2.32–725, p = .007), and CDA (−451G > A rs532545) CT genotype also was associated with MRD >0.1% after first chemotherapy cycle 3.8 OR (CI 95% 2.23–6.47, p = .027). Genotype GA in DCK (‐201C > A, rs2306744) is a protective factor to develop toxicity related to HSCT 0.8 OR (CI 95% 0.36–0.68, p = .046). CDA (−451G > A, rs532545) genotype CC was found to be a protective factor for colitis 0.2 OR (CI 95% 0.048–0.828, p = .019) in our cohort. Combined genotypes for CDA (−451G > A rs532545) and (79A > C, rs2072671) CC/AA were associated with increased risk for mucositis and liver toxicity after the first 7 × 3 cycle and after consolidation, while genotypes CT/CA were a protective factor. We did not find any association between GSTT1 and GSTM1 null alleles with clinical or toxicity events. A logistic regression model was performed to evaluate the presence of independent predictors associated with relapse, finding a positive association for event‐free survival (relapse or death) and for overall survival with the presence of ABCB1 1236/2677/3435 GA/CA/GA 9.086 OR (CI 95% 1.669–49.466, p = .011). No associated predictors were found for overall survival or MRD. DISCUSSION Few studies have analyzed pharmacogenetic risk associations in pediatric AML patients. Most patients with AML are classified, using conventional cytogenetics, recurrent mutations, and response at the end of induction using morphological or MRD counts at different timelines, there are no prognostic genomic or molecular criteria routinely used to identify patients at risk of chemotherapy failure. Genetic background for genes involved in pharmacological response represents an additional factor in treatment response. , , Identification of pharmacogenetic determinants are potential predictive markers for treatment‐related adverse events and toxicity and in establishing differences in treatment schemes or intensification of therapy in post‐induction phase. Green et al, informed association between ABCB1 (1236C > T, rs1128503), GG genotype with decreased survival when treated with cytarabine in AML patients as compared with AA + AG genotypes, in our cohort we found for ABCB1 (1236C > T, rs1128503) GG genotype an increased risk for cardiotoxicity 6.8 OR (CI 95% 1.08–42.73, p = .044), while AA or GA genotype were a protective factor against cardiotoxicity OR 0.14 (CI 95% 0.023–0.92, p = .044). In the same study, ABCB1 (2677C > A/T, rs2032582) CC genotype was associated with decreased survival when patients with AML were treated with cytarabine compared to genotypes AA + AC, we did not find any association between ABCB1 (2677C > A/T rs2032582) CC genotype and survival or toxicity. ABCB1 (3435G > A, rs1045642) GG genotype was associated with increased likelihood of complete remission when treated with cytarabine and idarubicin in AML patients as compared to AA + AG genotypes, other study showed for ABCB1 (3435G > A, rs1045642) AA + AG genotypes were associated with increased overall survival when treated with cytarabine in AML patients as compared to genotype GG. In our patients for ABCB1 (3435G > A, rs1045642) GG genotype we found an association with toxicity, and not with the response at the end of induction. While with AA genotype we have a protective factor for relapse 0.69 OR (CI 95% 0.56–0.85, p = .025) in contrast with the study mentioned previously. In a previous study, the ABCB1 triple variant haplotype ABCB1 (1236G > A rs1128503/2677C > A/T rs2032582/3435G > A rs1045642) TT/TT/TT was related to increased nephrotoxicity than other genotypes. For the combined genotypes ABCB1 (1236G > A rs1128503/2677C > A/T rs2032582/3435G > A rs1045642) in our patients, we found that the haplotype AA/AA/AA was a protective factor against relapse 0.632 OR (CI 95% 0.495–0.805, p = .040). However, the same genotypes were a risk factor for death after HSCT 13.73 OR (CI 95% 1.94–97.17, p = .009). On the other hand, ABCB1 (1236G > A rs1128503/2677C > A/T rs2032582/3435G > A rs1045642) combined genotypes GA/CA/GA was a risk factor for relapse 4.3 OR (CI 95% 1.09–17.10, p = .037) in our cohort. In a study of adult AML patients, ABCB1 (1236G > A rs1128503/2677C > A/T rs2032582/3435G > A rs1045642) AA/AA/AA genotypes analyzed with other SNV genes showed an increased risk for nephrotoxicity and liver toxicity. In a previous study, DCK gene (‐360C > T, rs377182313) and (‐201C > A, rs2306744) were evaluated among AML adult patients. They found that patients with (‐360C > T, rs377182313) CG and (‐201C > A, rs2306744) CT and (‐360C > T, rs377182313) GG and (‐201C > A, rs2306744) TT compound genotypes displayed a favorable response to chemotherapy and increased expression of dCK mRNA, whereas those with (‐360C > T, rs377182313) CC and (‐201C > A, rs2306744) CC tended to have a poor response and lower expression of mRNA ( p = .025 and p = .0034, respectively). Although (‐360C > T, rs377182313) was not included, no association was found for (‐201C > A, rs2306744) in this cohort. Previously, CDA (79A > C, rs2072671) CC and DCK (‐201C > A, rs2306744) CC genotypes were associated with increased risk of death and DCK (‐201C > A, rs2306744) CC genotype as a risk factor for toxicity grades >3 in a cohort of 27 Mexican patients. We did not find any of these associations, instead, DCK (‐201C > A, rs2306744) GA genotype was found to be a protective factor for toxicity after HSCT. In another study, the CDA (79A > C, rs2072671) CC genotype was associated with increased cytotoxicity when exposed to cytarabine in AML patients as compared with AA genotype. There was no association between this genotype and any toxicity in our cohort. Megias‐Vericat et al, found that CDA (79A > C, rs2072671) AC genotype was associated with overall survival at 5 years 2.2 OR (CI 95% 1.2–4.5, p = .015), event free survival 1.9 OR (CI 95% 1.01–3.4, p = .045) and relapse free survival 9.1 OR (CI 95% 1.2–68.6, p = .032). There was no association between this genotype and better clinical outcomes in our cohort. Also, CDA (‐451C > T, rs532545) TT genotype was associated with increased cytotoxicity when exposed to cytarabine in AML patients as compared to CC genotype. In our cohort, we found that CDA (‐451C > T, rs532545) CC genotype was a protective factor against colitis 0.2 OR (CI 95% 0.048–0.828, p = .019). Parmar et al. reported CDA 79A > C rs2072671 AC + CC genotypes associated with increased drug toxicity when treated with cytarabine as compared to AA genotype in an in vitro assay on healthy volunteers. In our cohort, no effects were found for any combined CDA genotypes. In our cohort, no association was found between ABCB1 (1236G > A rs1128503/2677C > A/T rs2032582/3435G > A rs1045642) AA/AA/AA genotypes and toxicity outcomes when combined with CDA (79A > C, rs2072671) or (‐451C > T, rs532545) and DCK (‐201C > A, rs2306744). However, ABCB1 (1236G > A rs1128503/2677C > A/T rs2032582/3435G > A rs1045642) GG/CC/GG genotype in addition to CDA (79A > C, rs2072671) CA or CDA (‐451C > T, rs532545) CT genotypes showed a higher risk for MRD >0.1% at the end of the first cycle of induction, an important prognostic factor for overall survival and event‐free survival. , Previously, GSTT1 null genotype was associated with an increased rate of early death after the initiation of chemotherapy in Japanese AML patients treated with cytarabine, mercaptopurine, prednisone, and daunorubicin. In our cohort, no association between GSTT1 and GSTM1 and toxicity effects were found, probably due to sample size (data not shown). There is scanty information published on pharmacogenetic associations in pediatric AML evaluating CDA, DCK , and ABCB1 genes. In our cohort, the majority of genotypic associations between these gene variants and toxicity or clinical outcomes were different from previous studies. However, most reports analyzing these gene variants have been reported mainly in adult patients, and in other admixed populations different from our cohort. Further studies are needed to evaluate the associations found here, since this is the first study made in colombian population, which is a highly structured population due to admixture between European derived, Amerindians, and African descent populations. For example, the reported genotype frequencies for CDA (79A > C, rs2072671) in a Mexican cohort are quite different to those reported for a Spanish cohort and our sample. For DCK (‐201C > A, rs2306744), our genotype frequencies were similar to those reported previously, but quite different from those reported in the Mexican cohort. CONCLUSIONS This is the first study of AML pharmacogenetics in Colombia, a country with a highly admix population structure. Here, we have used a SNaPShot™ assay to simultaneously analyze SNV in the CDA, DCK , and ABCB1 genes. With this assay, we can run one sample at a time at very low cost, and results could be obtained at the same time as the cytogenetics studies. Although some genetic associations were found, the low number of pediatric AML cases analyzed could be a limitation and further studies will be required to validate the associations found in an independent cohort. Pharmacogenomics might be useful as a future tool for patient stratification for treatment with different chemotherapy regimens. Luz K. Yunis: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (equal); investigation (equal); methodology (equal); writing – original draft (equal); writing – review and editing (equal). Adriana Linares‐Ballesteros: Conceptualization (lead); data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); supervision (lead); writing – original draft (lead); writing – review and editing (lead). Nelson Aponte: Formal analysis (supporting); methodology (supporting). Gisela Barros: Data curation (supporting); investigation (supporting). Johnny García: Data curation (supporting); investigation (supporting). Laura Niño: Data curation (supporting); investigation (supporting). Gloria Uribe: Methodology (supporting). Edna Quintero: Methodology (supporting). Juan J. Yunis: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); supervision (lead); writing – original draft (lead); writing – review and editing (lead). The authors have stated explicitly that there are no conflicts of interest in connection with this article. Informed consent was obtained from each patient or guardian. This protocol was approved by the institutional ethics committee of the participant institutions. This project was approved by the ethics committee of each institution, CEI 125‐18 and 243‐1 (HOMI Fundación Hospital Pediátrico La Misericordia and Clínica Infantil Colsubsidio Bogotá D.C., Colombia, respectively) and the ethics committee of Universidad Nacional de Colombia (007‐091‐18). Supplementary Figure S1. Schematic representation of genes analyzed in the anthracyclines (idarrubicine) and cytarabine pathway. Click here for additional data file. Supplemental Table S1 ABCB1, CDA, DCK, GSTT1 and GSTM1 genotypes of pediatric AML patients. Click here for additional data file. Supplemental Table S2 Toxicity definitions according to CTCAE version 5. Click here for additional data file. |
Evaluating the Efficacy of ChatGPT as a Patient Education Tool in Prostate Cancer: Multimetric Assessment | 67595713-7f26-4de4-a368-208d536c393d | 11358656 | Patient Education as Topic[mh] | Artificial intelligence (AI) chatbots have made significant strides in recent years . This was emphatically signposted with the launch of ChatGPT-3 (OpenAI) in November 2022, with ChatGPT becoming the most popular web-based tool for both patients and health care professionals . Now in its fourth iteration (ChatGPT-4), the AI language model can generate responses to a wide range of health questions and topics . AI chatbots, such as ChatGPT, have the potential to significantly impact patient education and disease experience by providing reliable, accessible, and personalized information . One patient population that stands to benefit from this is men who are concerned about prostate cancer. With the rising prevalence of prostate cancer globally—accounting for an estimated 1,414,259 new cases and over 375,304 deaths in 2020 alone—there is an urgent need for accurate and timely patient education information . The rate of prostate cancer survivorship is increasing, but this comes with its own challenges such as escalating health care costs and large numbers of survivors requiring ongoing care . In this context, shared decision-making becomes pivotal, particularly concerning prostate-specific antigen screening and prostate cancer treatment selection . Given the various treatments available, management decisions can be greatly influenced by a patient’s understanding of the anatomical, functional, and psychological impacts of treatment . Side effects, such as urinary incontinence and erectile dysfunction, can severely affect a patient’s quality of life, necessitating well-informed patients, and treatment choices . Furthermore, patient education has been shown to minimize psychological impacts such as depression and treatment regret . There are well-documented issues with unmet information needs of both men and their support networks throughout the prostate cancer care continuum . This includes challenges related to information quality and readability . The assessment of web-based health care information in prostate cancer has been well described through multiple domains including web page articles, YouTube (Google), and social media . The internet is now often the first source of information for men (and their stakeholders) seeking answers about diagnosis, treatment, and prognosis . Despite this trend, most long-term literature suggests that web-based health information is of moderate to poor quality . AI chatbots are a potential solution to fill the prostate cancer information quality gap . Given their scalability, AI chatbots can reach a wide demographic, including those in remote or underserved communities where medical resources are scarce . Natural language processing technologies (NLPTs) enable these platforms to present complex jargon in patient-specific terms, with the potential to address eHealth literacy variability, and to enhance patient understanding . Such platforms are also able to do this across a diverse number of languages . Despite these qualities, the accuracy and reliability of AI chatbot medical information must still be assessed using rigorous evaluation tools. Only a handful of studies have begun to test ChatGPT’s applicability in prostate cancer: one testing its knowledge directly with questions and statements and another assessing its appropriateness in screening recommendations . However, a significant knowledge gap persists in understanding the quality and safety of information patients receive from ChatGPT-4 for common internet queries. Ongoing evaluation is a necessary step to build health care provider confidence in these new technologies while ensuring that patients have access to vetted and safe health care and educational information. This study aims to demonstrate and assess the quality of ChatGPT responses to commonly asked patient education topics in prostate cancer care. By doing so, this study seeks to (1) illustrate to clinicians whether ChatGPT-4 is currently a reliable and safe patient education tool for prostate cancer information and (2) provide clinicians with a greater understanding of the current strengths and limitations of health-based queries which patients are likely to encounter when using technologies such as ChatGPT-4. A range of assessment tools will be applied to the AI-generated responses to assess output quality, safety, understandability, actionability, ease of use, readability, and reliability. A parallel assessment of the outputs by prostate cancer experts will also be conducted.
Question or Keyword Strategy Questions tested with the AI chatbot model (ChatGPT-4) were selected through an iterative process of literature and Google Keyword analysis. Literature concerning the information needs of men considering prostate cancer investigation and treatment was reviewed to determine the most common information topics and prostate cancer questions of interest to men . Subsequently, worldwide Google Trends data were analyzed to provide a more current public measure of prostate cancer information searches . Using “prostate cancer” as a keyword, both rising and top “related topics” and “related queries” of the past year were collected. Finally, while limited to training materials up to 2021, ChatGPT was itself queried, asking “What are the most common prostate cancer questions asked to ChatGPT?” . The 2 authors thematically analyzed this information to define the following eight questions to discuss with the AI model: (1) What are the symptoms of prostate cancer? (2) What are the risk factors for prostate cancer? (3) What is the survival rate of prostate cancer? (4) How is prostate cancer diagnosed? (5) What age should men start getting screened for prostate cancer? (6) What are the pros and cons of treatment options for prostate cancer? (7) How does prostate cancer affect sexual function? and (8) How does prostate cancer affect bladder function? Each question was posed to ChatGPT-4 with an additional request for references. A new ChatGPT account was established with a novel email address for each prompt in an effort to reduce the potential effects of each response on subsequent outputs of the AI model. Each output was recorded for individual quality and readability assessment . Quality Assessment Overview Due to the current absence of tools to evaluate the quality of AI natural language outputs, each conversation was evaluated using modified versions of pre-existing information quality assessment tools. These included the Patient Education Materials Assessment Tool (PEMAT) and DISCERN criteria . These tools were iteratively modified to accommodate the text-only nature and characteristics of AI natural language outputs. While DISCERN criteria have been adapted in literature, the PEMAT modification is new . Internal validity testing was undertaken by 4 reviewers using ChatGPT outputs from similar question sets for breast cancer and bowel cancer. The reliability of each tool tested was satisfactory, with Cronbach α>0.8 (DISCERN 0.852, PEMAT 0.82, and Global Quality Score [GQS] 0.85). The GQS was not modified . PEMAT-AI Tool The PEMAT tool evaluates and compares the understandability and actionability of patient education materials . The tool incorporates 17 items measuring understandability and 7 assessing actionability; these were reduced to 8 and 3, respectively, to suit the AI text-only outputs . Each item was given a single point if the criteria were met, and the total score was measured as a total percentage. Final scores were recorded as “pass” or “fail” based on the ≥70% cut‐off score set by the PEMAT guidelines . DISCERN-AI Tool The DISCERN criteria is a previously validated tool that aids health care consumers and health practitioners in appraising the quality of health care treatment information . To address the AI output, these criteria were modified to 7 questions (of the original 15) on a scale of 1 to 3, using questions 3-9 . Based on previous DISCERN quality assessment in the literature, each output was scored as very poor (6), poor (7-9), fair (8-12), good (13-15), and excellent (16-18) quality patient education material . GQS Tool The GQS is a 5-point Likert scale based on the quality of information, and the flow and ease of use of information presented via the web. The GQS encompasses a scale of 1 to 5; where 1 indicates “low quality” and 5 implies “high quality.” Results that received a score of 4 or 5 were rated high quality, those with a score of 3 were assessed as medium quality, and the ones with a score of 1 or 2 were categorized as low quality . Readability The readability of the AI responses was assessed using a battery of established algorithms: the Flesch Reading Ease score, Gunning Fog Index, Flesch-Kincaid Grade Level, Coleman-Liau Index, and Simple Measure of Gobbledygook (SMOG) Index . Multiple tools were used in an effort to limit the bias of each respective algorithm . Each AI output text was copied to Microsoft Word to maintain formatting, and then to Readable.com for analysis . Results from the answered questions were averaged across all outputs into a readability consensus . The Flesch Reading Ease score gauges text simplicity, where the score ranges from 0 to 100, with higher scores indicating easier readability. Texts with a score between 60 and 70 are generally considered to be at an eighth- to ninth-grade reading level and are usually easier for the average adult to read. The Gunning Fog Index and The Flesch-Kincaid Grade Level measure sentence complexity, the score represents the number of years of formal education a reader would need to understand the text on the first reading. For example, a score of 12 would mean the text is suitable for a 12th-grade reading level or higher. The Coleman-Liau Index is similar to Gunning Fog and The Flesch-Kincaid Grade Level but focuses on character count. This score also correlates with a US school grade level but is calculated using the number of characters instead of syllables, making it more suited for languages where syllable count is less indicative of complexity. The SMOG Index evaluates syllable density to assess readability and is often used for checking health messages. A score of 12 would mean the text is suitable for someone with at least a 12th-grade level of reading comprehension. Natural Language Assessment Tool for AI Expert review of each output was undertaken by 7 independent experienced urologists, using an assessment framework (Natural Language Assessment Tool for AI [NLAT- AI]) developed to assess the accuracy, safety, appropriateness, actionability, and effectiveness of information. Each domain was scored on a 5-point Likert scale (1=strongly disagree, to 5=“strongly agree”; ). All results were collated and presented as descriptive statistics. Qualitative feedback on each domain was sought regarding potential improvement and overall performance. References Assessment Due to known issues of AI hallucination: “the phenomenon of a machine, such as a chatbot, generating seemingly realistic sensory experiences that do not correspond to any real-world input,” a final brief tool (Reference Assessment AI [REF-AI]) was developed for analysis of the references provided by AI outputs . Each reference was reviewed by accessing the content via the direct link provided by the AI output, or a Google search of the reference. This tool assessed for reference hallucination (real or not), relevance (correlation between the references and AI output), and quality of references (type of institution linked to the reference). Each criterion was assessed with a score of 1-3, with a lower summative score indicating lower reference quality, and a higher score indicating high reference quality . Scores were averaged to yield a composite score for each axis of evaluation. The reliability of this tool tested similar question sets for breast cancer and bowel cancer was satisfactory (0.81). Ethical Considerations After consultation with the local institutional review board, it was determined that no formal ethical approval was required for this study as no human or animal participants were involved.
Questions tested with the AI chatbot model (ChatGPT-4) were selected through an iterative process of literature and Google Keyword analysis. Literature concerning the information needs of men considering prostate cancer investigation and treatment was reviewed to determine the most common information topics and prostate cancer questions of interest to men . Subsequently, worldwide Google Trends data were analyzed to provide a more current public measure of prostate cancer information searches . Using “prostate cancer” as a keyword, both rising and top “related topics” and “related queries” of the past year were collected. Finally, while limited to training materials up to 2021, ChatGPT was itself queried, asking “What are the most common prostate cancer questions asked to ChatGPT?” . The 2 authors thematically analyzed this information to define the following eight questions to discuss with the AI model: (1) What are the symptoms of prostate cancer? (2) What are the risk factors for prostate cancer? (3) What is the survival rate of prostate cancer? (4) How is prostate cancer diagnosed? (5) What age should men start getting screened for prostate cancer? (6) What are the pros and cons of treatment options for prostate cancer? (7) How does prostate cancer affect sexual function? and (8) How does prostate cancer affect bladder function? Each question was posed to ChatGPT-4 with an additional request for references. A new ChatGPT account was established with a novel email address for each prompt in an effort to reduce the potential effects of each response on subsequent outputs of the AI model. Each output was recorded for individual quality and readability assessment .
Overview Due to the current absence of tools to evaluate the quality of AI natural language outputs, each conversation was evaluated using modified versions of pre-existing information quality assessment tools. These included the Patient Education Materials Assessment Tool (PEMAT) and DISCERN criteria . These tools were iteratively modified to accommodate the text-only nature and characteristics of AI natural language outputs. While DISCERN criteria have been adapted in literature, the PEMAT modification is new . Internal validity testing was undertaken by 4 reviewers using ChatGPT outputs from similar question sets for breast cancer and bowel cancer. The reliability of each tool tested was satisfactory, with Cronbach α>0.8 (DISCERN 0.852, PEMAT 0.82, and Global Quality Score [GQS] 0.85). The GQS was not modified . PEMAT-AI Tool The PEMAT tool evaluates and compares the understandability and actionability of patient education materials . The tool incorporates 17 items measuring understandability and 7 assessing actionability; these were reduced to 8 and 3, respectively, to suit the AI text-only outputs . Each item was given a single point if the criteria were met, and the total score was measured as a total percentage. Final scores were recorded as “pass” or “fail” based on the ≥70% cut‐off score set by the PEMAT guidelines . DISCERN-AI Tool The DISCERN criteria is a previously validated tool that aids health care consumers and health practitioners in appraising the quality of health care treatment information . To address the AI output, these criteria were modified to 7 questions (of the original 15) on a scale of 1 to 3, using questions 3-9 . Based on previous DISCERN quality assessment in the literature, each output was scored as very poor (6), poor (7-9), fair (8-12), good (13-15), and excellent (16-18) quality patient education material . GQS Tool The GQS is a 5-point Likert scale based on the quality of information, and the flow and ease of use of information presented via the web. The GQS encompasses a scale of 1 to 5; where 1 indicates “low quality” and 5 implies “high quality.” Results that received a score of 4 or 5 were rated high quality, those with a score of 3 were assessed as medium quality, and the ones with a score of 1 or 2 were categorized as low quality .
Due to the current absence of tools to evaluate the quality of AI natural language outputs, each conversation was evaluated using modified versions of pre-existing information quality assessment tools. These included the Patient Education Materials Assessment Tool (PEMAT) and DISCERN criteria . These tools were iteratively modified to accommodate the text-only nature and characteristics of AI natural language outputs. While DISCERN criteria have been adapted in literature, the PEMAT modification is new . Internal validity testing was undertaken by 4 reviewers using ChatGPT outputs from similar question sets for breast cancer and bowel cancer. The reliability of each tool tested was satisfactory, with Cronbach α>0.8 (DISCERN 0.852, PEMAT 0.82, and Global Quality Score [GQS] 0.85). The GQS was not modified .
The PEMAT tool evaluates and compares the understandability and actionability of patient education materials . The tool incorporates 17 items measuring understandability and 7 assessing actionability; these were reduced to 8 and 3, respectively, to suit the AI text-only outputs . Each item was given a single point if the criteria were met, and the total score was measured as a total percentage. Final scores were recorded as “pass” or “fail” based on the ≥70% cut‐off score set by the PEMAT guidelines .
The DISCERN criteria is a previously validated tool that aids health care consumers and health practitioners in appraising the quality of health care treatment information . To address the AI output, these criteria were modified to 7 questions (of the original 15) on a scale of 1 to 3, using questions 3-9 . Based on previous DISCERN quality assessment in the literature, each output was scored as very poor (6), poor (7-9), fair (8-12), good (13-15), and excellent (16-18) quality patient education material .
The GQS is a 5-point Likert scale based on the quality of information, and the flow and ease of use of information presented via the web. The GQS encompasses a scale of 1 to 5; where 1 indicates “low quality” and 5 implies “high quality.” Results that received a score of 4 or 5 were rated high quality, those with a score of 3 were assessed as medium quality, and the ones with a score of 1 or 2 were categorized as low quality .
The readability of the AI responses was assessed using a battery of established algorithms: the Flesch Reading Ease score, Gunning Fog Index, Flesch-Kincaid Grade Level, Coleman-Liau Index, and Simple Measure of Gobbledygook (SMOG) Index . Multiple tools were used in an effort to limit the bias of each respective algorithm . Each AI output text was copied to Microsoft Word to maintain formatting, and then to Readable.com for analysis . Results from the answered questions were averaged across all outputs into a readability consensus . The Flesch Reading Ease score gauges text simplicity, where the score ranges from 0 to 100, with higher scores indicating easier readability. Texts with a score between 60 and 70 are generally considered to be at an eighth- to ninth-grade reading level and are usually easier for the average adult to read. The Gunning Fog Index and The Flesch-Kincaid Grade Level measure sentence complexity, the score represents the number of years of formal education a reader would need to understand the text on the first reading. For example, a score of 12 would mean the text is suitable for a 12th-grade reading level or higher. The Coleman-Liau Index is similar to Gunning Fog and The Flesch-Kincaid Grade Level but focuses on character count. This score also correlates with a US school grade level but is calculated using the number of characters instead of syllables, making it more suited for languages where syllable count is less indicative of complexity. The SMOG Index evaluates syllable density to assess readability and is often used for checking health messages. A score of 12 would mean the text is suitable for someone with at least a 12th-grade level of reading comprehension.
Expert review of each output was undertaken by 7 independent experienced urologists, using an assessment framework (Natural Language Assessment Tool for AI [NLAT- AI]) developed to assess the accuracy, safety, appropriateness, actionability, and effectiveness of information. Each domain was scored on a 5-point Likert scale (1=strongly disagree, to 5=“strongly agree”; ). All results were collated and presented as descriptive statistics. Qualitative feedback on each domain was sought regarding potential improvement and overall performance.
Due to known issues of AI hallucination: “the phenomenon of a machine, such as a chatbot, generating seemingly realistic sensory experiences that do not correspond to any real-world input,” a final brief tool (Reference Assessment AI [REF-AI]) was developed for analysis of the references provided by AI outputs . Each reference was reviewed by accessing the content via the direct link provided by the AI output, or a Google search of the reference. This tool assessed for reference hallucination (real or not), relevance (correlation between the references and AI output), and quality of references (type of institution linked to the reference). Each criterion was assessed with a score of 1-3, with a lower summative score indicating lower reference quality, and a higher score indicating high reference quality . Scores were averaged to yield a composite score for each axis of evaluation. The reliability of this tool tested similar question sets for breast cancer and bowel cancer was satisfactory (0.81).
After consultation with the local institutional review board, it was determined that no formal ethical approval was required for this study as no human or animal participants were involved.
ChatGPT Outputs The responses generated by the AI model, ChatGPT-4, provided broad, medically aligned information . The assessment of the ChatGPT-4 output using PEMAT-AI, DISCERN-AI, and GQS patient education material assessment tools demonstrated high results across all tools. The pooled PEMAT-AI understandability score easily passed the acceptability threshold of >70% (mean 79.44%, SD 10.44%); only question 3 failed the >70% threshold at 66.67% while the remaining were 76% or greater . The pooled DISCERN-AI rating was scored as “good” quality 77% (mean 13.88, SD 0.93), and all individual questions rated “good” on the DISCERN-AI except for question 5, which scored excellent (mean 15.67; ). The pooled GQS was rated as high (mean 4.46, SD 0.50 out of 5; ). Assessment tool results for each question are tabulated and graphed ( and - ). Reliability testing was high with Cronbach α=0.846. NLAT-AI Assessment Expert assessment of the AI outputs with NLAT-AI was consistent with a mean >3.0 out of 5.0 (neutral) in all domains across all question replies. NLAT-AI pooled means included accuracy of 3.96 (SD 0.91), safety of 4.32 (SD 0.86), appropriateness of 4.45 (SD 0.81), actionability of 4.05 (SD 1.15), and effectiveness of 4.09 (SD 0.98). Descriptive statistics for each question are tabulated and graphed ( and ). Internal validity testing demonstrated high reliability with Cronbach α=0.906. Qualitative feedback via NLAT-AI on questions 1 through 8 indicates some areas for improvement despite the generally accurate and easy-to-understand nature of responses. Common themes were a need for greater specificity, updated and comprehensive information, and a more globally inclusive perspective . Outputs were often characterized as good starting points or overviews which could benefit patients. Natural Language Assessment Tool for Artificial Intelligence qualitative feedback. Question 1: Symptoms Overall, a reasonable answer to the question... more emphasis should be put on the fact that prostate cancer is usually asymptomatic, usually detected on screening, only symptomatic when advanced. Could have been better if discussed symptoms of locally advanced prostate cancer (LUTS, haematuria, etc) and symptoms of metastatic prostate cancer (bone pain, weight loss, etc) Need to strongly emphasize that most prostate cancers are asymptomatic so prostate-specific antigen testing is necessary Question 2: Risk factors Considering that this is tailored for Americans, it may not be actionable for others. From a safety perspective, I would emphasize the importance of seeking medical review in the event of family history. Remove the modifiable risk factors as it makes patients think they can prevent it Question 3: Survival rates There is...no mention of the impact of treatment on survival so a patient could be forgiven for thinking this was survival rates in the event of no treatment being given. “Relative survival” is not clearly explained. The survival rate [is] overestimated in organ-confined disease as this is far more complex. It should be more clarified. When talking about prostate cancer survival 10 years is the minimum that should be discussed Fairly good- this is what I would tell my patients. Question 4: Diagnosis Overall reasonable answer from ChatGPT CT and bone scans are used for staging; but now in Australia is superseded by PSMA Reasonable answer. Some inaccuracies in how the tests are used, as well as their sequencing. PSMA PET not mentioned which is an important part of diagnosis and staging. These deficiencies likely reflect the rapidly evolving nature of prostate cancer diagnosis. The answer is easy to understand and general principles of diagnosis sound. Question 5: Screening Point 2 is very contentious and...gives a very one-sided view of prostate cancer screening. This is only appropriate for American audience. Point 2 is concerning as this represents one [clinician] group who is very much against prostate cancer screening... therefore may risk not giving a balanced view. No mention of any local guidelines, and no EAU [European Association of Urology] guidelines. Question 6: Treatment Very useful summary for patients immediately after diagnosis. No mention of novel tx [treatments] eg: focal therapy, cryo, HIFU No mention of robotic surgery versus open surgery This is a very simple table about the pros and cons. Question 7: Sexual function Overall a very good answer—misses minor points Very well written Would also mention that erectile function improves over time. Surgery does not damage the vessels for erection Question 8: Bladder function Nice summary Accurate and easy to understand Minor issues only with the discussion on stress or urge incontinence Hormone therapy should not causes bladder dysfunction. In fact, it might improve it Readability Assessment The readability algorithm consensus was “difficult to read” (Flesch Reading Ease score mean 45.97, SD 8.69; Gunning Fog Index mean 14.55, SD 4.79), averaging an 11th-grade reading level, equivalent to 15- to 17-year-olds (Flesch-Kincaid Grade Level mean 12.12, SD 4.34; The Coleman-Liau Index mean 12.75, SD 1.98; SMOG Index mean 11.06, SD 3.20). Questions 1 and 2 were the easiest to read scoring an 8th-grade level, while questions 6 (grade 23 level), 7 (grade 12 level), and 8 (grade 13 level) were very difficult to read . REF-AI Assessment REF-AI identified 2 reference hallucinations from 30 total references across all questions (pooled REF-AI Real mean 2.86). Most references effectively supported the text, while 4 questions had 1 or 2 citations that did not directly support the information provided ( ; pooled REF-AI supporting mean 2.75). A total of 86% (26/30) of references were from reputable government organizations, while 2 were direct citations from scientific literature (pooled REF-AI source mean 2.13). Individual statements were provided a direct reference in only 3 outputs. The remaining outputs instead provided a list of references at the bottom of the text. Some direct links to references were not complete, instead delivering the user to the organization’s primary website URL, likely reflecting updated website directories since the 2021 ChatGPT indexation. The 2 hallucinated references were present in questions 7 and 8, where weblinks did not connect and despite extensive Google and library searches, the original material was unable to be located.
The responses generated by the AI model, ChatGPT-4, provided broad, medically aligned information . The assessment of the ChatGPT-4 output using PEMAT-AI, DISCERN-AI, and GQS patient education material assessment tools demonstrated high results across all tools. The pooled PEMAT-AI understandability score easily passed the acceptability threshold of >70% (mean 79.44%, SD 10.44%); only question 3 failed the >70% threshold at 66.67% while the remaining were 76% or greater . The pooled DISCERN-AI rating was scored as “good” quality 77% (mean 13.88, SD 0.93), and all individual questions rated “good” on the DISCERN-AI except for question 5, which scored excellent (mean 15.67; ). The pooled GQS was rated as high (mean 4.46, SD 0.50 out of 5; ). Assessment tool results for each question are tabulated and graphed ( and - ). Reliability testing was high with Cronbach α=0.846.
Expert assessment of the AI outputs with NLAT-AI was consistent with a mean >3.0 out of 5.0 (neutral) in all domains across all question replies. NLAT-AI pooled means included accuracy of 3.96 (SD 0.91), safety of 4.32 (SD 0.86), appropriateness of 4.45 (SD 0.81), actionability of 4.05 (SD 1.15), and effectiveness of 4.09 (SD 0.98). Descriptive statistics for each question are tabulated and graphed ( and ). Internal validity testing demonstrated high reliability with Cronbach α=0.906. Qualitative feedback via NLAT-AI on questions 1 through 8 indicates some areas for improvement despite the generally accurate and easy-to-understand nature of responses. Common themes were a need for greater specificity, updated and comprehensive information, and a more globally inclusive perspective . Outputs were often characterized as good starting points or overviews which could benefit patients. Natural Language Assessment Tool for Artificial Intelligence qualitative feedback. Question 1: Symptoms Overall, a reasonable answer to the question... more emphasis should be put on the fact that prostate cancer is usually asymptomatic, usually detected on screening, only symptomatic when advanced. Could have been better if discussed symptoms of locally advanced prostate cancer (LUTS, haematuria, etc) and symptoms of metastatic prostate cancer (bone pain, weight loss, etc) Need to strongly emphasize that most prostate cancers are asymptomatic so prostate-specific antigen testing is necessary Question 2: Risk factors Considering that this is tailored for Americans, it may not be actionable for others. From a safety perspective, I would emphasize the importance of seeking medical review in the event of family history. Remove the modifiable risk factors as it makes patients think they can prevent it Question 3: Survival rates There is...no mention of the impact of treatment on survival so a patient could be forgiven for thinking this was survival rates in the event of no treatment being given. “Relative survival” is not clearly explained. The survival rate [is] overestimated in organ-confined disease as this is far more complex. It should be more clarified. When talking about prostate cancer survival 10 years is the minimum that should be discussed Fairly good- this is what I would tell my patients. Question 4: Diagnosis Overall reasonable answer from ChatGPT CT and bone scans are used for staging; but now in Australia is superseded by PSMA Reasonable answer. Some inaccuracies in how the tests are used, as well as their sequencing. PSMA PET not mentioned which is an important part of diagnosis and staging. These deficiencies likely reflect the rapidly evolving nature of prostate cancer diagnosis. The answer is easy to understand and general principles of diagnosis sound. Question 5: Screening Point 2 is very contentious and...gives a very one-sided view of prostate cancer screening. This is only appropriate for American audience. Point 2 is concerning as this represents one [clinician] group who is very much against prostate cancer screening... therefore may risk not giving a balanced view. No mention of any local guidelines, and no EAU [European Association of Urology] guidelines. Question 6: Treatment Very useful summary for patients immediately after diagnosis. No mention of novel tx [treatments] eg: focal therapy, cryo, HIFU No mention of robotic surgery versus open surgery This is a very simple table about the pros and cons. Question 7: Sexual function Overall a very good answer—misses minor points Very well written Would also mention that erectile function improves over time. Surgery does not damage the vessels for erection Question 8: Bladder function Nice summary Accurate and easy to understand Minor issues only with the discussion on stress or urge incontinence Hormone therapy should not causes bladder dysfunction. In fact, it might improve it
The readability algorithm consensus was “difficult to read” (Flesch Reading Ease score mean 45.97, SD 8.69; Gunning Fog Index mean 14.55, SD 4.79), averaging an 11th-grade reading level, equivalent to 15- to 17-year-olds (Flesch-Kincaid Grade Level mean 12.12, SD 4.34; The Coleman-Liau Index mean 12.75, SD 1.98; SMOG Index mean 11.06, SD 3.20). Questions 1 and 2 were the easiest to read scoring an 8th-grade level, while questions 6 (grade 23 level), 7 (grade 12 level), and 8 (grade 13 level) were very difficult to read .
REF-AI identified 2 reference hallucinations from 30 total references across all questions (pooled REF-AI Real mean 2.86). Most references effectively supported the text, while 4 questions had 1 or 2 citations that did not directly support the information provided ( ; pooled REF-AI supporting mean 2.75). A total of 86% (26/30) of references were from reputable government organizations, while 2 were direct citations from scientific literature (pooled REF-AI source mean 2.13). Individual statements were provided a direct reference in only 3 outputs. The remaining outputs instead provided a list of references at the bottom of the text. Some direct links to references were not complete, instead delivering the user to the organization’s primary website URL, likely reflecting updated website directories since the 2021 ChatGPT indexation. The 2 hallucinated references were present in questions 7 and 8, where weblinks did not connect and despite extensive Google and library searches, the original material was unable to be located.
Principal Findings In the digital information age, understanding what patient health information is accessed and the quality of this information is crucial. This study demonstrates several examples of information that patients (and their caregivers) may encounter when conducting searches related to prostate cancer management. In our analysis, ChatGPT-4 provided generally comprehensive answers to prostate cancer questions, mostly in line with current medical guidelines and literature. ChatGPT-4 demonstrated promise when assessed with a range of patient education and information quality assessment tools, as well as expert review. Robust scores and expert feedback indicate that the generated content was reliable, safe, and actionable for patients, albeit with room for improvement in minor nuanced details, global applicability, and readability. Current evidence indicates that 75% of people turn to the internet for decision-making during a health crisis . Despite the abundance of available patient information, studies assessing the quality of digital health information indicate significant shortcomings . For prostate cancer, the quality of information that reaches the patient is known to be inconsistent . For example, a previous assessment of the top 100 “prostate cancer” web page results identified via search engine query showed that only 11.1% of sites demonstrate an excellent on the original DISCERN criteria . While our analysis has used necessarily disparate methods, a comparison of our DISCERN-AI results (good-excellent) to static web page DISCERN scores suggests that ChatGPT prostate cancer information outputs may be of a higher quality than many traditional web pages . ChatGPT4 appears capable of providing broad and largely accurate information which may further augment self-directed patient or stakeholder enquiry. Nevertheless, a direct comparison of ChatGPT outputs to established gold standard information sources is necessary to clearly define the role of this new communication technology as part of patient care and education. Our findings appear to differ from Coskun et al , where ChatGPT-3 had accuracy issues using queries generated by the European Association of Urology Patient Information. Interestingly, Zheng et al discovered that ChatGPT-4 can offer suitable counseling on disease prevention and screening for prostate cancer patients. These differences may represent the rapid evolution of the algorithm as our testing used the newer model. Exclusive use of US-centric guidelines raised questions of bias among our experts. Others have also highlighted such bias, noting that 51% of training data for major large language models is US sourced . The disparities between ChatGPT-3 and ChatGPT-4 highlight the continual advancement and refinement of the underlying technology, reinforcing the need for periodic assessment and validation as newer models emerge . Conversely, a lack of validated and reproducible tools to make reliable quality assessments of NPLTs is likely to play a role in varied results within this juvenile domain of clinical research . While the methods used in our study were an effort to standardize output assessment in our work, we recognize and encourage further rigorous work to develop validated and reproducible assessment tools that can be applied to a range of NPLT outputs and platforms. Despite the NLAT-AI rating, and general appropriateness of the language across all questions, the objective readability from algorithms demonstrated a high reading level and difficulty. This is likely reflective of the literacy bias present among our highly educated expert pool . While the recommended reading level for patient education material varies between organizations, the consensus is that it should generally lie between grade 6 and 8 reading levels . The readability algorithms thus suggest that the generated content may be challenging for some readers. These findings are of importance given that lower readability may limit accessibility for certain socioeconomic or minority groups . Literacy is a known negative correlate of prostate cancer health outcomes . Compounding this concern is the effect of the user’s overarching eHealth literacy, which is likely to affect chatbot engagement behaviors and patterns of information comprehension and use . Effects of both traditional literacy and eHealth literacy on the end user experience of NLPTs require urgent investigation due to the pervasiveness that these technologies are already presenting within society and in web-based health communication . The digital nature of the ChatGPT-4 model, where users can continuously engage and seek clarifications, offers a potential advantage and solution to static patient information materials. Although beyond this study’s scope, the ChatGPT-4 model permits ongoing discussions, enabling patients to seek clarifications of information. These conversations allow for personalized explanations related to patient health results, the opportunity to simplify language, and may ultimately address some concerns raised by our expert assessors. This is an extremely powerful and unique component of this new digital technology. Future iterations of such models may benefit by incorporating clear adaptability features, where the complexity and specificity of the content can be adjusted based on user preferences or needs. Further studies are required to explore how the longitudinal and dynamic features of NLPTs affect information quality and patient comprehension. This will be particularly important in comparison to traditional website and social media-based information sources which currently dominate the landscape of self-educative information sourcing in prostate cancer care . NPLTs with predetermined or flexible user settings attuned to patient preference, needs, or literacy level are a potential futurist pathway to cost-effective and scalable forms of tailored patient health education materials. Hallucination, where information is fabricated by the NLPT and presented as valid, is a well-documented phenomenon specific to NLPTs and ChatGPT . This study demonstrated that hallucinations could occur when searching for prostate cancer with NPLT or chatbots. While only occurring in 2 instances of 30, these findings continue. Designation between hallucination and faux hallucination should also be considered. Faux-hallucination results from modified references after ChatGPT-4’s indexation, leading to broken links or lost references. Website redesign or content that no longer exists after the 2021 indexation is a potential etiology for hallucination that has not been fully explored. Equally, such disappearance of content with time may also match the definition of hallucination in the future. While not a prominent issue in this study, these findings continue to demonstrate the potential for fabricated information, which can be easily overlooked by the unassuming clinician, patient, or researcher. While still in its infancy, large language models must continue to solve the issue of hallucination before integration into high-risk systems, such as health care, can be considered. While hallucinations are a notable concern, there are several other limitations of current NPLTs that need to be considered. Despite malleability, it is unknown whether the ChatGPT-4 model may fully replicate the nuance of human communication necessary for effective patient health education . Additionally, the most significant limitation of ChatGPT is its potential for biased, outdated, or misleading content generation . Even with relatively high-quality scores, this study shows that ChatGPT can still produce misleading or biased content under discriminatory and expert scrutiny, posing some element of risk for those with poor eHealth literacy . However, while expert reviewers identified minor inaccuracies, none of these points were considered to be significantly concerning safety issues. Nevertheless, there is currently a lack of evidence to predict the impact of these technologies on patients’ understanding, decision-making, or health, without further inquiry and consideration of patients’ ability to interact with these new eHealth technologies. We strongly recommend clinicians report these concerns to prostate cancer patients and their stakeholders when guiding patient use of web-based information in their care. Furthermore, the opaque and dynamic nature of this technology’s private enterprise proprietary algorithms is also a concern . Algorithm development will likely outpace quality assurance efforts and raise questions about the necessity of clinician involvement in NPLT model development that aims to present health-based information . The effectively unknown and vast array of sources from which ChatGPT’s training data are derived raises ethical concerns. Without knowing the origins and credibility of such data, it is difficult for clinicians to fully trust generated content, presenting us with a modernized but perpetual issue of distrust in web-based information which may ultimately hinder adoption and progress . Finally, there are also financial considerations; the cost of using ChatGPT-4 (as opposed to the currently free ChatGPT-3.5) or other NPLTs may form a barrier to widespread adoption in health care settings and has the potential to drive disparate levels of health care if not effectively managed and regulated. Limitations Limitations of this study include the sample size of assessors, which may skew the evaluation of the included tool’s reliability and efficacy. The qualitative assessments of experts are at inherent risk of bias for or against the use of novel technology and ChatGPT-4. However, these experts are also deeply aware of the nature and quality of current prostate cancer education materials, providing additional insight that is of value to this work. It is important to note this assessment was purposefully narrow in scope and may not reflect the myriad of interactions under the vast topics of prostate cancer. It is unknown how applicable these interactions are in wider prostate cancer education scenarios and ongoing investigation is required. Work is currently underway to assess an expanded question set with a comparison to currently accepted patient education gold standards in prostate cancer. While not an explicit purpose of this study, the exploratory assessments used in this work (DISCERN-AI, PEMAT-AI, NLAT-AI, and REF-AI) demonstrate interreliability and replicability across several cancer-type information outputs. They may thus have potential use for clinicians and researchers interested in reviewing the quality of other cancer-based outputs of ChatGPT-4 or other NPLTs. Nevertheless, their validity requires further testing and greater investigation is necessary to develop specific tools to assess NPLT output quality in the long term. Conclusion Our analysis found ChatGPT-4’s responses to common prostate cancer queries were of good quality, and a potentially useful patient education adjunct for prostate cancer care. Objective quality assessment tools were reflective of NPLT outputs, which were generally reliable and appropriate, although with room for improvement. Our expert panel was impressed by the appropriateness and safety of the language and information given. However, clinicians should be aware that there are several limitations to ChatGPT-4 prostate cancer outputs including hallucination, specificity issues, and difficult readability. Future studies are required to assess whether more longitudinal (back-and-forth) ChatGPT-4 discourse may offset some of the concerns highlighted in this analysis, and how patients of differing eHealth literacy levels may engage with and have care affected by such technologies.
In the digital information age, understanding what patient health information is accessed and the quality of this information is crucial. This study demonstrates several examples of information that patients (and their caregivers) may encounter when conducting searches related to prostate cancer management. In our analysis, ChatGPT-4 provided generally comprehensive answers to prostate cancer questions, mostly in line with current medical guidelines and literature. ChatGPT-4 demonstrated promise when assessed with a range of patient education and information quality assessment tools, as well as expert review. Robust scores and expert feedback indicate that the generated content was reliable, safe, and actionable for patients, albeit with room for improvement in minor nuanced details, global applicability, and readability. Current evidence indicates that 75% of people turn to the internet for decision-making during a health crisis . Despite the abundance of available patient information, studies assessing the quality of digital health information indicate significant shortcomings . For prostate cancer, the quality of information that reaches the patient is known to be inconsistent . For example, a previous assessment of the top 100 “prostate cancer” web page results identified via search engine query showed that only 11.1% of sites demonstrate an excellent on the original DISCERN criteria . While our analysis has used necessarily disparate methods, a comparison of our DISCERN-AI results (good-excellent) to static web page DISCERN scores suggests that ChatGPT prostate cancer information outputs may be of a higher quality than many traditional web pages . ChatGPT4 appears capable of providing broad and largely accurate information which may further augment self-directed patient or stakeholder enquiry. Nevertheless, a direct comparison of ChatGPT outputs to established gold standard information sources is necessary to clearly define the role of this new communication technology as part of patient care and education. Our findings appear to differ from Coskun et al , where ChatGPT-3 had accuracy issues using queries generated by the European Association of Urology Patient Information. Interestingly, Zheng et al discovered that ChatGPT-4 can offer suitable counseling on disease prevention and screening for prostate cancer patients. These differences may represent the rapid evolution of the algorithm as our testing used the newer model. Exclusive use of US-centric guidelines raised questions of bias among our experts. Others have also highlighted such bias, noting that 51% of training data for major large language models is US sourced . The disparities between ChatGPT-3 and ChatGPT-4 highlight the continual advancement and refinement of the underlying technology, reinforcing the need for periodic assessment and validation as newer models emerge . Conversely, a lack of validated and reproducible tools to make reliable quality assessments of NPLTs is likely to play a role in varied results within this juvenile domain of clinical research . While the methods used in our study were an effort to standardize output assessment in our work, we recognize and encourage further rigorous work to develop validated and reproducible assessment tools that can be applied to a range of NPLT outputs and platforms. Despite the NLAT-AI rating, and general appropriateness of the language across all questions, the objective readability from algorithms demonstrated a high reading level and difficulty. This is likely reflective of the literacy bias present among our highly educated expert pool . While the recommended reading level for patient education material varies between organizations, the consensus is that it should generally lie between grade 6 and 8 reading levels . The readability algorithms thus suggest that the generated content may be challenging for some readers. These findings are of importance given that lower readability may limit accessibility for certain socioeconomic or minority groups . Literacy is a known negative correlate of prostate cancer health outcomes . Compounding this concern is the effect of the user’s overarching eHealth literacy, which is likely to affect chatbot engagement behaviors and patterns of information comprehension and use . Effects of both traditional literacy and eHealth literacy on the end user experience of NLPTs require urgent investigation due to the pervasiveness that these technologies are already presenting within society and in web-based health communication . The digital nature of the ChatGPT-4 model, where users can continuously engage and seek clarifications, offers a potential advantage and solution to static patient information materials. Although beyond this study’s scope, the ChatGPT-4 model permits ongoing discussions, enabling patients to seek clarifications of information. These conversations allow for personalized explanations related to patient health results, the opportunity to simplify language, and may ultimately address some concerns raised by our expert assessors. This is an extremely powerful and unique component of this new digital technology. Future iterations of such models may benefit by incorporating clear adaptability features, where the complexity and specificity of the content can be adjusted based on user preferences or needs. Further studies are required to explore how the longitudinal and dynamic features of NLPTs affect information quality and patient comprehension. This will be particularly important in comparison to traditional website and social media-based information sources which currently dominate the landscape of self-educative information sourcing in prostate cancer care . NPLTs with predetermined or flexible user settings attuned to patient preference, needs, or literacy level are a potential futurist pathway to cost-effective and scalable forms of tailored patient health education materials. Hallucination, where information is fabricated by the NLPT and presented as valid, is a well-documented phenomenon specific to NLPTs and ChatGPT . This study demonstrated that hallucinations could occur when searching for prostate cancer with NPLT or chatbots. While only occurring in 2 instances of 30, these findings continue. Designation between hallucination and faux hallucination should also be considered. Faux-hallucination results from modified references after ChatGPT-4’s indexation, leading to broken links or lost references. Website redesign or content that no longer exists after the 2021 indexation is a potential etiology for hallucination that has not been fully explored. Equally, such disappearance of content with time may also match the definition of hallucination in the future. While not a prominent issue in this study, these findings continue to demonstrate the potential for fabricated information, which can be easily overlooked by the unassuming clinician, patient, or researcher. While still in its infancy, large language models must continue to solve the issue of hallucination before integration into high-risk systems, such as health care, can be considered. While hallucinations are a notable concern, there are several other limitations of current NPLTs that need to be considered. Despite malleability, it is unknown whether the ChatGPT-4 model may fully replicate the nuance of human communication necessary for effective patient health education . Additionally, the most significant limitation of ChatGPT is its potential for biased, outdated, or misleading content generation . Even with relatively high-quality scores, this study shows that ChatGPT can still produce misleading or biased content under discriminatory and expert scrutiny, posing some element of risk for those with poor eHealth literacy . However, while expert reviewers identified minor inaccuracies, none of these points were considered to be significantly concerning safety issues. Nevertheless, there is currently a lack of evidence to predict the impact of these technologies on patients’ understanding, decision-making, or health, without further inquiry and consideration of patients’ ability to interact with these new eHealth technologies. We strongly recommend clinicians report these concerns to prostate cancer patients and their stakeholders when guiding patient use of web-based information in their care. Furthermore, the opaque and dynamic nature of this technology’s private enterprise proprietary algorithms is also a concern . Algorithm development will likely outpace quality assurance efforts and raise questions about the necessity of clinician involvement in NPLT model development that aims to present health-based information . The effectively unknown and vast array of sources from which ChatGPT’s training data are derived raises ethical concerns. Without knowing the origins and credibility of such data, it is difficult for clinicians to fully trust generated content, presenting us with a modernized but perpetual issue of distrust in web-based information which may ultimately hinder adoption and progress . Finally, there are also financial considerations; the cost of using ChatGPT-4 (as opposed to the currently free ChatGPT-3.5) or other NPLTs may form a barrier to widespread adoption in health care settings and has the potential to drive disparate levels of health care if not effectively managed and regulated.
Limitations of this study include the sample size of assessors, which may skew the evaluation of the included tool’s reliability and efficacy. The qualitative assessments of experts are at inherent risk of bias for or against the use of novel technology and ChatGPT-4. However, these experts are also deeply aware of the nature and quality of current prostate cancer education materials, providing additional insight that is of value to this work. It is important to note this assessment was purposefully narrow in scope and may not reflect the myriad of interactions under the vast topics of prostate cancer. It is unknown how applicable these interactions are in wider prostate cancer education scenarios and ongoing investigation is required. Work is currently underway to assess an expanded question set with a comparison to currently accepted patient education gold standards in prostate cancer. While not an explicit purpose of this study, the exploratory assessments used in this work (DISCERN-AI, PEMAT-AI, NLAT-AI, and REF-AI) demonstrate interreliability and replicability across several cancer-type information outputs. They may thus have potential use for clinicians and researchers interested in reviewing the quality of other cancer-based outputs of ChatGPT-4 or other NPLTs. Nevertheless, their validity requires further testing and greater investigation is necessary to develop specific tools to assess NPLT output quality in the long term.
Our analysis found ChatGPT-4’s responses to common prostate cancer queries were of good quality, and a potentially useful patient education adjunct for prostate cancer care. Objective quality assessment tools were reflective of NPLT outputs, which were generally reliable and appropriate, although with room for improvement. Our expert panel was impressed by the appropriateness and safety of the language and information given. However, clinicians should be aware that there are several limitations to ChatGPT-4 prostate cancer outputs including hallucination, specificity issues, and difficult readability. Future studies are required to assess whether more longitudinal (back-and-forth) ChatGPT-4 discourse may offset some of the concerns highlighted in this analysis, and how patients of differing eHealth literacy levels may engage with and have care affected by such technologies.
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Molecular landscape and subtype-specific therapeutic response of nasopharyngeal carcinoma revealed by integrative pharmacogenomics | fa410f0a-b3d6-4ce1-b013-1dc3cc22c813 | 8144567 | Pharmacology[mh] | Nasopharyngeal carcinoma (NPC) is a type of malignant tumor that is commonly found in specific geographical locations including Southeast Asia, North Africa, southern provinces of China, Hong Kong, and Taiwan – . In 2015, 60,600 new NPC cases were identified, and 34,100 patients died in China . Many risk factors are involved in NPC, including environmental factors, Epstein–Barr virus (EBV) infection, smoking, diet, and personal lifestyle of peoples, etc. , yet how these factors contribute to NPC formation is poorly understood. Genomic variations and familial risks are other important causes for NPC, with more young people being affected by NPC than other cancer types . Unlike other head and neck cancers, the asymptomatic nature of NPC is a major challenge, which hinders the early diagnosis of this disease. Therefore, many patients are diagnosed at advanced stages, which reduces the patient’s overall survival (OS) , . Earlier studies proposed an array of genetic factors and genetic aberrations leading to the development of NPC, including NF-κB pathway activating mutations, chromatin modification-related mutations, ERBB-PI3K signaling activating mutations, etc. , – . However, many questions, such as those regarding the major causative factors, the key driving pathways, druggable genetic targets in NPC, especially in different histological subtypes, are still unanswered clearly. According to the current World Health Organization (WHO) classification system, NPC is classified into three major histological subtypes: keratinizing squamous cell carcinoma (KSCC), non-keratinizing carcinoma, and basaloid squamous cell carcinoma. Non-keratinizing tumors are further subcategorized as non-keratinizing undifferentiated carcinoma (NKUC) and non-keratinizing differentiated carcinoma (NKDC) . Although the WHO subtype system is the most common form of NPC clinical classification, an increasing number of clinicians realize that the current WHO classification is insufficient for predicting chemotherapy and radiotherapy (RT) outcomes , . The prognosis does not differ significantly between NKUC and NKDC, which are the two major subtypes of NPC and account for ~95% of all cases in China . A new prognostic histopathologic classification system of NPC has emerged that classifies NPC into four subtypes based on morphologic characteristics: epithelial carcinoma (EC), sarcomatoid carcinoma (SC), mixed sarcomatoid-epithelial carcinoma (MSEC), and squamous cell carcinoma (SCC), and these NPC subtypes could be linked to prognosis . However, further information about the genomic features and therapeutic response among different subtypes is worthy of investigation. RT is established as the definitive treatment for nonmetastatic NPC at an early stage, which leads to favorable clinical and survival outcomes with a 5-year OS rate of 87.3–93%, for stage I NPC patients – . However, due to the intrinsic invasiveness and asymptomatic nature of the disease, the majority of NPC patients (60–70%) are diagnosed at an advanced stage, with local spread or regional lymph node metastasis . For NPC patients with recurrent/metastatic tumor, the outcome is very poor, with a median OS of ~20 months , and chemoradiotherapy (CRT) is the standard-of-care treatment at this stage as recommended by the National Comprehensive Cancer Network guidelines (v2.2018). If the cancer cells have spread to distant organs, chemotherapy is the only option , . The commonly used chemotherapeutics for treating NPC includes cisplatin, fluorouracil, docetaxel, paclitaxel, gemcitabine, capecitabine, irinotecan, doxorubicin, vinorelbine, carboplatin and oxaliplatin. Several studies have indicated that CRT can significantly improve therapeutic outcome compared with RT alone , . To investigate new strategies identifying the personalized optimal CRT and chemotherapy regimens is promising to improve the prognosis of NPC patients. Furthermore, since only a few chemical drugs have been approved for NPC, to explore more therapeutic drugs that can be used for the treatment of NPC is necessary. Recent studies have employed the patient-derived organoid (PDO) culture system for drug screening against various cancers, including breast , colorectal , gastric , gastrointestinal , prostate , esophageal , liver , pancreatic , and bladder cancers etc. Vlachogiannis et al., had demonstrated the good potential of this system to accurately predict the clinical responses of cancer drugs using organoids derived from metastatic, heavily pretreated colorectal and gastroesophageal cancer patients. Their data indicated that the PDOs showed as high as 88% positive predictive value and 100% negative predictive value in cancer patients . Similar high accuracy in predicting clinical outcomes were also revealed by other studies – . In this study, to explore the application of the PDO system in screening chemotherapy drugs that could be used alone or in CRT for NPC, we have conducted a pharmacogenomics-based precision medicine approach by integrating genomics with a drug sensitivity test on PDOs to comprehensively investigate effective regimens for individual patients. Our integrative approach uncovers potential subtype-related and patient-specific strategies for the use of drugs and CRT combinations against this deadly disease.
Clinical information and histological subtyping of NPCs A total of 106 NPC tumors were collected from the hospital with routine records (Supplementary Table ; Supplementary Data , ), and we further studied their histological features. All NPC patients were of Chinese origin with a median diagnosed age of 48 years. EBV status was examined on 40 tumor samples, the results indicated that 36 samples showed strongly positive, and 4 samples were low positive (Supplementary Data ). With characterization on WHO subtypes, 73.58% tumors were NKUC, 23.58% were NKDC, and 2.83% were KSCC (Supplementary Table ; Supplementary Data ). Based on the standard of the new NPC classification system , all tumors were classified into four subtypes: (1) epithelial carcinoma (EC) (57/106, 53.77%), which is mainly composed of morphologically round epithelial cells; (2) sarcomatoid carcinoma (SC) (20/106, 18.87%), which contains large proportions of spindle sarcomatoid cells; (3) mixed sarcomatoid-epithelial carcinoma (MSEC) (26/106, 24.53%), which shares both features of EC and SC; and (4) squamous cell carcinoma (SCC) (3/106, 2.83%), which is characterized by keratinizing phenotype that is rarely found in other subtypes (Fig. ). More percent of distant metastasis was diagnosed in SC subtype patients (35%) than in EC subtype patients (12.28%) (Supplementary Data ). Next, we conducted the immunofluorescence assay using several antibodies against tumor subtype markers. The data indicated that the EC subtype extensively expressed pan-epithelial markers, including AE1/3, CK5/6, and P63, and the SC subtype was largely positive for vimentin, while MSEC exhibited a mixed pattern of both epithelial and sarcomatoid cell markers (Fig. ; Supplementary Fig. ). These data uncovered distinct molecular features among EC, SC, and MSEC subtypes. Notably, highly proliferating Ki67-positive tumor cells and CD3e-positive lymphocytes were found across all subtypes, with no clear difference among subtypes (Supplementary Fig. ). Mutational landscapes of NPC To further investigate subtype-specific genomic features and therapeutic prognosis, we performed whole-exome sequencing (WES) to study the genomic landscape of NPC subtypes. A total of 2662 somatic mutations including 2306 missense mutations, 191 nonsense mutations, 82 deletions, 31 insertions, and 52 other types of mutations were detected from 88 paired tumors by overlapping results from three different callers (MuTect2, Strelka2 and LANCET), and 2148 genes were affected (Fig. ; Supplementary Fig. ; Supplementary Data ). Validation of candidate mutations with Sanger sequencing showed that a true positive rate of 100% was achieved (Supplementary Fig. ; Supplementary Data ). The somatic mutation rate in NPC is relatively low compared to other types of cancers, with the somatic mutation rate of less than one per megabase (Supplementary Fig. ), which is consistent with previous studies – . Averagely, we identified 30.3 somatic SNVs per sample. The recurred mutations presented in Fig. revealed the oncogenic drivers of NPC, such as TP53 (mutational frequency at 8.0%), CYLD (10.2%), KMT2C (5.7%), NOTCH2 (6.8%), NFKBIA (4.5%), FBXW7 (4.5%), ARID1A (2.3%), PTEN (2.3%), and BAP1 (2.3%). The mutation frequencies identified here were comparable with those reported by several previous studies (Supplementary Table ) , – , although they were, in general, lower than that of some other cancer types . The recurrently mutated genes aggregated into signaling pathways involving in cell cycle, NF-κB signaling, receptor tyrosine kinase (RTK), chromatin remodeling, apoptosis, microtubule polymerization, mitosis regulation, DNA repair and EMT/invasion (Fig. ). Among these top mutated pathways, NF-κB signaling ( CYLD (mutational frequency at 15.2% in EC) , NFKBIA (8.7% in EC) , TRAF3 (6.5% in EC), and RIPK2 (4.3% in EC)), mitosis regulation ( WEE1 (4.3% in EC), NOTCH2 (8.7% in EC), and FBXW7 (8.7% in EC)), DNA repair ( ATM (6.5% in EC) and NBN/NBS1 (4.3% in EC)), and microtubule polymerization ( FMN2 (4.3% in EC)) exhibited relatively high mutational frequencies in EC subtype, which indicated that they might be the contributive factors to subtype-specific oncogenic mechanisms. Mutational signatures analysis revealed that C > T base substitutions was the predominant signatures in NPC without obvious subtype difference (Fig. ; Supplementary Fig. ). The second frequent signature in our NPC cohort was the C > A transition, which was associated with smoking exposure (Supplementary Fig. ) . Consistent conclusion was also demonstrated by previous report . Among top frequent COSMIC signatures in NPC, signatures 2 and 13 were related to APOBEC family, signatures 6, 15, 20, and 16 were related to DNA mismatch repair, signature 5 was of unknown aetiology, signatures 4 and 29 were due to tobacco, and signature 1 was associated with methylcytosine (Supplementary Fig. ). There was no significant difference on these NPC-related COSMIC mutational signatures among subtypes, but slightly higher APOBEC family and methylcytosine related signatures were observed in EC subtype than MSEC and SC subtypes, whereas tobacco associated signatures were higher in SC subtype than other subtypes (Supplementary Fig. ). Driver pathways and networks revealed by copy number variation (CNV) analysis Further investigation on somatic CNVs revealed that frequent chromosomal deletions of Chr. 3p, 9p, 14q, 16q, and amplifications of 3q, 8q, 12p, 12q, and 18q were the major features of NPC across all subtypes (Fig. ; Supplementary Fig. ), which suggested that such recurrent changes were critical genetic events leading to NPC tumorigenesis. The highest frequency of chromosome 3p deletion, locus of MST1R and BAP1 , indicated that the inactivation of tumor suppressor genes in this chromosome might be an early event contributing to transformation from nasopharyngeal epithelium to NPC. There were no significant differences on overall chromosomal gain and loss frequencies among EC, MSEC and SC subtypes, except EC vs. SC on chromosomal gain frequencies (Fig. ). Age also acted as an important contributive factor during NPC progression. Accumulation of chromosomal abnormality was observed with increasing age, and several unique chromosomal variations presented in patients over 50 years of age, such as chromosomal gain of Chr. 3q and losses of Chr. 16q and 19p (Supplementary Fig. ). Somatic CNVs in gene level were called by Sequenza and CNVkit, and only consensus results shown in both callers were retained for further analysis (Fig. ; Supplementary Fig. ). The detected driver mutational events aggregated into several molecular mechanisms, including defective G1-S checkpoint surveillance ( CDKN2A/B , TP53, and CCND1 ), activated NF-κB signaling ( CYLD , TRAF3 , NFKBIA , NLRC5 , LTBR , TNFRSF1A , RELA , NIBP , RELA , RIPK2 , IKBKB , and BIRC2/3 ), aberrant RTK ( PIK3CA , PTEN , ERBB3 , KRAS , MET , BRAF , and MST1R ), and chromatin remodeling ( KMT2C/D, BAP1, ARID1A , and TET1 ) (Fig. ). The mutation frequencies of these NPC drivers were in consistent with previous studies (Supplementary Tables , ) . They were the most frequently mutated pathways across all subtypes uncovered by CNV analysis. One of the interesting findings on these top frequent mutations was that macrophage stimulating 1 receptor ( MST1R ) was detected with recurrent deletions in 55.6% of tumors (Fig. ). MST1R expression in wild-type and mutant samples was further confirmed by IHC staining (Supplementary Fig. ). MST1R was also known as c-Met-related tyrosine kinase, and normally harbored activation/gain mutations and/or overexpression in other cancer types – . In addition to playing an oncogenic role as tyrosine kinase to enhance activation of Ras/MAPK and other signaling cascades, MST1R also plays a vital function in host defense against viral infection, including Epstein–Barr virus (EBV) and human immunodeficiency virus (HIV) , , . Considering the high frequent loss of MST1R had no obvious subtype and age preferences (Supplementary Fig. ), it was suggested that MST1R loss may act as an early event of NPC by increasing oncogenic susceptibility associated with EBV infection, although the action of MST1R in other types of cancers might be opposite. Consistently, when we applied CRISPR-Cas9 system to knockout MST1R in wild-type PDOs, loss of MST1R in NPC neither promoted nor decreased organoid growth (Supplementary Fig. ). Network analysis revealed that MST1R had close interaction with 14-3-3, and MST1R/14-3-3 complex was believed to contribute to the NPC susceptibility (Supplementary Fig. ) . Since subtypes among NPC exhibited distinct morphological phenotypes (Fig. ), they should be driven by different oncogenic factors. To obtain further insights into subtype-specific oncogenic drivers, we next sorted detected somatic CNVs by subtype-specific mutational frequencies and mapped the important subtype-specific mutations to known functional annotations (Fig. ), finally summarized as a pathway diagram to explain subtype-specific driver mechanisms (Fig. ). Chromosomal amplifications of Chr. 1q, 5p, 7p, 15q, 17q, 20q, 21q, 22q and deletions in Chr. 5q, 13q exhibited significant differences among NPC subtypes (Fig. ; Supplementary Fig. ). Further gene-level analysis revealed the EC subtype harbored more frequent mutations involved in microtubule polymerization ( STK11/LKB1 , TPPP , MAP2 , PHLDB2/LL5ß , DNAH5 , KIF2A , and KIF3A ), defective mitotic spindle checkpoint regulation ( PLK2 , SKA1 , SKP2 , MYC , TRIP13 , CEP72 , and YWHAZ ), DNA repair ( APLF , RAD1 , FANCL , and NBN/NBS1 ) (Fig. ). Although several mutations related to NF-κB signaling activation were found across all subtypes as listed above, more activators ( NIBP , IKBKB , and RIPK2 ) were shown at higher frequencies in the EC subtype than in MSEC and SC (Fig. ). Moreover, several important EMT/invasion promoting CNVs ( ETV1 , MACC1 , MAPKAPK2 , COL6A1 , RUNX1 , TIAM1 , and TWIST1 ) were also detected in SC with relatively high frequencies (Fig. ), which might account for the invasive phenotype of SC subtype (Fig. ). Thus, as revealed by genomics analysis, some somatic CNVs and SNVs with distinct subtype-specific mutational frequencies were exhibited in different NPC subtypes, and they aggregated into several important signaling pathways, including microtubule polymerization, mitosis regulation, NF-κB signaling, EMT/invasion, etc., which might contribute to the distinct histological and molecular features among NPC subtypes. NPC organoid model recapitulated morphological signature of NPC subtypes To develop a patient-specific therapeutic approach, we performed 3-dimensional (3D) organotypic culture for fresh NPCs by adapting well-developed methods established for other cancer types. Distinguished from earlier organoid culture studies, we incorporated drug sensitivity with genomic features to establish a precision oncology pipeline for integrative pharmacogenomics studies. We successfully generated patient-derived organoid (PDO) models for 40 out of 43 (93%) patients under our optimized conditions, including 23 EC, 10 MSEC, and 7 SC (Supplementary Table ). Among 40 tumors with PDOs, four organoid lines were derived from two MSEC tumors. In total, we have 42 PDO lines available for further drug treatment study. All established PDOs could readily expanded at least five passages, and later were stored in our living biobank. Organoids derived from EC subtype NPC commonly showed solid sphere-like structures with smooth surfaces, exhibiting few invading cells found at the surface of organoids (Fig. ). In contrast, tumor cells originating from the SC subtype were unable to grow dense spheres under 3D culture conditions, instead, they exhibited a loose discohesive phenotype with spreading spindle cells. To better mimic cell–cell contacts of the tumor microenvironment, we adjusted our culture method slightly, specifically for SC-type organoids. Initially, we allowed cells to aggregate into dense spheres in low-attachment plates, then the formed spheres were embedded into Matrigel as regular organoid culture. Following this improvement, we clearly observed the matrix-invading capability of spindle tumor cells derived from the SC subtype with extensive spike-like protrusions displayed on the organoids surface (Fig. ). Organoids from MSEC subtype usually showed a spindle organoid phenotype similar to SC after stable culture, but smooth solid spheres could still be observed at an early stage (1–2 passages) and were progressively overtaken by spreading of spindle cells during passaging. In general, PDOs well recapitulated the subtype-specific morphological features of the corresponding original tumors. Except for those of the MSEC subtype, PDOs could only preserved the mixed sarcomatoid-epithelial pattern for up to 2 weeks under unified culture conditions. We made further efforts to establish separated cultures for two distinct populations of cells within individual MSEC tumor. After manually separating smooth solid spheres and spindle cells at the first passage of organoid culture from one MSEC tumor, we subsequently established several pairs of organoid lines with EC-type organoids and SC-type organoids (Fig. ). EC-type organoids and SC-type organoids derived from MSEC tumors shared similar morphological phenotypes with organoids derived from EC and SC tumors, respectively (Fig. ) . Our earlier data revealed that EMT probably serves as an important molecular feature to distinguish subtypes among NPC (Fig. ), therefore, we stained PDOs with the pan-epithelial marker AE1/3 and the mesenchymal marker vimentin. In consistent with the parental subtype-specific features, EC subtype organoids showed extensive AE1/3 expression (Fig. ), while organoids derived from MSEC and SC subtypes were positive for vimentin expression (Fig. ). Moreover, PDOs also retained the EBV latency status as evidenced by LMP1 staining on paired tumors and PDOs (Fig. ). Further, we conducted transcriptome sequencing for 14 PDOs, including 8 EC, 4 MSEC, and 2 SC PDOs. Unsurprisingly, EMT promoting genes were extensively enriched with high expression in SC-type organoids, including VIM , ZEB1/2 , S100A4 , FN1 , MMP2 , and TWIST1/2 (Fig. ). Distinct expression patterns of EMT genes were observed in PDO63E and PDO63S organoids derived from the same MSEC tumor, suggesting the mixed sarcomatoid-epithelial feature of MSEC (Fig. 6d). These data supported that PDO model could faithfully recapitulate subtype-specific morphological and molecular signatures of NPC and further confirmed the EMT signature as a discriminative molecular feature for NPC subtypes. To examine whether the PDOs preserved genomic features of parental tumors, WES was performed on 15 pairs of tumors and PDOs. Genome-wide CNV analysis demonstrated that chromosomal gains and losses of parental tumors were well retained in PDOs for the majority of pairs (Fig. ). Similarly, paired tumors and PDOs maintained consistent mutational signature profiles (Supplementary Fig. ). Furthermore, the cancer related gene mutations in parental tumors, thought to be tumor-specific drivers, were preserved in most paired PDOs (Fig. ). Since there were two morphologically distinct cell populations observed in same MSEC tumor, it was of interest to explore whether they originated from same clone or polyclone. WES analysis on paired P66, PDO66E, and PDO66S indicated that EC-type PDO66E share a number of common mutations with SC-type PDO66S. This suggested that the heterogeneity of two distinct populations within MSEC might originate from the same cell during tumor evolution (Supplementary Fig. ). Altogether, our data demonstrated that NPC organoid model faithfully recapitulated the signatures of parental tumors, including morphological, molecular and genomic features. PDOs may serve as good proxies to evaluate treatment responses of potential therapeutics for individual patients. Subtype-specific drug response revealed by PDO-based drug screening Next, we conducted drug screening to identify effective therapeutic candidates using PDOs. 42 PDOs were successfully screened with a drug library containing 48 drugs, including several first-line chemotherapeutic drugs for NPC, such as docetaxel, paclitaxel and bleomycin, in real-time at passages 1–3 within 2–3 weeks after biopsy collection (Supplementary Fig. ; Supplementary Data ). Each drug was screened at six concentrations from 0.08 to 20 μM at 3-fold dilutions, and the drug sensitivity was represented by an inhibitory concentration of 50% (IC50). In general, none of the PDOs exhibited identical drug responses, reflecting individual differences, although subtype-specific responses were noticed to some drugs (Fig. ; Supplementary Fig. ). For example, organoids derived from SC and MSEC, except EC-type PDO63E and PDO66E, were sensitive to microtubule inhibitors (MTi), while organoids derived from EC exhibited resistance to MTi at various levels (Fig. ). Some representative drug responses were repeated and shown in Fig. . As mentioned earlier, PDO63E and PDO63S were derived from a single MSEC tumor and exhibited distinct EC-type and SC-type morphology, respectively. EC-type PDO63E organoids showed the same resistance response to MTi as EC PDOs, while SC-type PDO63S organoids were sensitive to MTi, similar to SC PDOs (Fig. ). On the other hand, for EGFR inhibitors (EGFRi), 22 of 23 PDOs derived from EC and EC-type PDO63E were highly sensitive, while all organoids derived from SC and 9 out of 12 PDOs derived from MSEC showed resistance to EGFRi. Of note, MSEC PDOs displayed diverse responses to EGFRi (Fig. ), indicating that MSEC contains both SC and EC features. In general, EC subtype PDOs were sensitive to EGFRi and resistant to MTi, while opposite phenotypes were shown in PDOs derived from SC and over half of MSEC. To understand the regulating key molecular signaling responsible for this distinct responsiveness, we firstly treated several resistant and sensitive cell lines with gefitinib, a known EGFRi, and examined key EGFR downstream targets by Western blot analysis. Our data revealed that gefitinib significantly reduced both p-AKT and p-ERK in sensitive PDOs PDO63E and PDO4, p-STAT3 also showed a reduction in PDO4 but not in PDO63E (Fig. ), and in the other four sensitive PDOs, three detected downstream signaling pathways of EGFR exhibited blocked phosphorylation to varying degrees, which were seldom found in resistant PDOs (Fig. ; Supplementary Fig. ). In general, ERK and AKT phosphorylation were blocked in all tested sensitive PDOs under gefitinib treatment, except PDO14, which did not show a decrease in AKT phosphorylation. Combinational treatment with AKT inhibitor MK2206, ERK inhibitor GDC0994, STAT3 inhibitor C188-9 and gefitinib could overcome the EGFRi resistance in PDO63S (Supplementary Fig. , ). These data indicate that the MAPK and PI3K-AKT signaling pathways might play crucial roles in the treatment efficiency of EGFRi in NPC. Furthermore, MTi (docetaxel) treatment increased the expression of mitotic checkpoint proteins (cyclin A and cyclin B), degraded the antiapoptotic protein Bcl2 through phosphorylation, and activated proapoptotic proteins such as Bax and cleaved caspase-3 (CASP3) in sensitive PDOs, while no significant changes in these proteins were observed in resistant PDOs (Fig. ; Supplementary Fig. ). Cyclin B is expressed during the late G2 and early M phases of the cell cycle and drives cells into the M phase. Docetaxel treatment blocked the formation of the spindle, leading to accumulation of cells in M phase, which was consistent with the increased level of cyclin B. Suppression of the apoptosis-blocking function through phosphorylation and degradation of Bcl2 was one of the important established MTi-affecting mechanisms of tumor cells , . In all six sensitive PDOs, docetaxel strongly induced Bcl2 degradation and activated the apoptotic death marker Bax, which indicated the importance of Bcl2-mediated antiapoptosis in NPC. In the resistant PDOs, none of these events were occurred (Fig. ; Supplementary Fig. ), which was consistent with their resistance to MTi. As representative MSEC-derived PDOs, EC-type PDO63E, and SC-type PDO63S exhibited opposite responses to MTi. SC-type PDO63S responded well to MTi treatment with mitotic checkpoint protein blockage and Bcl2 degradation, followed by induction of apoptosis activation, whereas EC-type PDO63E showed no significant changes in these signaling pathways. Together with their distinct responses to EGFRi mentioned earlier, these results further demonstrated the heterogeneity of MSEC subtype tumor, at least two distinct tumor populations were occurred in one tumor: one population shared a similar drug response pattern similar to that of EC, and the other shared similarity to SC. Individualized synergistic therapeutic combinations identified from PDO-based chemoradiotherapy (CRT) screening In addition to chemotherapy, which is the widely accepted regimen for advanced NPC with metastasis, chemoradiotherapy (CRT) is recognized as the standard therapy for stage II and more advanced NPC, yet only limited chemical drugs were approved for NPC treatment. To investigate whether PDOs could be used for identifying more drugs for CRT with enhanced therapeutic effect towards individual patients, we conducted sensitivity tests for radiotherapy (RT) and CRT. Through treatment on eight PDO lines encompassing three major NPC subtypes with increased ionizing radiation (IR) dose from 2, 4, 6, 8, and 10 Gy, we found that the tested EC subtype PDOs were more sensitive to RT than the SC and MSEC subtype PDOs (Fig. ). Our RT results reflected the previously reported clinical outcome that SC subtype patients had an ~20% lower 5-year OS rate than EC subtype patients . For recurrent and/or metastatic NPC, CRT is the standard-of-care treatment. We next examined the efficacy of commonly using CRT combinations on 20 PDOs derived from individual NPCs (Fig. ; Supplementary Data ). In general, CRT exhibited better sensitivities on NPC PDOs than chemotherapeutics alone, especially the most commonly used CRT regimens, RT + cisplatin and RT + fluorouracil (Fig. ), which generally reflected the clinical treatment outcomes , , . When comparing efficacy among NPC subtypes, CRT demonstrated better sensitivities on EC-type organoids than SC-type organoids for the majority of cases, including CRT regimens combined with cisplatin and fluorouracil (Fig. ). These results were consistent with the previously reported clinical outcome that CRT contributed to longer survival time for EC subtype patients than that for SC subtype patients . To identify more drugs that could sensitize PDOs to RT, we further screened 48 anticancer drugs on eight PDOs combined with IR at a dose of approximately IC20-30 (4 Gy) (Supplementary Data ). One of the most striking difference was demonstrated by CRT combinations with EGFRi on organoids derived from EC subtype (Fig. ; Supplementary Fig. ). For 4 tested EC subtype PDO lines, CRT exhibited synergistically better inhibitory effect than EGFRi treatment alone, with the average IC50 value fold increases at 7.09, 5.87, 6.01, and 8.17 in each of these PDOs, respectively (Fig. ; Supplementary Fig. ). Since EGFRi was identified as EC subtype-sensitizing drug, as demonstrated by our earlier data (Fig. ), our CRT screening results further suggested that the EGFRi and RT combination could be a good clinical CRT choice to achieve synergistic killing effect for EC subtype patients. In addition, an improvement on MTi efficacy was also found when combining with RT, but there was no observation on drug synergy (Fig. ). Furthermore, a few other CRT combinations with enhanced sensitivity were also revealed by our screening data, such as combinations with arsenic trioxide (Supplementary Fig. ), olaparib (Supplementary Fig. ), vorinostat (Supplementary Fig. ), sunitinib (Supplementary Fig. ), and zoledronate (Supplementary Fig. ). Thus, our analysis uncovered the subtype-related and patient-specific responses to RT and CRT and potentially provided more choice of drugs for CRT to treat patients suffering from advanced or metastatic stage cancer. Transcriptome analysis revealed genomic driver mechanisms of NPC subtypes Transcriptome sequencing was performed for 14 PDOs and subsequently assigned for unsupervised clustering by principal component analysis (PCA) to evaluate the overall transcriptional pattern of the samples. The results clearly demonstrated that organoids derived from the same subtypes were well clustered into the corresponding subtypes (Fig. ), indicating that PDOs maintained their subtype differences at the transcriptional level. As revealed by our earlier genomic sequencing analysis, EC exhibited distinct driver mechanisms with SC and MSEC, including microtubule polymerization, G2-M checkpoint regulation, NF-κB signaling, and EMT. We wondered whether these subtype-specific genomic mutational pathways could be converted to functional transcriptional signaling. The unbiased geneset enrichment analysis (GSEA) was performed to investigate top differentiated pathways between EC- and SC-type PDOs (absolute NES value > 1, p -value < 0.05, and FDR value < 0.05) (Fig. ). Significantly differentiated expressed genes were filtered by DESeq2 and limma (log 2 fold change > 1, p- value < 0.05, FDR (padj) value < 0.05) (Fig. ; Supplementary Fig. ; Supplementary Data ), then were presented in heatmap (Fig. ). Microtubule-targeting drugs associated mechanisms, including microtubule signaling, mitotic cell cycle, apoptosis, NF-κB signaling, and androgen response, were among the top enriched pathways in EC-type organoids (Fig. , Supplementary Fig. ) – . Meanwhile, multiple EGFR signaling related pathways and cellular response to radiation were also among top differentiated pathways (Fig. ; Supplementary Figs. a-l, ), which might contribute to the subtype-differentiated responses to EGFR inhibitors and RT. EMT and invasion related pathways, including EMT, extracellular matrix and response to TGF-β, were enriched in SC-type organoids (Fig. , Supplementary Fig. ). Of note, differentially expressed pathway genes were also present between PDO63E and PDO63S (Fig. ; Supplementary Fig. ), which was consistent with earlier results on their drug response and protein expression differences, and further demonstrated the heterogeneity feature of MSEC. In addition, the stemness score was calculated for each NPC subtype PDOs, and the results indicated that EC subtype PDOs maintained high stemness properties, whereas the opposite phenotype was shown in SC subtype PDOs (Supplementary Fig. ). Distinctly to EC and SC, a diverse stemness distribution was observed in MSEC PDOs (Supplementary Fig. ). Moreover, the immune score analysis was also performed to determine whether there were subtype differences among immune cell types, and the results indicated no obvious differences in CD4 + T cells, CD8 + T cells, B cells, or macrophages among the three subtypes (Supplementary Fig. ) Pharmacogenomics-based precision medicine During past decade, next-generation sequencing has been extensively applied for precision oncology and has made great progress in matching targeted therapy with predictive gene signatures. However, several essential limitations have also come to surface, including the lack of identified druggable mutations for the majority of patient in current clinical practice and the reliability of drug prediction value, which was controversial . By following conventional practice focusing on targetable mutations identification, we performed genomics sequencing for tumors of individual patients, but unfortunately, well-known drug sensitizing hotspot mutations were seldom found in the majority of NPC tumors as expected , for example, the frequency of EGFR SNVs was only 0.94% (1/106) in our NPC cohort (Supplementary Data ), which kept us from directly identifying corresponding targeted drugs. To better deliver precision medicine to NPC patients, we next proposed to develop a pharmacogenomics-based precision medicine (PBPM) approach that integrated genomics/transcriptomics and drug tests to better provide faithful precision treatment guidance. On the drug test side, we applied PDOs as the proxies to examine drug responsiveness for individual patients and found that many EC subtype PDOs were resistant to MTi, while opposite response of MTi was observed in SC and MSEC subtype derived organoids (Figs. a, ). The results indicated that some subtype-associated gene signatures might contribute to the subtype-specific MTi responses, thus uncovering these gene signatures might be valuable for predicting MTi sensitivity. Next, we incorporated genomic variations of all protein coding genes into our analysis, in addition to only druggable mutations. By grouping samples with MTi sensitivity on PDOs, we revealed that consistent subtype-specific mutations pattern was potentially involved in microtubule-targeting drug responses (Fig. ). Microtubule-targeting drugs were known to disrupt microtubule dynamics, invoke the mitotic checkpoint, lead to cell cycle arrest, and subsequently induce apoptosis – . Thus, activation of key mutations involved in antagonizing these processes may help tumor gain resistance to MTi. Of note, we observed an obvious positive correlation between EC-specific MTi resistance and EC-specific genomic activations of microtubule polymerization, mitosis regulation, NF-κB and DNA repair (Fig. ). A representative personalized case was demonstrated with P13, which help to illustrate the intrinsic correlation mechanism (Fig. ). To further validate these mutations as predictive signatures for MTi response, we applied CRISPR-Cas9 to knockout TPPP (tubulin polymerization-promoting protein), MAP2 (microtubule-associated protein 2), and SKP2 (S-phase kinase-associated protein 2) in PDO9 and PDO4, respectively (Supplementary Fig. ; Supplementary Data ). TPPP , MAP2 , and SKP2 knockout organoids showed sensitive responses to docetaxel treatment when comparing to parental wild-type organoids (Fig. ; Supplementary Fig. ), indicating mutations involving in microtubule polymerization and mitosis regulation could serve as potential predictive signatures for MTi response in NPC. Another personalized case was demonstrated with P86, which acquired EGFRi resistance. As shown in Supplementary Fig. , copy number gains of MET and KRAS were observed in P86 tumors. Activation of oncogenic MET and KRAS potentially induced EGFRi resistance through bypassing conventional EGFR signaling. To further confirm their contributive effect on EGFRi resistance, we applied crizotinib and vorinostat to perform combinational inhibition together with gefitinib in PDO86 (Supplementary Fig. ). Crizotinib is a specific inhibitor of c-MET, and HDAC inhibitor vorinostat was previously reported to overcome EGFRi resistance by blocking KRAS – . The result showed that after dual-blocking of c-MET and KRAS, EGFRi resistance in PDO86 was significantly overcome (Supplementary Fig. ), indicating that copy number gains of MET and KRAS might contributed to EGFRi resistance in PDO86. Based on our previous practice on genomics driven PBPM strategy, although as many as all protein coding mutations were involved into analysis, only a portion of patients could be identified with the discriminative genomic signatures associated to drug response and got the benefit. Integrating transcriptomics with drug response seems to be another good option to develop PBPM. As shown in Fig. , microtubule signaling, mitotic cell cycle, apoptosis, NF-κB signaling, and androgen response, were among top subtype-differentiated pathways. They were reported to contribute to MTi response mechanisms – . We assembled gene cohorts containing signature genes involved in these pathways with differential expression patterns between EC and SC/MSEC subtypes, and these genesets was further applied to analyze the correlation between subtypes and MTi response (Supplementary Data ). EC subtype PDOs showed a positive correlation with the docetaxel resistance, while SC and MSEC subtype PDOs exhibited a positive correlation with the docetaxel sensitivity (Fig. ). We next examined the impact of individual subtype-differentiated pathways on discriminating the MTi responses. The results demonstrated that individual pathways could well distinguish docetaxel-sensitive and docetaxel-resistant PDOs (Fig. ). This data led us to believe that these five subtype-differentiating pathway signatures were the candidate contributive factors for the NPC subtype-specific MTi response. To further validate our hypotheses as well as test the drug prediction value of these pathway signatures, we download public data of both transcriptome sequencing and MTi treatment results from the Genomics of Drug Sensitivity in Cancer (GDSC) database . After sorting MTi-sensitive and MTi-resistant cell lines with the bottom 10% and top 10% IC50 values of docetaxel, paclitaxel, vinorelbine, and vincristine, we applied our pathway signatures to GDSC data to perform receiver operating characteristic (ROC) analysis. As shown in Fig. , the individual pathway signature obtained area under curve (AUC) values ranging from 0.83 to 0.914 for predicting docetaxel sensitivity, indicating significant contributive impacts of these pathway signatures on MTi sensitivity, while the predictive AUC value of the random geneset was 0.5. Of note, when combining all 5 pathway signatures together, we achieved the best drug predictive value with AUC values of 0.918 (docetaxel) (Fig. ), 0.949 (paclitaxel) (Supplementary Fig. ), 0.906 (vincristine) (Supplementary Fig. ), and 0.915 (vinorelbine) (Supplementary Fig. ). To further confirm the efficiency of prediction power in clinical practice, we applied datasets of human cancer patients treated with paclitaxel and docetaxel to evaluate the predictive value. Both GSE22513 and GSE6434 human dataset achieved good prediction accuracy for MTi response with AUC values of 0.914 and 0.841 using the 105 combined gene signatures (Fig. ). Similar result was also demonstrated with patient-derived xenografts (PDXs) (AUC = 0.872) (Supplementary Fig. ). Through our proposed PBPM approach by integrating genomics/transcriptomics and PDO-based drug tests, the multidimensionally confirmed results could serve as promising indications to guide individual treatment, even to those subjects without any identified druggable mutations from genomic analysis. More importantly, guided by the integrated PBPM analysis results, we further demonstrated the histological subtype-related drug responsiveness of MTi on PDOs, which could significantly facilitate precision oncology for NPC to be subtype-guided therapy in the future.
A total of 106 NPC tumors were collected from the hospital with routine records (Supplementary Table ; Supplementary Data , ), and we further studied their histological features. All NPC patients were of Chinese origin with a median diagnosed age of 48 years. EBV status was examined on 40 tumor samples, the results indicated that 36 samples showed strongly positive, and 4 samples were low positive (Supplementary Data ). With characterization on WHO subtypes, 73.58% tumors were NKUC, 23.58% were NKDC, and 2.83% were KSCC (Supplementary Table ; Supplementary Data ). Based on the standard of the new NPC classification system , all tumors were classified into four subtypes: (1) epithelial carcinoma (EC) (57/106, 53.77%), which is mainly composed of morphologically round epithelial cells; (2) sarcomatoid carcinoma (SC) (20/106, 18.87%), which contains large proportions of spindle sarcomatoid cells; (3) mixed sarcomatoid-epithelial carcinoma (MSEC) (26/106, 24.53%), which shares both features of EC and SC; and (4) squamous cell carcinoma (SCC) (3/106, 2.83%), which is characterized by keratinizing phenotype that is rarely found in other subtypes (Fig. ). More percent of distant metastasis was diagnosed in SC subtype patients (35%) than in EC subtype patients (12.28%) (Supplementary Data ). Next, we conducted the immunofluorescence assay using several antibodies against tumor subtype markers. The data indicated that the EC subtype extensively expressed pan-epithelial markers, including AE1/3, CK5/6, and P63, and the SC subtype was largely positive for vimentin, while MSEC exhibited a mixed pattern of both epithelial and sarcomatoid cell markers (Fig. ; Supplementary Fig. ). These data uncovered distinct molecular features among EC, SC, and MSEC subtypes. Notably, highly proliferating Ki67-positive tumor cells and CD3e-positive lymphocytes were found across all subtypes, with no clear difference among subtypes (Supplementary Fig. ).
To further investigate subtype-specific genomic features and therapeutic prognosis, we performed whole-exome sequencing (WES) to study the genomic landscape of NPC subtypes. A total of 2662 somatic mutations including 2306 missense mutations, 191 nonsense mutations, 82 deletions, 31 insertions, and 52 other types of mutations were detected from 88 paired tumors by overlapping results from three different callers (MuTect2, Strelka2 and LANCET), and 2148 genes were affected (Fig. ; Supplementary Fig. ; Supplementary Data ). Validation of candidate mutations with Sanger sequencing showed that a true positive rate of 100% was achieved (Supplementary Fig. ; Supplementary Data ). The somatic mutation rate in NPC is relatively low compared to other types of cancers, with the somatic mutation rate of less than one per megabase (Supplementary Fig. ), which is consistent with previous studies – . Averagely, we identified 30.3 somatic SNVs per sample. The recurred mutations presented in Fig. revealed the oncogenic drivers of NPC, such as TP53 (mutational frequency at 8.0%), CYLD (10.2%), KMT2C (5.7%), NOTCH2 (6.8%), NFKBIA (4.5%), FBXW7 (4.5%), ARID1A (2.3%), PTEN (2.3%), and BAP1 (2.3%). The mutation frequencies identified here were comparable with those reported by several previous studies (Supplementary Table ) , – , although they were, in general, lower than that of some other cancer types . The recurrently mutated genes aggregated into signaling pathways involving in cell cycle, NF-κB signaling, receptor tyrosine kinase (RTK), chromatin remodeling, apoptosis, microtubule polymerization, mitosis regulation, DNA repair and EMT/invasion (Fig. ). Among these top mutated pathways, NF-κB signaling ( CYLD (mutational frequency at 15.2% in EC) , NFKBIA (8.7% in EC) , TRAF3 (6.5% in EC), and RIPK2 (4.3% in EC)), mitosis regulation ( WEE1 (4.3% in EC), NOTCH2 (8.7% in EC), and FBXW7 (8.7% in EC)), DNA repair ( ATM (6.5% in EC) and NBN/NBS1 (4.3% in EC)), and microtubule polymerization ( FMN2 (4.3% in EC)) exhibited relatively high mutational frequencies in EC subtype, which indicated that they might be the contributive factors to subtype-specific oncogenic mechanisms. Mutational signatures analysis revealed that C > T base substitutions was the predominant signatures in NPC without obvious subtype difference (Fig. ; Supplementary Fig. ). The second frequent signature in our NPC cohort was the C > A transition, which was associated with smoking exposure (Supplementary Fig. ) . Consistent conclusion was also demonstrated by previous report . Among top frequent COSMIC signatures in NPC, signatures 2 and 13 were related to APOBEC family, signatures 6, 15, 20, and 16 were related to DNA mismatch repair, signature 5 was of unknown aetiology, signatures 4 and 29 were due to tobacco, and signature 1 was associated with methylcytosine (Supplementary Fig. ). There was no significant difference on these NPC-related COSMIC mutational signatures among subtypes, but slightly higher APOBEC family and methylcytosine related signatures were observed in EC subtype than MSEC and SC subtypes, whereas tobacco associated signatures were higher in SC subtype than other subtypes (Supplementary Fig. ).
Further investigation on somatic CNVs revealed that frequent chromosomal deletions of Chr. 3p, 9p, 14q, 16q, and amplifications of 3q, 8q, 12p, 12q, and 18q were the major features of NPC across all subtypes (Fig. ; Supplementary Fig. ), which suggested that such recurrent changes were critical genetic events leading to NPC tumorigenesis. The highest frequency of chromosome 3p deletion, locus of MST1R and BAP1 , indicated that the inactivation of tumor suppressor genes in this chromosome might be an early event contributing to transformation from nasopharyngeal epithelium to NPC. There were no significant differences on overall chromosomal gain and loss frequencies among EC, MSEC and SC subtypes, except EC vs. SC on chromosomal gain frequencies (Fig. ). Age also acted as an important contributive factor during NPC progression. Accumulation of chromosomal abnormality was observed with increasing age, and several unique chromosomal variations presented in patients over 50 years of age, such as chromosomal gain of Chr. 3q and losses of Chr. 16q and 19p (Supplementary Fig. ). Somatic CNVs in gene level were called by Sequenza and CNVkit, and only consensus results shown in both callers were retained for further analysis (Fig. ; Supplementary Fig. ). The detected driver mutational events aggregated into several molecular mechanisms, including defective G1-S checkpoint surveillance ( CDKN2A/B , TP53, and CCND1 ), activated NF-κB signaling ( CYLD , TRAF3 , NFKBIA , NLRC5 , LTBR , TNFRSF1A , RELA , NIBP , RELA , RIPK2 , IKBKB , and BIRC2/3 ), aberrant RTK ( PIK3CA , PTEN , ERBB3 , KRAS , MET , BRAF , and MST1R ), and chromatin remodeling ( KMT2C/D, BAP1, ARID1A , and TET1 ) (Fig. ). The mutation frequencies of these NPC drivers were in consistent with previous studies (Supplementary Tables , ) . They were the most frequently mutated pathways across all subtypes uncovered by CNV analysis. One of the interesting findings on these top frequent mutations was that macrophage stimulating 1 receptor ( MST1R ) was detected with recurrent deletions in 55.6% of tumors (Fig. ). MST1R expression in wild-type and mutant samples was further confirmed by IHC staining (Supplementary Fig. ). MST1R was also known as c-Met-related tyrosine kinase, and normally harbored activation/gain mutations and/or overexpression in other cancer types – . In addition to playing an oncogenic role as tyrosine kinase to enhance activation of Ras/MAPK and other signaling cascades, MST1R also plays a vital function in host defense against viral infection, including Epstein–Barr virus (EBV) and human immunodeficiency virus (HIV) , , . Considering the high frequent loss of MST1R had no obvious subtype and age preferences (Supplementary Fig. ), it was suggested that MST1R loss may act as an early event of NPC by increasing oncogenic susceptibility associated with EBV infection, although the action of MST1R in other types of cancers might be opposite. Consistently, when we applied CRISPR-Cas9 system to knockout MST1R in wild-type PDOs, loss of MST1R in NPC neither promoted nor decreased organoid growth (Supplementary Fig. ). Network analysis revealed that MST1R had close interaction with 14-3-3, and MST1R/14-3-3 complex was believed to contribute to the NPC susceptibility (Supplementary Fig. ) . Since subtypes among NPC exhibited distinct morphological phenotypes (Fig. ), they should be driven by different oncogenic factors. To obtain further insights into subtype-specific oncogenic drivers, we next sorted detected somatic CNVs by subtype-specific mutational frequencies and mapped the important subtype-specific mutations to known functional annotations (Fig. ), finally summarized as a pathway diagram to explain subtype-specific driver mechanisms (Fig. ). Chromosomal amplifications of Chr. 1q, 5p, 7p, 15q, 17q, 20q, 21q, 22q and deletions in Chr. 5q, 13q exhibited significant differences among NPC subtypes (Fig. ; Supplementary Fig. ). Further gene-level analysis revealed the EC subtype harbored more frequent mutations involved in microtubule polymerization ( STK11/LKB1 , TPPP , MAP2 , PHLDB2/LL5ß , DNAH5 , KIF2A , and KIF3A ), defective mitotic spindle checkpoint regulation ( PLK2 , SKA1 , SKP2 , MYC , TRIP13 , CEP72 , and YWHAZ ), DNA repair ( APLF , RAD1 , FANCL , and NBN/NBS1 ) (Fig. ). Although several mutations related to NF-κB signaling activation were found across all subtypes as listed above, more activators ( NIBP , IKBKB , and RIPK2 ) were shown at higher frequencies in the EC subtype than in MSEC and SC (Fig. ). Moreover, several important EMT/invasion promoting CNVs ( ETV1 , MACC1 , MAPKAPK2 , COL6A1 , RUNX1 , TIAM1 , and TWIST1 ) were also detected in SC with relatively high frequencies (Fig. ), which might account for the invasive phenotype of SC subtype (Fig. ). Thus, as revealed by genomics analysis, some somatic CNVs and SNVs with distinct subtype-specific mutational frequencies were exhibited in different NPC subtypes, and they aggregated into several important signaling pathways, including microtubule polymerization, mitosis regulation, NF-κB signaling, EMT/invasion, etc., which might contribute to the distinct histological and molecular features among NPC subtypes.
To develop a patient-specific therapeutic approach, we performed 3-dimensional (3D) organotypic culture for fresh NPCs by adapting well-developed methods established for other cancer types. Distinguished from earlier organoid culture studies, we incorporated drug sensitivity with genomic features to establish a precision oncology pipeline for integrative pharmacogenomics studies. We successfully generated patient-derived organoid (PDO) models for 40 out of 43 (93%) patients under our optimized conditions, including 23 EC, 10 MSEC, and 7 SC (Supplementary Table ). Among 40 tumors with PDOs, four organoid lines were derived from two MSEC tumors. In total, we have 42 PDO lines available for further drug treatment study. All established PDOs could readily expanded at least five passages, and later were stored in our living biobank. Organoids derived from EC subtype NPC commonly showed solid sphere-like structures with smooth surfaces, exhibiting few invading cells found at the surface of organoids (Fig. ). In contrast, tumor cells originating from the SC subtype were unable to grow dense spheres under 3D culture conditions, instead, they exhibited a loose discohesive phenotype with spreading spindle cells. To better mimic cell–cell contacts of the tumor microenvironment, we adjusted our culture method slightly, specifically for SC-type organoids. Initially, we allowed cells to aggregate into dense spheres in low-attachment plates, then the formed spheres were embedded into Matrigel as regular organoid culture. Following this improvement, we clearly observed the matrix-invading capability of spindle tumor cells derived from the SC subtype with extensive spike-like protrusions displayed on the organoids surface (Fig. ). Organoids from MSEC subtype usually showed a spindle organoid phenotype similar to SC after stable culture, but smooth solid spheres could still be observed at an early stage (1–2 passages) and were progressively overtaken by spreading of spindle cells during passaging. In general, PDOs well recapitulated the subtype-specific morphological features of the corresponding original tumors. Except for those of the MSEC subtype, PDOs could only preserved the mixed sarcomatoid-epithelial pattern for up to 2 weeks under unified culture conditions. We made further efforts to establish separated cultures for two distinct populations of cells within individual MSEC tumor. After manually separating smooth solid spheres and spindle cells at the first passage of organoid culture from one MSEC tumor, we subsequently established several pairs of organoid lines with EC-type organoids and SC-type organoids (Fig. ). EC-type organoids and SC-type organoids derived from MSEC tumors shared similar morphological phenotypes with organoids derived from EC and SC tumors, respectively (Fig. ) . Our earlier data revealed that EMT probably serves as an important molecular feature to distinguish subtypes among NPC (Fig. ), therefore, we stained PDOs with the pan-epithelial marker AE1/3 and the mesenchymal marker vimentin. In consistent with the parental subtype-specific features, EC subtype organoids showed extensive AE1/3 expression (Fig. ), while organoids derived from MSEC and SC subtypes were positive for vimentin expression (Fig. ). Moreover, PDOs also retained the EBV latency status as evidenced by LMP1 staining on paired tumors and PDOs (Fig. ). Further, we conducted transcriptome sequencing for 14 PDOs, including 8 EC, 4 MSEC, and 2 SC PDOs. Unsurprisingly, EMT promoting genes were extensively enriched with high expression in SC-type organoids, including VIM , ZEB1/2 , S100A4 , FN1 , MMP2 , and TWIST1/2 (Fig. ). Distinct expression patterns of EMT genes were observed in PDO63E and PDO63S organoids derived from the same MSEC tumor, suggesting the mixed sarcomatoid-epithelial feature of MSEC (Fig. 6d). These data supported that PDO model could faithfully recapitulate subtype-specific morphological and molecular signatures of NPC and further confirmed the EMT signature as a discriminative molecular feature for NPC subtypes. To examine whether the PDOs preserved genomic features of parental tumors, WES was performed on 15 pairs of tumors and PDOs. Genome-wide CNV analysis demonstrated that chromosomal gains and losses of parental tumors were well retained in PDOs for the majority of pairs (Fig. ). Similarly, paired tumors and PDOs maintained consistent mutational signature profiles (Supplementary Fig. ). Furthermore, the cancer related gene mutations in parental tumors, thought to be tumor-specific drivers, were preserved in most paired PDOs (Fig. ). Since there were two morphologically distinct cell populations observed in same MSEC tumor, it was of interest to explore whether they originated from same clone or polyclone. WES analysis on paired P66, PDO66E, and PDO66S indicated that EC-type PDO66E share a number of common mutations with SC-type PDO66S. This suggested that the heterogeneity of two distinct populations within MSEC might originate from the same cell during tumor evolution (Supplementary Fig. ). Altogether, our data demonstrated that NPC organoid model faithfully recapitulated the signatures of parental tumors, including morphological, molecular and genomic features. PDOs may serve as good proxies to evaluate treatment responses of potential therapeutics for individual patients.
Next, we conducted drug screening to identify effective therapeutic candidates using PDOs. 42 PDOs were successfully screened with a drug library containing 48 drugs, including several first-line chemotherapeutic drugs for NPC, such as docetaxel, paclitaxel and bleomycin, in real-time at passages 1–3 within 2–3 weeks after biopsy collection (Supplementary Fig. ; Supplementary Data ). Each drug was screened at six concentrations from 0.08 to 20 μM at 3-fold dilutions, and the drug sensitivity was represented by an inhibitory concentration of 50% (IC50). In general, none of the PDOs exhibited identical drug responses, reflecting individual differences, although subtype-specific responses were noticed to some drugs (Fig. ; Supplementary Fig. ). For example, organoids derived from SC and MSEC, except EC-type PDO63E and PDO66E, were sensitive to microtubule inhibitors (MTi), while organoids derived from EC exhibited resistance to MTi at various levels (Fig. ). Some representative drug responses were repeated and shown in Fig. . As mentioned earlier, PDO63E and PDO63S were derived from a single MSEC tumor and exhibited distinct EC-type and SC-type morphology, respectively. EC-type PDO63E organoids showed the same resistance response to MTi as EC PDOs, while SC-type PDO63S organoids were sensitive to MTi, similar to SC PDOs (Fig. ). On the other hand, for EGFR inhibitors (EGFRi), 22 of 23 PDOs derived from EC and EC-type PDO63E were highly sensitive, while all organoids derived from SC and 9 out of 12 PDOs derived from MSEC showed resistance to EGFRi. Of note, MSEC PDOs displayed diverse responses to EGFRi (Fig. ), indicating that MSEC contains both SC and EC features. In general, EC subtype PDOs were sensitive to EGFRi and resistant to MTi, while opposite phenotypes were shown in PDOs derived from SC and over half of MSEC. To understand the regulating key molecular signaling responsible for this distinct responsiveness, we firstly treated several resistant and sensitive cell lines with gefitinib, a known EGFRi, and examined key EGFR downstream targets by Western blot analysis. Our data revealed that gefitinib significantly reduced both p-AKT and p-ERK in sensitive PDOs PDO63E and PDO4, p-STAT3 also showed a reduction in PDO4 but not in PDO63E (Fig. ), and in the other four sensitive PDOs, three detected downstream signaling pathways of EGFR exhibited blocked phosphorylation to varying degrees, which were seldom found in resistant PDOs (Fig. ; Supplementary Fig. ). In general, ERK and AKT phosphorylation were blocked in all tested sensitive PDOs under gefitinib treatment, except PDO14, which did not show a decrease in AKT phosphorylation. Combinational treatment with AKT inhibitor MK2206, ERK inhibitor GDC0994, STAT3 inhibitor C188-9 and gefitinib could overcome the EGFRi resistance in PDO63S (Supplementary Fig. , ). These data indicate that the MAPK and PI3K-AKT signaling pathways might play crucial roles in the treatment efficiency of EGFRi in NPC. Furthermore, MTi (docetaxel) treatment increased the expression of mitotic checkpoint proteins (cyclin A and cyclin B), degraded the antiapoptotic protein Bcl2 through phosphorylation, and activated proapoptotic proteins such as Bax and cleaved caspase-3 (CASP3) in sensitive PDOs, while no significant changes in these proteins were observed in resistant PDOs (Fig. ; Supplementary Fig. ). Cyclin B is expressed during the late G2 and early M phases of the cell cycle and drives cells into the M phase. Docetaxel treatment blocked the formation of the spindle, leading to accumulation of cells in M phase, which was consistent with the increased level of cyclin B. Suppression of the apoptosis-blocking function through phosphorylation and degradation of Bcl2 was one of the important established MTi-affecting mechanisms of tumor cells , . In all six sensitive PDOs, docetaxel strongly induced Bcl2 degradation and activated the apoptotic death marker Bax, which indicated the importance of Bcl2-mediated antiapoptosis in NPC. In the resistant PDOs, none of these events were occurred (Fig. ; Supplementary Fig. ), which was consistent with their resistance to MTi. As representative MSEC-derived PDOs, EC-type PDO63E, and SC-type PDO63S exhibited opposite responses to MTi. SC-type PDO63S responded well to MTi treatment with mitotic checkpoint protein blockage and Bcl2 degradation, followed by induction of apoptosis activation, whereas EC-type PDO63E showed no significant changes in these signaling pathways. Together with their distinct responses to EGFRi mentioned earlier, these results further demonstrated the heterogeneity of MSEC subtype tumor, at least two distinct tumor populations were occurred in one tumor: one population shared a similar drug response pattern similar to that of EC, and the other shared similarity to SC.
In addition to chemotherapy, which is the widely accepted regimen for advanced NPC with metastasis, chemoradiotherapy (CRT) is recognized as the standard therapy for stage II and more advanced NPC, yet only limited chemical drugs were approved for NPC treatment. To investigate whether PDOs could be used for identifying more drugs for CRT with enhanced therapeutic effect towards individual patients, we conducted sensitivity tests for radiotherapy (RT) and CRT. Through treatment on eight PDO lines encompassing three major NPC subtypes with increased ionizing radiation (IR) dose from 2, 4, 6, 8, and 10 Gy, we found that the tested EC subtype PDOs were more sensitive to RT than the SC and MSEC subtype PDOs (Fig. ). Our RT results reflected the previously reported clinical outcome that SC subtype patients had an ~20% lower 5-year OS rate than EC subtype patients . For recurrent and/or metastatic NPC, CRT is the standard-of-care treatment. We next examined the efficacy of commonly using CRT combinations on 20 PDOs derived from individual NPCs (Fig. ; Supplementary Data ). In general, CRT exhibited better sensitivities on NPC PDOs than chemotherapeutics alone, especially the most commonly used CRT regimens, RT + cisplatin and RT + fluorouracil (Fig. ), which generally reflected the clinical treatment outcomes , , . When comparing efficacy among NPC subtypes, CRT demonstrated better sensitivities on EC-type organoids than SC-type organoids for the majority of cases, including CRT regimens combined with cisplatin and fluorouracil (Fig. ). These results were consistent with the previously reported clinical outcome that CRT contributed to longer survival time for EC subtype patients than that for SC subtype patients . To identify more drugs that could sensitize PDOs to RT, we further screened 48 anticancer drugs on eight PDOs combined with IR at a dose of approximately IC20-30 (4 Gy) (Supplementary Data ). One of the most striking difference was demonstrated by CRT combinations with EGFRi on organoids derived from EC subtype (Fig. ; Supplementary Fig. ). For 4 tested EC subtype PDO lines, CRT exhibited synergistically better inhibitory effect than EGFRi treatment alone, with the average IC50 value fold increases at 7.09, 5.87, 6.01, and 8.17 in each of these PDOs, respectively (Fig. ; Supplementary Fig. ). Since EGFRi was identified as EC subtype-sensitizing drug, as demonstrated by our earlier data (Fig. ), our CRT screening results further suggested that the EGFRi and RT combination could be a good clinical CRT choice to achieve synergistic killing effect for EC subtype patients. In addition, an improvement on MTi efficacy was also found when combining with RT, but there was no observation on drug synergy (Fig. ). Furthermore, a few other CRT combinations with enhanced sensitivity were also revealed by our screening data, such as combinations with arsenic trioxide (Supplementary Fig. ), olaparib (Supplementary Fig. ), vorinostat (Supplementary Fig. ), sunitinib (Supplementary Fig. ), and zoledronate (Supplementary Fig. ). Thus, our analysis uncovered the subtype-related and patient-specific responses to RT and CRT and potentially provided more choice of drugs for CRT to treat patients suffering from advanced or metastatic stage cancer.
Transcriptome sequencing was performed for 14 PDOs and subsequently assigned for unsupervised clustering by principal component analysis (PCA) to evaluate the overall transcriptional pattern of the samples. The results clearly demonstrated that organoids derived from the same subtypes were well clustered into the corresponding subtypes (Fig. ), indicating that PDOs maintained their subtype differences at the transcriptional level. As revealed by our earlier genomic sequencing analysis, EC exhibited distinct driver mechanisms with SC and MSEC, including microtubule polymerization, G2-M checkpoint regulation, NF-κB signaling, and EMT. We wondered whether these subtype-specific genomic mutational pathways could be converted to functional transcriptional signaling. The unbiased geneset enrichment analysis (GSEA) was performed to investigate top differentiated pathways between EC- and SC-type PDOs (absolute NES value > 1, p -value < 0.05, and FDR value < 0.05) (Fig. ). Significantly differentiated expressed genes were filtered by DESeq2 and limma (log 2 fold change > 1, p- value < 0.05, FDR (padj) value < 0.05) (Fig. ; Supplementary Fig. ; Supplementary Data ), then were presented in heatmap (Fig. ). Microtubule-targeting drugs associated mechanisms, including microtubule signaling, mitotic cell cycle, apoptosis, NF-κB signaling, and androgen response, were among the top enriched pathways in EC-type organoids (Fig. , Supplementary Fig. ) – . Meanwhile, multiple EGFR signaling related pathways and cellular response to radiation were also among top differentiated pathways (Fig. ; Supplementary Figs. a-l, ), which might contribute to the subtype-differentiated responses to EGFR inhibitors and RT. EMT and invasion related pathways, including EMT, extracellular matrix and response to TGF-β, were enriched in SC-type organoids (Fig. , Supplementary Fig. ). Of note, differentially expressed pathway genes were also present between PDO63E and PDO63S (Fig. ; Supplementary Fig. ), which was consistent with earlier results on their drug response and protein expression differences, and further demonstrated the heterogeneity feature of MSEC. In addition, the stemness score was calculated for each NPC subtype PDOs, and the results indicated that EC subtype PDOs maintained high stemness properties, whereas the opposite phenotype was shown in SC subtype PDOs (Supplementary Fig. ). Distinctly to EC and SC, a diverse stemness distribution was observed in MSEC PDOs (Supplementary Fig. ). Moreover, the immune score analysis was also performed to determine whether there were subtype differences among immune cell types, and the results indicated no obvious differences in CD4 + T cells, CD8 + T cells, B cells, or macrophages among the three subtypes (Supplementary Fig. )
During past decade, next-generation sequencing has been extensively applied for precision oncology and has made great progress in matching targeted therapy with predictive gene signatures. However, several essential limitations have also come to surface, including the lack of identified druggable mutations for the majority of patient in current clinical practice and the reliability of drug prediction value, which was controversial . By following conventional practice focusing on targetable mutations identification, we performed genomics sequencing for tumors of individual patients, but unfortunately, well-known drug sensitizing hotspot mutations were seldom found in the majority of NPC tumors as expected , for example, the frequency of EGFR SNVs was only 0.94% (1/106) in our NPC cohort (Supplementary Data ), which kept us from directly identifying corresponding targeted drugs. To better deliver precision medicine to NPC patients, we next proposed to develop a pharmacogenomics-based precision medicine (PBPM) approach that integrated genomics/transcriptomics and drug tests to better provide faithful precision treatment guidance. On the drug test side, we applied PDOs as the proxies to examine drug responsiveness for individual patients and found that many EC subtype PDOs were resistant to MTi, while opposite response of MTi was observed in SC and MSEC subtype derived organoids (Figs. a, ). The results indicated that some subtype-associated gene signatures might contribute to the subtype-specific MTi responses, thus uncovering these gene signatures might be valuable for predicting MTi sensitivity. Next, we incorporated genomic variations of all protein coding genes into our analysis, in addition to only druggable mutations. By grouping samples with MTi sensitivity on PDOs, we revealed that consistent subtype-specific mutations pattern was potentially involved in microtubule-targeting drug responses (Fig. ). Microtubule-targeting drugs were known to disrupt microtubule dynamics, invoke the mitotic checkpoint, lead to cell cycle arrest, and subsequently induce apoptosis – . Thus, activation of key mutations involved in antagonizing these processes may help tumor gain resistance to MTi. Of note, we observed an obvious positive correlation between EC-specific MTi resistance and EC-specific genomic activations of microtubule polymerization, mitosis regulation, NF-κB and DNA repair (Fig. ). A representative personalized case was demonstrated with P13, which help to illustrate the intrinsic correlation mechanism (Fig. ). To further validate these mutations as predictive signatures for MTi response, we applied CRISPR-Cas9 to knockout TPPP (tubulin polymerization-promoting protein), MAP2 (microtubule-associated protein 2), and SKP2 (S-phase kinase-associated protein 2) in PDO9 and PDO4, respectively (Supplementary Fig. ; Supplementary Data ). TPPP , MAP2 , and SKP2 knockout organoids showed sensitive responses to docetaxel treatment when comparing to parental wild-type organoids (Fig. ; Supplementary Fig. ), indicating mutations involving in microtubule polymerization and mitosis regulation could serve as potential predictive signatures for MTi response in NPC. Another personalized case was demonstrated with P86, which acquired EGFRi resistance. As shown in Supplementary Fig. , copy number gains of MET and KRAS were observed in P86 tumors. Activation of oncogenic MET and KRAS potentially induced EGFRi resistance through bypassing conventional EGFR signaling. To further confirm their contributive effect on EGFRi resistance, we applied crizotinib and vorinostat to perform combinational inhibition together with gefitinib in PDO86 (Supplementary Fig. ). Crizotinib is a specific inhibitor of c-MET, and HDAC inhibitor vorinostat was previously reported to overcome EGFRi resistance by blocking KRAS – . The result showed that after dual-blocking of c-MET and KRAS, EGFRi resistance in PDO86 was significantly overcome (Supplementary Fig. ), indicating that copy number gains of MET and KRAS might contributed to EGFRi resistance in PDO86. Based on our previous practice on genomics driven PBPM strategy, although as many as all protein coding mutations were involved into analysis, only a portion of patients could be identified with the discriminative genomic signatures associated to drug response and got the benefit. Integrating transcriptomics with drug response seems to be another good option to develop PBPM. As shown in Fig. , microtubule signaling, mitotic cell cycle, apoptosis, NF-κB signaling, and androgen response, were among top subtype-differentiated pathways. They were reported to contribute to MTi response mechanisms – . We assembled gene cohorts containing signature genes involved in these pathways with differential expression patterns between EC and SC/MSEC subtypes, and these genesets was further applied to analyze the correlation between subtypes and MTi response (Supplementary Data ). EC subtype PDOs showed a positive correlation with the docetaxel resistance, while SC and MSEC subtype PDOs exhibited a positive correlation with the docetaxel sensitivity (Fig. ). We next examined the impact of individual subtype-differentiated pathways on discriminating the MTi responses. The results demonstrated that individual pathways could well distinguish docetaxel-sensitive and docetaxel-resistant PDOs (Fig. ). This data led us to believe that these five subtype-differentiating pathway signatures were the candidate contributive factors for the NPC subtype-specific MTi response. To further validate our hypotheses as well as test the drug prediction value of these pathway signatures, we download public data of both transcriptome sequencing and MTi treatment results from the Genomics of Drug Sensitivity in Cancer (GDSC) database . After sorting MTi-sensitive and MTi-resistant cell lines with the bottom 10% and top 10% IC50 values of docetaxel, paclitaxel, vinorelbine, and vincristine, we applied our pathway signatures to GDSC data to perform receiver operating characteristic (ROC) analysis. As shown in Fig. , the individual pathway signature obtained area under curve (AUC) values ranging from 0.83 to 0.914 for predicting docetaxel sensitivity, indicating significant contributive impacts of these pathway signatures on MTi sensitivity, while the predictive AUC value of the random geneset was 0.5. Of note, when combining all 5 pathway signatures together, we achieved the best drug predictive value with AUC values of 0.918 (docetaxel) (Fig. ), 0.949 (paclitaxel) (Supplementary Fig. ), 0.906 (vincristine) (Supplementary Fig. ), and 0.915 (vinorelbine) (Supplementary Fig. ). To further confirm the efficiency of prediction power in clinical practice, we applied datasets of human cancer patients treated with paclitaxel and docetaxel to evaluate the predictive value. Both GSE22513 and GSE6434 human dataset achieved good prediction accuracy for MTi response with AUC values of 0.914 and 0.841 using the 105 combined gene signatures (Fig. ). Similar result was also demonstrated with patient-derived xenografts (PDXs) (AUC = 0.872) (Supplementary Fig. ). Through our proposed PBPM approach by integrating genomics/transcriptomics and PDO-based drug tests, the multidimensionally confirmed results could serve as promising indications to guide individual treatment, even to those subjects without any identified druggable mutations from genomic analysis. More importantly, guided by the integrated PBPM analysis results, we further demonstrated the histological subtype-related drug responsiveness of MTi on PDOs, which could significantly facilitate precision oncology for NPC to be subtype-guided therapy in the future.
In the present study, we have conducted a comprehensive integrative analysis of genomic and transcriptomic data, and drug sensitivity on PDOs to study the biological features of different subtypes of NPC, their responses to therapeutic drugs, and different combinations of CRT. Our data uncovered several notable findings, including (1) EC, MSEC, SC, and SCC subtypes of NPC have some distinct characteristics in terms of genomics, gene expression, organoid growth pattern, and drug responses, which have not been recognized before; (2) under our optimized culture conditions, PDOs of NPC were established with a high successful rate (93%); (3) anticancer drug screening using PDOs identifies remarkable differential responses of different subtypes to the treatment; and (4) our integrative analysis revealed correlations between subtype-specific molecular characterizations and subtype-specific drug and CRT responses of NPC, which demonstrates a promising pipeline for subtype-guided precision medicine. Heterogeneity is observed in all cancer types, including NPC, which often leads to treatment failure and tumor recurrence whenever fractions of tumor cells survived from initial therapy , . Recurrent NPC usually has a poor prognosis with a median OS of ~20 months . Using precision medicine in the initial treatment would significantly improve prognosis and implementing subtype-guided therapeutic regimens is a good attempt. By analyzing genomic mutations and transcriptional expressions of three main subtypes of NPC, including EC, MSEC, and SC, which account for approximately 97% of NPC, we identified aberrant driver pathways of each subtype: EC is driven by microtubule polymerization activations, defective mitotic spindle checkpoint regulation and DNA repair; SC is associated with EMT/invasion promoting signaling; and MSEC is a more heterogeneous subtype with molecular features of both EC and SC. Of note, both EC-type epithelial and SC-type sarcomatoid tumor cells, such as PDO63E and PDO63S in MSEC subtype, although displaying distinct transcriptional expression patterns and diverse responses to certain type of cancer drugs, they share some key common genomic features. This suggests that the heterogeneity of two distinct populations within MSEC might originate from the same cell during tumor evolution. However, different subtypes of NPC also share some common features, such as defective G1-S checkpoint surveillance, activated NF-κB signaling, aberrant RTKs and chromatin remodeling. Of note, one of the most notable common features is that they all lose MST1R at an extreme high frequency (55.6%), indicating that MST1R may act as a tumor suppressor in NPC, contrary to the carcinogenic effects of MST1R in other types of cancer – . It has been reported that MST1R is an NPC susceptibility gene associated with MST1R/14-3-3 interaction networks in the response to EBV infection, rather than functions as a tyrosine kinase that promotes proliferation and migration . Loss of MST1R function may be a cancer susceptible marker for NPC. Previous studies revealed significant prognostic variation between EC and SC subtypes. . Generally, the SC subtype has an ~20% lower OS rate than the EC subtype, and the OS rate of MSEC is between EC and SC . Other independent studies also demonstrated that NPC with a high proportion of vimentin-positive spindle tumor cells showed poor prognosis, including survival, clinical stage, lymphatic invasion, and recurrence, rather than nonspindle subtypes , , . However, the underlying molecular basis contributing to this subtype difference remains a gap in the field until our present study. Early-stage NPC is sensitive to RT, and ~90% of patients at stage I benefit from RT. However, 15–58% of NPC patients experience recurrence, and most of them develop metastases and therefore must undergo retreatment , . The outcome for patients with recurrent or metastatic NPC is very poor, with a median OS of only approximately 20 months . Although CRT and chemotherapy usually provide better treatment outcomes for advanced NPC patients than RT alone , , and have been widely applied as standard care for patients with advanced stage NPC, it remains a huge challenge to improve prognosis by deciding a suitable regimen for the corresponding patient. By classifying NPC into subtypes and identifying effective drugs for each subtype, this strategy offers a good option to facilitate precision medicine. In this study, by utilizing PDOs, we identified several drugs that were efficacious in specific subtypes, including MTi for SC and EGFRi for EC. Among these drugs, docetaxel and paclitaxel are FDA-approved microtubule-targeting drugs for NPC and have been widely applied in clinical practice. EGFR inhibition has also emerged as a new effective strategy against advanced NPC, with options including approved anti-EGFR monoclonal antibodies, cetuximab and nimotuzumab, and chemical EGFR inhibitors, gefitinib and neratinib, in phase III clinical trials for NPC – . Importantly, our study identified a dozen of CRT combinations with good synergistic effects against NPC PDOs with individual- or subtype-specific response patterns. One of the most effective CRTs was the combination of RT with EGFRi, which exhibited a synergistic and potent killing effect on EC subtype PDOs. Precision medicine is a promising strategy for treating the right patient with right drugs, which is urgently needed for cancer patients. It was estimated that only a 25% response rate, on the average, is achieved with each round of conventional cancer chemotherapy across cancer types , and the combination with druggable mutation detection may significantly increase the therapy response rate to ~50% . Nevertheless, for tumors without known druggable targets or cancer types with less genomic characterization, such as NPC, it remains difficult to pursue a suitable regimen for individuals. Organotypic culture opened a door to fill this need, as this system faithfully recapitulates the original tumor and works as a proxy for testing drugs for individual patients – . Researchers have demonstrated a series of perfect examples for personalized medicine applying organotypic culture in various cancer types , – . We are one of the pioneering groups to establish this platform for treating NPC. Of note, in contrast to the majority of previous reports studying precision medicine approaches either from the genomics/transcriptomics perspective or from the PDX/PDO-based drug screening aspect, we integrated advantages from both sides and proposed the PBPM approach. Genomics/transcriptomics analysis and drug response assay on PDOs mutually validated each other to provide higher confidence in the therapeutic suggestions for physicians and patients than conventional methods used previously. We proposed that the PBPM approach could be a feasible strategy for precision medicine in NPC. Nevertheless, some limitations on the proposed PBPM approach still requires necessary attentions for the field at current stage. First, more cases of clinical validation are essentially needed to evaluate the efficacy and safety of PBPM. Second, there is a potential risk that the heterogenous tumor linages might gradually evolve or lose during a long-term organotypic culture, subsequently affect the accuracy of PBPM. So, to screen for effective drugs using early-passages PDOs is highly recommended. Third, for some tumors of mixed subtypes, the possibility of selective killing effect of PBPM-identified drugs might occur. Thus, further studies on developing the approach to find drug combinations against various tumor heterogenous clones are necessary. In summary, we analyzed EC, MSEC, SC, and SCC subtypes and identified subtype-specific driver pathways for NPC. We have established PDOs with high efficiency and uncovered subtype-specific drug and CRT responses by screening a library of anticancer drugs. We elucidated that drug responses are well correlated with corresponding genomic mutations, transcriptional expression and protein changes (Supplementary Fig. ). Thus, our study provides an example of applying integrative pharmacogenomics to establish a personalized treatment strategy for NPC subtype-guided therapies.
Sample collection Fresh tumor biopsies and paired blood samples pathologically diagnosed with NPC were collected from the Kiang Wu Hospital, Macau from August 2016 to December 2019. Formalin fixed, paraffin-embedded (FFPE) NPC tumors and adjacent tissue sections samples were collected at the Affiliated Hospital, Southwest Medical University, Luzhou, Sichuan. Prior patient written consents were obtained from donors with informing the use for genomics sequencing, organoid culture, drug test, publication, and associated scientific studies. All collected tumors were further confirmed, classified, and stage assessed through TNM staging. The protocol of this study was assessed and approved by the ethics committees of University of Macau, Kiang Wu Hospital and the Affiliated Hospital of Southwest Medical University. Detailed clinicopathological characteristics of all NPC patients included in this study are summarized in Supplementary Table and Supplementary Data , the prior consents to publish information of gender, age, ethnicity and associated clinical characteristics were obtained from subjects. Tissue dissociation Upon arrival, tumor tissues were firstly minced into 1–3 mm 3 pieces. Two random pieces were picked up for DNA isolation and formalin fixing. The remaining portions were digested with collagenase buffer (Supplementary Data ) at 37 °C for about 1 h with gentle shaking. The material was further digested with dispase II (5 mg/ml)/deoxyribonuclease (0.1 mg/ml) solution for 5 min at 37 °C and later dissociated with 0.25% trypsin for 1–2 min at 37 °C. After treating with RBC lysis buffer (eBiosciences) for 3 min, the remaining tumor cells were collected for organoid culture and cryopreservation. Organoid culture Dissociated tumor cells were resuspended in Matrigel solution, then seeded in prewarmed 24-well culture plates at 30 μL per drop. Once cell-Matrigel drops were solidified at 37 °C, 400 μL/well organoid culture medium (Supplementary Data ) was added in to initiate continuous culture. Medium were refreshed every 2–3 days and passage developed every 5–10 days depending on organoid density and size. Upon passaging organoids, organoid-Matrigel drops were firstly mechanically disrupted with 0.25% trypsin by pipetting, later were transferred to 37 °C allowing further dissociation to smaller cell aggregates. During continuous culture, tumor organoids originated from different subtypes eventually developed to two distinct phenotypes: EC-type and SC-type. For EC-type organoids, dissociated organoids were directly resuspended in Matrigel solution and seeded for amplifying culture. For SC-type organoid passage, passaging cells were firstly cultured in low-attachment plates to allow aggregation to dense spheres, then formed spheres were setup culture as same as EC-type organoids. Histology and immunostaining Tumor tissues and organoids were fixed in 10% neutral buffered formalin followed by gradient dehydration, wax immersion, paraffin embedding, and sectioning. For processing organoids, prestaining of eosin was performed during dehydration in order to locate organoids in paraffin blocks and sections. Haematoxylin–eosin (HE) staining and immunostaining was performed using standard protocols on 5 μm sections , . The antibodies used were listed as following: AE1/3 (1:50, Abcam, ab27988), Vimentin (1:100, CST, 5741 S; 1:100, Santa Cruz, sc-6260), CK5/6 (1:50, Abcam, ab17133), p63 (1:50, Abcam, ab124762), MST1R (1:100, ATLAS, HPA008180), LMP1 (1:100, Abcam, ab78113), Ki67 (1:400, CST, 9449 S), and CD3e (1:100, Dako, A0452). Tumor subtyping NPC are histologically classified into the following four subtypes based on morphologic characteristics: epithelial carcinoma (EC), sarcomatoid carcinoma (SC), mixed sarcomatoid-epithelial carcinoma (MSEC), and squamous cell carcinoma (SCC) . Most of tumor regions in EC subtype are identified with round epithelial cells and vesicular nuclei phenotype appearance. SC subtype has a large proportion of spindle-shaped and fusiform cells. MSEC subtype encompasses with both round epithelial tumor cells and spindle sarcomatoid tumor cells, with scattered or nest infiltration of spindle cells in round epithelial cell region. SCC is characterized by keratinizing squamous tumor cells. DNA and RNA extraction Of 106 samples assigned for WES, DNA of 43 samples were isolated from bulk fresh primary tumors, 4 from bulk FFPE samples, 59 from microdissected FFPE samples (Supplementary Data ). The tumor purities for bulk samples were assessed by H&E stanning and calculated by Sequenza and Absolute methods (Supplementary Data , ) , . Genomic DNA and paired blood were extracted using DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s instructions. LASER-capture microdissection of FFPE samples was conducted under Leica LMD 7000 Laser MicroDissection system so that the clusters of tumor cells and adjacent normal tissues could be separately dissected for genomic DNA preparation by QIAamp DNA Micro kit (Qiagen). RNA from organoids was extracted using RNeasy Micro kit (Qiagen). Whole exome and transcriptome libraries preparation and sequencing DNA library was prepared for Illumina platform using NEBNext Ultra DNA library Prep kit for Illumina (NEB) and subjected to whole-exome enrichment using Nextera Rapid Capture Exome kit (Illumina) following the standard protocol. RNA libraries were prepared using NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB) following the manufacturer’s protocol. Barcoded DNA and RNA libraries were mixed to pool libraries, then run sequencing performed by Novogene Co. Ltd. Whole-exome sequencing (WES) data analysis Whole-exome sequencing was performed on tumor, paired normal and paired organoid samples with average sequencing coverage ~100× (Supplementary Data ), including 43 fresh tumor, 63 FFPE tumor and 15 organoid samples. The FASTQ raw files are available at Sequence Read Archive (SRA) database of NCBI under accession number PRJNA716262 ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA716262/ ) and the National Omics Data Encyclopedia (NODE) database under accession number OEP001733 ( https://www.biosino.org/node/project/detail/OEP001733/ ). The data deposition was complied with the Regulations on Management of Human Genetic Resources in China. The pipeline used for data processing was summarized in Supplementary Fig. . In brief, sequence reads were aligned to human genome build 38 (Hg38) using Burrows-Wheeler Alignment tool (BWA-version 0.7.12) (20080505) with maximal exact matches (v0.7.17) followed by reducing duplicates with Sambamba (v0.6.8) , realignment of indels and base recalibration with Genome Analysis ToolKit (v4.1.0.0) according to the best practice guidelines. Somatic SNVs and INDELs were called by three independent programs with default parameters, including MuTect2 (v4.1.0.0), Strelka2 (v2.9.10) , and LANCET (v1.1.0) (Supplementary Fig. ). Paired blood control or adjacent control samples were applied as the reference to call somatic mutations of each tumor independently. The results of each caller were stored in VCF format and further filtered for PASS variants. The vcf2maf tools ( https://github.com/mskcc/vcf2maf ) was used to convert VCF into MAF with performing annotation by ENSEMBL Variant Effect Predictor (v100.0) according to reference release 93. Only mutations detected by at least two callers were kept as true positive ones and assigned for further analysis. Somatic SNVs and INDELs were further filtered by ngs-filters ( https://github.com/mskcc/ngs-filters ) to determine high-confident variants. The following criteria were applied on candidate mutations identification: (i) minor allele frequency in ExAC less than 0.0004. (ii) tumor sample total depth more than 20, and support reads for the reference less than 1 while for alteration more than 3. (iii) variants located in low-mappability regions were filtered. (iv) mutations with allele frequency more than 5% were retained. SNVs and INDELs called by three tools were merged into a single nonredundant MAF file which contained 2662 somatic mutations including 2148 genes for downstream analysis. Mutation signature analysis was performed by MutationalPatterns (v2.0.0) R package and further compared with COSMIC mutational signatures version 2 using deconstructSigs (v1.8.0) R package applying ‘exome2genome’ normalized method. Copy number analysis was performed in both Sequenza (v3.0.0) , and CNVkit (v0.9.0) with default parameter. Further chromosome arm-level and gene-level variations were detected by GISTIC 2.0 . Consensus CNVs detected by both CNVkit and Sequenza were considered as tune positive ones. Tumor purity was assessed using Sequenza and Absolute method , . Transcriptome sequencing and data analysis RNA sequencing was performed for 14 PDOs and the quality check was carried out using MultiQC (v1.5) . The FASTQ raw files are available at SRA database of NCBI under accession number PRJNA682500 ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA682500/ ). The data deposition was complied with the Regulations on Management of Human Genetic Resources in China. RNA reads were aligned to human reference genome GRCh38 using HISAT2 (v2.1.0) followed by counting RNA reads by featureCounts , one of programs in Subread package (v1.6.4). Genes with zero read counts for all samples were removed. Then differentially expressed genes between EC- and SC-type PDOs were identified using two tools, DESeq2 (v1.26.0) and limma (v3.42.2) , respectively. Significantly differential expressed genes were defined based on overlapped genes between two tools. The cutoff criteria for both tools were the absolute value of log 2 fold change > 1, p- value < 0.05, FDR (padj) value < 0.05. The multiple hypothesis testing was performed using Benjamin–Hochberg correction implemented in the DESeq2 and limma package. DESeq2 found 3853 significantly differential expressed genes while limma detected 2791. There were 2506 common genes between two callers. Pathway enrichment analysis and geneset enrichment analysis were performed using R package clusterProfiler (v3.14.3), results were filtered by absolute NES value > 1, p -value < 0.05, and FDR value < 0.05. The multiple hypothesis testing was performed using Benjamin–Hochberg correction implemented in clusterProfiler package. Evaluation on drug prediction accuracy with signature geneset Public data source of transcriptional expression and drug treatment responses on cancer cell lines, PDXs and cancer patients were download from Genomics of Drug Sensitivity in Cancer (GDSC) database ( https://www.cancerrxgene.org/ ) and Gene Expression Omnibus (GEO) database ( https://www.ncbi.nlm.nih.gov/geo/ ). Cancer cell line data treated with docetaxel (880 lines), paclitaxel (758 lines), vinorelbine (751 lines), and vincristine (727 lines) were download, and samples with IC50 values of bottom 10% and top 10% were defined as drug sensitive and resistant ones. PDX data treated with docetaxel were download from GSE110153 dataset. Data of human breast cancer patients treated with paclitaxel and docetaxel were download from GSE22513 and GSE6434 dataset, respectively. The receiver operating characteristic (ROC) analysis was used for evaluating drug prediction accuracy of in-house signature geneset on downloaded public data. ROC analysis was performed by online tool MetaboAnalyst (v4.0) ( https://www.metaboanalyst.ca ). Validation of gene expression by real-time PCR Important differentially expressed genes among NPC subtypes including BIRC3, AR, FN1 , and MMP2 were further confirmed using real-time PCR. The details of primers used were listed in Supplementary Table . Drug screening After preliminarily testing 146 chemical drugs on PDOs, we organized a drug library containing 48 therapeutic drugs with good efficacy on NPC for large-scale screening (Supplementary Data ). The drug library was assembled with 40 cancer-approved drugs in clinical use, four repurposed noncancer drugs and four nonapproved compounds, but now in phase II/III clinical trials. Compounds were stored in DMSO at the concentration of 10 mM, then diluted in sterile PBS and arrayed in 384-well plates at 6-point serial dilutions (0.82–200 μM, as 10× working dilution). Organoids at early passages were assigned for drug screening. Initially, organoids were enzymatically dissociated into single cells, then diluted with the medium for drug screening (organoid culture medium minus Y-27632, SB202190, and A83-01; supplemented with 2.5% Matrigel). Cells were seeded in type-I collagen gel precoated 384-well plate at a density of about 1200-1500 cells per well. On the other day, drugs were added into each well with 6-point dilutions and cells were incubated with drugs for 4 days. To examine organoid viability, quantifying ATP content in each well as the proxy for metabolically viable cells using the CellTiter-Glo 2.0 assay (Promega) or CellTiter-Lumi Plus assay (Beyotime) was applied. Luminescence readout from drug treated wells were normalized against control wells and expressed as percentage cell viability, IC50 values were calculated using GraphPad Prism software. Chemoradiotherapy (CRT) screening and synergistic effect determination Organoid viability was firstly examined with ionizing radiation (IR) treatment alone at dose (Gy) of 0, 2, 4, 6, 8, 10 by following same protocol with previous drug screening. For CRT screening, organoids were treated by either chemical drug alone or chemical drug combining with 4 Gy IR, and IR treatment was prior to adding chemical drug in treated wells. When evaluating the combinational effect of each drug with IR, the CRT combination with fold change of IC50 over 3 was recognized as the candidate having good combinational effect. Fold change was calculated by dividing IC50 value of chemical drug treatment alone by IC50 value of combinational treatment. If IC50 value (μM) is >20 or <0.08, the maximum treatment dose (μM) 20 or minimum treatment dose (μM) 0.08 would be served as proxy respectively for calculating fold change. To demonstrate whether CRT combination has synergistic effect or not, we compared three dose response curves, including curves of chemical drug alone treatment, combinational treatment and theoretical additive effect. The predictive additive killing effect is calculated as E total = E 1 + E 2 − E 1 × E 2 (where E 1 is inhibitory effect of IR at dose of 4 Gy and E 2 is inhibitory effect of drug X at defined dose). This classifies a synergistic CRT combination if dose response curve of combinational treatment is lower than additive effect curve. Western blot analysis Organoids were passaged and cultured in 6-well plate for one day before adding drugs (5 μM gefitinib or 50 nM docetaxel or vehicle). For gefitinib inhibitor experiment, organoids were collected for total protein extraction at 12 and 24 h after treatment, and for docetaxel, at 24 h and 48 h. The protein bands were visualized by using Immobilon Western Chemiluminescent HRP Substrate (Millipore) under ChemiDoc Touch Imaging System (Bio-Rad). The antibodies used were listed as following: Phospho-AKT (1:1000, CST, 4060 S), AKT (1:1000, CST, 9272 S), Phospho-ERK (1:1000, CST, 4370 S), ERK (1:1000, CST, 4695 S), Phospho-STAT3 (1:1000, CST, 9145 S), STAT3 (1:1000, CST, 9139 S), Cyclin A (1:200, Santa Cruz, sc-751), Cyclin B (1:200, Santa Cruz, sc-752), Phospho-Bcl-2 (1:1000, CST, 2827 S), Bcl-2 (1:1000, ProteinTech, 12789-1-ap), Bax (1:1000, ProteinTech, 50599-2-lg), Cleaved Caspase-3 (1:1000, CST, 9661 S), MST1R (1:1000, ATLAS, HPA008180), and β-Actin (1:1000, Sigma, A5316). Generation of Cas9-sgRNA plasmid and knockout (KO) PDO lines Sequences of sgRNA targeting human TPPP , MAP2 , and SKP2 were obtained Human CRISPR Knockout Pooled Library (GeCKO v2) (Supplementary Data ), and were cloned into lentiCRISPR v2 vector (Addgene plasmid # 52961). To generate the KO PDO lines, lentivirus system was employed to transduce organoids. Briefly, lentiviruses were produced by co-transfection of a lentivirus vector plasmid, pCMV delta R8.2, and pCMV-VSV-G into HEK 293 T cells. Fresh culture medium was changed 12 h later and culture supernatant was collected at 72 h post-transfection and concentrated by ultracentrifugation. For organoid transfection, the concentrated lentiviruses were firstly mixed with Matrigel at 1:1 volume ratio, then organoids were embedded into the Matrigel containing lentiviruses and cultured with regular organoid culture medium. To select for mutated cells, the culture medium was supplemented with 2 μg/ml puromycin at 72 h post-transfection and continued for 1 week. The remaining organoids after selection were assigned for growth curve and drug treatment evaluation. Statistics and reproductivity All representative experiments were performed in triplicates or duplicates independently. The results were reported as the mean ± SEM or mean ± SD. Statistical significance was calculated using the two-sided t -test, unless otherwise indicated. The p values were considered statistically significant if the p -value ≤ 0.05. Reporting summary Further information on research design is available in the linked to this article.
Fresh tumor biopsies and paired blood samples pathologically diagnosed with NPC were collected from the Kiang Wu Hospital, Macau from August 2016 to December 2019. Formalin fixed, paraffin-embedded (FFPE) NPC tumors and adjacent tissue sections samples were collected at the Affiliated Hospital, Southwest Medical University, Luzhou, Sichuan. Prior patient written consents were obtained from donors with informing the use for genomics sequencing, organoid culture, drug test, publication, and associated scientific studies. All collected tumors were further confirmed, classified, and stage assessed through TNM staging. The protocol of this study was assessed and approved by the ethics committees of University of Macau, Kiang Wu Hospital and the Affiliated Hospital of Southwest Medical University. Detailed clinicopathological characteristics of all NPC patients included in this study are summarized in Supplementary Table and Supplementary Data , the prior consents to publish information of gender, age, ethnicity and associated clinical characteristics were obtained from subjects.
Upon arrival, tumor tissues were firstly minced into 1–3 mm 3 pieces. Two random pieces were picked up for DNA isolation and formalin fixing. The remaining portions were digested with collagenase buffer (Supplementary Data ) at 37 °C for about 1 h with gentle shaking. The material was further digested with dispase II (5 mg/ml)/deoxyribonuclease (0.1 mg/ml) solution for 5 min at 37 °C and later dissociated with 0.25% trypsin for 1–2 min at 37 °C. After treating with RBC lysis buffer (eBiosciences) for 3 min, the remaining tumor cells were collected for organoid culture and cryopreservation.
Dissociated tumor cells were resuspended in Matrigel solution, then seeded in prewarmed 24-well culture plates at 30 μL per drop. Once cell-Matrigel drops were solidified at 37 °C, 400 μL/well organoid culture medium (Supplementary Data ) was added in to initiate continuous culture. Medium were refreshed every 2–3 days and passage developed every 5–10 days depending on organoid density and size. Upon passaging organoids, organoid-Matrigel drops were firstly mechanically disrupted with 0.25% trypsin by pipetting, later were transferred to 37 °C allowing further dissociation to smaller cell aggregates. During continuous culture, tumor organoids originated from different subtypes eventually developed to two distinct phenotypes: EC-type and SC-type. For EC-type organoids, dissociated organoids were directly resuspended in Matrigel solution and seeded for amplifying culture. For SC-type organoid passage, passaging cells were firstly cultured in low-attachment plates to allow aggregation to dense spheres, then formed spheres were setup culture as same as EC-type organoids.
Tumor tissues and organoids were fixed in 10% neutral buffered formalin followed by gradient dehydration, wax immersion, paraffin embedding, and sectioning. For processing organoids, prestaining of eosin was performed during dehydration in order to locate organoids in paraffin blocks and sections. Haematoxylin–eosin (HE) staining and immunostaining was performed using standard protocols on 5 μm sections , . The antibodies used were listed as following: AE1/3 (1:50, Abcam, ab27988), Vimentin (1:100, CST, 5741 S; 1:100, Santa Cruz, sc-6260), CK5/6 (1:50, Abcam, ab17133), p63 (1:50, Abcam, ab124762), MST1R (1:100, ATLAS, HPA008180), LMP1 (1:100, Abcam, ab78113), Ki67 (1:400, CST, 9449 S), and CD3e (1:100, Dako, A0452).
NPC are histologically classified into the following four subtypes based on morphologic characteristics: epithelial carcinoma (EC), sarcomatoid carcinoma (SC), mixed sarcomatoid-epithelial carcinoma (MSEC), and squamous cell carcinoma (SCC) . Most of tumor regions in EC subtype are identified with round epithelial cells and vesicular nuclei phenotype appearance. SC subtype has a large proportion of spindle-shaped and fusiform cells. MSEC subtype encompasses with both round epithelial tumor cells and spindle sarcomatoid tumor cells, with scattered or nest infiltration of spindle cells in round epithelial cell region. SCC is characterized by keratinizing squamous tumor cells.
Of 106 samples assigned for WES, DNA of 43 samples were isolated from bulk fresh primary tumors, 4 from bulk FFPE samples, 59 from microdissected FFPE samples (Supplementary Data ). The tumor purities for bulk samples were assessed by H&E stanning and calculated by Sequenza and Absolute methods (Supplementary Data , ) , . Genomic DNA and paired blood were extracted using DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s instructions. LASER-capture microdissection of FFPE samples was conducted under Leica LMD 7000 Laser MicroDissection system so that the clusters of tumor cells and adjacent normal tissues could be separately dissected for genomic DNA preparation by QIAamp DNA Micro kit (Qiagen). RNA from organoids was extracted using RNeasy Micro kit (Qiagen).
DNA library was prepared for Illumina platform using NEBNext Ultra DNA library Prep kit for Illumina (NEB) and subjected to whole-exome enrichment using Nextera Rapid Capture Exome kit (Illumina) following the standard protocol. RNA libraries were prepared using NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB) following the manufacturer’s protocol. Barcoded DNA and RNA libraries were mixed to pool libraries, then run sequencing performed by Novogene Co. Ltd.
Whole-exome sequencing was performed on tumor, paired normal and paired organoid samples with average sequencing coverage ~100× (Supplementary Data ), including 43 fresh tumor, 63 FFPE tumor and 15 organoid samples. The FASTQ raw files are available at Sequence Read Archive (SRA) database of NCBI under accession number PRJNA716262 ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA716262/ ) and the National Omics Data Encyclopedia (NODE) database under accession number OEP001733 ( https://www.biosino.org/node/project/detail/OEP001733/ ). The data deposition was complied with the Regulations on Management of Human Genetic Resources in China. The pipeline used for data processing was summarized in Supplementary Fig. . In brief, sequence reads were aligned to human genome build 38 (Hg38) using Burrows-Wheeler Alignment tool (BWA-version 0.7.12) (20080505) with maximal exact matches (v0.7.17) followed by reducing duplicates with Sambamba (v0.6.8) , realignment of indels and base recalibration with Genome Analysis ToolKit (v4.1.0.0) according to the best practice guidelines. Somatic SNVs and INDELs were called by three independent programs with default parameters, including MuTect2 (v4.1.0.0), Strelka2 (v2.9.10) , and LANCET (v1.1.0) (Supplementary Fig. ). Paired blood control or adjacent control samples were applied as the reference to call somatic mutations of each tumor independently. The results of each caller were stored in VCF format and further filtered for PASS variants. The vcf2maf tools ( https://github.com/mskcc/vcf2maf ) was used to convert VCF into MAF with performing annotation by ENSEMBL Variant Effect Predictor (v100.0) according to reference release 93. Only mutations detected by at least two callers were kept as true positive ones and assigned for further analysis. Somatic SNVs and INDELs were further filtered by ngs-filters ( https://github.com/mskcc/ngs-filters ) to determine high-confident variants. The following criteria were applied on candidate mutations identification: (i) minor allele frequency in ExAC less than 0.0004. (ii) tumor sample total depth more than 20, and support reads for the reference less than 1 while for alteration more than 3. (iii) variants located in low-mappability regions were filtered. (iv) mutations with allele frequency more than 5% were retained. SNVs and INDELs called by three tools were merged into a single nonredundant MAF file which contained 2662 somatic mutations including 2148 genes for downstream analysis. Mutation signature analysis was performed by MutationalPatterns (v2.0.0) R package and further compared with COSMIC mutational signatures version 2 using deconstructSigs (v1.8.0) R package applying ‘exome2genome’ normalized method. Copy number analysis was performed in both Sequenza (v3.0.0) , and CNVkit (v0.9.0) with default parameter. Further chromosome arm-level and gene-level variations were detected by GISTIC 2.0 . Consensus CNVs detected by both CNVkit and Sequenza were considered as tune positive ones. Tumor purity was assessed using Sequenza and Absolute method , .
RNA sequencing was performed for 14 PDOs and the quality check was carried out using MultiQC (v1.5) . The FASTQ raw files are available at SRA database of NCBI under accession number PRJNA682500 ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA682500/ ). The data deposition was complied with the Regulations on Management of Human Genetic Resources in China. RNA reads were aligned to human reference genome GRCh38 using HISAT2 (v2.1.0) followed by counting RNA reads by featureCounts , one of programs in Subread package (v1.6.4). Genes with zero read counts for all samples were removed. Then differentially expressed genes between EC- and SC-type PDOs were identified using two tools, DESeq2 (v1.26.0) and limma (v3.42.2) , respectively. Significantly differential expressed genes were defined based on overlapped genes between two tools. The cutoff criteria for both tools were the absolute value of log 2 fold change > 1, p- value < 0.05, FDR (padj) value < 0.05. The multiple hypothesis testing was performed using Benjamin–Hochberg correction implemented in the DESeq2 and limma package. DESeq2 found 3853 significantly differential expressed genes while limma detected 2791. There were 2506 common genes between two callers. Pathway enrichment analysis and geneset enrichment analysis were performed using R package clusterProfiler (v3.14.3), results were filtered by absolute NES value > 1, p -value < 0.05, and FDR value < 0.05. The multiple hypothesis testing was performed using Benjamin–Hochberg correction implemented in clusterProfiler package.
Public data source of transcriptional expression and drug treatment responses on cancer cell lines, PDXs and cancer patients were download from Genomics of Drug Sensitivity in Cancer (GDSC) database ( https://www.cancerrxgene.org/ ) and Gene Expression Omnibus (GEO) database ( https://www.ncbi.nlm.nih.gov/geo/ ). Cancer cell line data treated with docetaxel (880 lines), paclitaxel (758 lines), vinorelbine (751 lines), and vincristine (727 lines) were download, and samples with IC50 values of bottom 10% and top 10% were defined as drug sensitive and resistant ones. PDX data treated with docetaxel were download from GSE110153 dataset. Data of human breast cancer patients treated with paclitaxel and docetaxel were download from GSE22513 and GSE6434 dataset, respectively. The receiver operating characteristic (ROC) analysis was used for evaluating drug prediction accuracy of in-house signature geneset on downloaded public data. ROC analysis was performed by online tool MetaboAnalyst (v4.0) ( https://www.metaboanalyst.ca ).
Important differentially expressed genes among NPC subtypes including BIRC3, AR, FN1 , and MMP2 were further confirmed using real-time PCR. The details of primers used were listed in Supplementary Table .
After preliminarily testing 146 chemical drugs on PDOs, we organized a drug library containing 48 therapeutic drugs with good efficacy on NPC for large-scale screening (Supplementary Data ). The drug library was assembled with 40 cancer-approved drugs in clinical use, four repurposed noncancer drugs and four nonapproved compounds, but now in phase II/III clinical trials. Compounds were stored in DMSO at the concentration of 10 mM, then diluted in sterile PBS and arrayed in 384-well plates at 6-point serial dilutions (0.82–200 μM, as 10× working dilution). Organoids at early passages were assigned for drug screening. Initially, organoids were enzymatically dissociated into single cells, then diluted with the medium for drug screening (organoid culture medium minus Y-27632, SB202190, and A83-01; supplemented with 2.5% Matrigel). Cells were seeded in type-I collagen gel precoated 384-well plate at a density of about 1200-1500 cells per well. On the other day, drugs were added into each well with 6-point dilutions and cells were incubated with drugs for 4 days. To examine organoid viability, quantifying ATP content in each well as the proxy for metabolically viable cells using the CellTiter-Glo 2.0 assay (Promega) or CellTiter-Lumi Plus assay (Beyotime) was applied. Luminescence readout from drug treated wells were normalized against control wells and expressed as percentage cell viability, IC50 values were calculated using GraphPad Prism software.
Organoid viability was firstly examined with ionizing radiation (IR) treatment alone at dose (Gy) of 0, 2, 4, 6, 8, 10 by following same protocol with previous drug screening. For CRT screening, organoids were treated by either chemical drug alone or chemical drug combining with 4 Gy IR, and IR treatment was prior to adding chemical drug in treated wells. When evaluating the combinational effect of each drug with IR, the CRT combination with fold change of IC50 over 3 was recognized as the candidate having good combinational effect. Fold change was calculated by dividing IC50 value of chemical drug treatment alone by IC50 value of combinational treatment. If IC50 value (μM) is >20 or <0.08, the maximum treatment dose (μM) 20 or minimum treatment dose (μM) 0.08 would be served as proxy respectively for calculating fold change. To demonstrate whether CRT combination has synergistic effect or not, we compared three dose response curves, including curves of chemical drug alone treatment, combinational treatment and theoretical additive effect. The predictive additive killing effect is calculated as E total = E 1 + E 2 − E 1 × E 2 (where E 1 is inhibitory effect of IR at dose of 4 Gy and E 2 is inhibitory effect of drug X at defined dose). This classifies a synergistic CRT combination if dose response curve of combinational treatment is lower than additive effect curve.
Organoids were passaged and cultured in 6-well plate for one day before adding drugs (5 μM gefitinib or 50 nM docetaxel or vehicle). For gefitinib inhibitor experiment, organoids were collected for total protein extraction at 12 and 24 h after treatment, and for docetaxel, at 24 h and 48 h. The protein bands were visualized by using Immobilon Western Chemiluminescent HRP Substrate (Millipore) under ChemiDoc Touch Imaging System (Bio-Rad). The antibodies used were listed as following: Phospho-AKT (1:1000, CST, 4060 S), AKT (1:1000, CST, 9272 S), Phospho-ERK (1:1000, CST, 4370 S), ERK (1:1000, CST, 4695 S), Phospho-STAT3 (1:1000, CST, 9145 S), STAT3 (1:1000, CST, 9139 S), Cyclin A (1:200, Santa Cruz, sc-751), Cyclin B (1:200, Santa Cruz, sc-752), Phospho-Bcl-2 (1:1000, CST, 2827 S), Bcl-2 (1:1000, ProteinTech, 12789-1-ap), Bax (1:1000, ProteinTech, 50599-2-lg), Cleaved Caspase-3 (1:1000, CST, 9661 S), MST1R (1:1000, ATLAS, HPA008180), and β-Actin (1:1000, Sigma, A5316).
Sequences of sgRNA targeting human TPPP , MAP2 , and SKP2 were obtained Human CRISPR Knockout Pooled Library (GeCKO v2) (Supplementary Data ), and were cloned into lentiCRISPR v2 vector (Addgene plasmid # 52961). To generate the KO PDO lines, lentivirus system was employed to transduce organoids. Briefly, lentiviruses were produced by co-transfection of a lentivirus vector plasmid, pCMV delta R8.2, and pCMV-VSV-G into HEK 293 T cells. Fresh culture medium was changed 12 h later and culture supernatant was collected at 72 h post-transfection and concentrated by ultracentrifugation. For organoid transfection, the concentrated lentiviruses were firstly mixed with Matrigel at 1:1 volume ratio, then organoids were embedded into the Matrigel containing lentiviruses and cultured with regular organoid culture medium. To select for mutated cells, the culture medium was supplemented with 2 μg/ml puromycin at 72 h post-transfection and continued for 1 week. The remaining organoids after selection were assigned for growth curve and drug treatment evaluation.
All representative experiments were performed in triplicates or duplicates independently. The results were reported as the mean ± SEM or mean ± SD. Statistical significance was calculated using the two-sided t -test, unless otherwise indicated. The p values were considered statistically significant if the p -value ≤ 0.05.
Further information on research design is available in the linked to this article.
Peer Review File Reporting Summary Supplementary information Description of Additional Supplementary Files Supplementary Data 1 Supplementary Data 2 Supplementary Data 3 Supplementary Data 4 Supplementary Data 5 Supplementary Data 6 Supplementary Data 7 Supplementary Data 8 Supplementary Data 9 Supplementary Data 10 Supplementary Data 11 Supplementary Data 12 Supplementary Data 13 Reporting summary
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The healing power of language: caring for patients with limited english proficiency and COVID-19 | b11ba4bd-4947-4741-b39d-a602cfc814aa | 8010487 | Pediatrics[mh] | |
Tomographic diagnosis of alveolar bone coverage impact in orthodontic planning: cross-sectional study | 49a5e3d2-5e4a-4535-8987-525eed5f1e83 | 11457962 | Dentistry[mh] | Radiography examinations are essential in orthodontic diagnosis and treatment planning - for example, two-dimensional (2D) imaging was used satisfactorily for many years. However, some limitations of 2D examinations make it difficult to analyze some structures, such as: overlapping images, poor sharpness, magnification, and distortion. - The advent of cone-beam computed tomography (CBCT) allowed the evaluation of previously inaccessible areas and structures, like the buccal and lingual alveolar plates and resorptive lesions. - CBCT is recommended to analyze root resorption, supernumerary teeth, temporomandibular joint pathology, and limits of alveolar bone. - Orthodontists increasingly rely on three-dimensional (3D) imaging techniques such as CBCT for comprehensive treatment planning. However, the utilization of 3D imaging presents several challenges. Firstly, interpreting volumetric data requires specialized training and expertise, which may not be readily available to all orthodontists. Secondly, there are concerns regarding radiation exposure associated with CBCT scans. Additionally, the cost and accessibility of CBCT machines pose logistical challenges for some orthodontic practices. Alveolar bone coverage is a crucial diagnostic parameter for orthodontic treatment planning, especially in cases in which small inclinations of teeth can cause irreversible damage to the periodontal tissue, and is not routinely assessed through imaging. - One of the most relevant aspects of CBCT examinations is the evaluation of buccal and lingual bone thicknesses before orthodontic movement. , , Hence, detailed orthodontic diagnosis ensures accurate treatment planning and long-term maintenance of periodontal health. , Treatment planning decisions should be made based on the patient’s documentation. However, there is limited information focusing on the importance of CBCT images in the decision-making process among specialists. To the best of our knowledge, no previous study has evaluated the importance of CBCT scanning in the diagnosis of BC for orthodontic treatment planning. Thus, the present study aimed to evaluate the impact of alveolar bone coverage assessed through CBCT images on treatment planning by Brazilian orthodontists. The hypothesis was that the CBCT information regarding anterior BC would influence the treatment plan formulated by Brazilian orthodontists.
STUDY DESIGN An observational and descriptive study, blinded to patient factors, was designed to compare orthodontic treatment plans for malocclusions with critical anterior bone coverage (BC) conditions, based on either 2D information alone or complemmented with additional 3D radiographic data. Treatment plans were simulated using two sequential questionnaires, with the second questionnaire sent immediately after receiving the response for the first. Panoramic radiographs and CBCT images of anterior teeth were used. The study was approved by the Ethical Committee of Federal University of Bahia/Brazil (CAAE: 26024913.0.0000.5024), and involved Brazilian orthodontists recruited from September 2017 to September 2019 via Google Forms questionnaires. Informed consent was obtained from patients to use their photographs and radiographs. Based on the total number of 31.194 registered orthodontic specialists in the Federal Council of Dentistry in 2017, a representative sample of 38 was computed. Two hundred specialists were invited via email, of whom 159 accepted the invitation and were included in the study. CASE SELECTION Critical bone coverage was defined as bone thickness of 1 mm or less measured in tomographic axial and sagittal sections. Six patient records, four with critical bone coverage detected in CBCT images and two with adequate bone coverage, were selected for the study. The six selected cases were carefully chosen to provide a diversity of clinical situations. Among the cases included, there were two men and four women, with a mean age of approximately 20.83 years and a standard deviation of 5.93 years. CBCT images and reports were included to simulate clinical scenarios. Five cases were from patients without prior orthodontic treatment, and one was a retreatment case. Informed consent was obtained from all patients. QUESTIONNAIRES FORMULATION AND APPLICATION Two questionnaires were formulated using the Google Forms platform. The first questionnaire presented the selected cases randomly, each containing extraoral and intraoral photographs, panoramic and lateral cephalometric radiographs. Orthodontists were asked to choose a treatment plan for each case. The provided options included the expansion of dental arches, vestibularization (flaring) of the anterior teeth, interproximal reduction, extraction of tooth/teeth (excluding third molars or supernumerary teeth), orthognathic surgery, and others. These were multiple-choice answers . The second questionnaire included the same cases, with additional slides displaying CBCT images and radiologist reports on bone coverage. The options for answers were the same as those in the first questionnaire. Orthodontists were also asked about the criteria for requesting CBCT in their practice . STATISTICAL ANALYSIS Data from 159 orthodontists who responded to both questionnaires were analyzed descriptively. Participants’ characteristics and CBCT indications were assessed using frequencies. Orthodontists were divided into two groups based on experience, and a chi-square test was used to compare treatment plan changes with and without CBCT information. The McNemar test evaluated differences between the first and second treatment plans proposed by each orthodontist. Statistical analysis was conducted using SPSS version 19.0.
An observational and descriptive study, blinded to patient factors, was designed to compare orthodontic treatment plans for malocclusions with critical anterior bone coverage (BC) conditions, based on either 2D information alone or complemmented with additional 3D radiographic data. Treatment plans were simulated using two sequential questionnaires, with the second questionnaire sent immediately after receiving the response for the first. Panoramic radiographs and CBCT images of anterior teeth were used. The study was approved by the Ethical Committee of Federal University of Bahia/Brazil (CAAE: 26024913.0.0000.5024), and involved Brazilian orthodontists recruited from September 2017 to September 2019 via Google Forms questionnaires. Informed consent was obtained from patients to use their photographs and radiographs. Based on the total number of 31.194 registered orthodontic specialists in the Federal Council of Dentistry in 2017, a representative sample of 38 was computed. Two hundred specialists were invited via email, of whom 159 accepted the invitation and were included in the study.
Critical bone coverage was defined as bone thickness of 1 mm or less measured in tomographic axial and sagittal sections. Six patient records, four with critical bone coverage detected in CBCT images and two with adequate bone coverage, were selected for the study. The six selected cases were carefully chosen to provide a diversity of clinical situations. Among the cases included, there were two men and four women, with a mean age of approximately 20.83 years and a standard deviation of 5.93 years. CBCT images and reports were included to simulate clinical scenarios. Five cases were from patients without prior orthodontic treatment, and one was a retreatment case. Informed consent was obtained from all patients.
Two questionnaires were formulated using the Google Forms platform. The first questionnaire presented the selected cases randomly, each containing extraoral and intraoral photographs, panoramic and lateral cephalometric radiographs. Orthodontists were asked to choose a treatment plan for each case. The provided options included the expansion of dental arches, vestibularization (flaring) of the anterior teeth, interproximal reduction, extraction of tooth/teeth (excluding third molars or supernumerary teeth), orthognathic surgery, and others. These were multiple-choice answers . The second questionnaire included the same cases, with additional slides displaying CBCT images and radiologist reports on bone coverage. The options for answers were the same as those in the first questionnaire. Orthodontists were also asked about the criteria for requesting CBCT in their practice .
Data from 159 orthodontists who responded to both questionnaires were analyzed descriptively. Participants’ characteristics and CBCT indications were assessed using frequencies. Orthodontists were divided into two groups based on experience, and a chi-square test was used to compare treatment plan changes with and without CBCT information. The McNemar test evaluated differences between the first and second treatment plans proposed by each orthodontist. Statistical analysis was conducted using SPSS version 19.0.
Among the 159 included participants (mean age ± standard deviation = 42.91 ± 9.47 years, 51% male), most had more than ten years of experience in orthodontic practice (66.7%) and 72.3% had a graduate degree only in orthodontics. The majority of orthodontists who participated in the survey completed their postgraduate studies in orthodontics in the Southeast (51.6%) or in the Northeast (39.6%) of the country. Additionally, they did not advise CBCT routinely (95.6%) for diagnostic purposes. The indications for CBCT used by orthodontists were tooth ankylosis, followed by surgery, tooth impaction, root resorption, periodontal evaluations, and determination of the amount of opening in the intermaxillary suture . There was a significant statistical difference between the proposed treatment plans in questionnaires 1 and 2. In all six cases, most orthodontists changed the treatment plan after assessing the CBCT images and the radiologist’s report presented in the second questionnaire, as seen in 93.7% ( p <0.001) in case 5, 78.6% ( p <0.001) in case 4, 74.2% ( p <0.001) in case 3, 69.8% ( p <0.001) in case 6, 66% ( p <0.001) in case 2 and 61% ( p =0.01) in case 1. Cases 1 and 3 showed adequate BC in the anterior teeth. Differently, cases 2, 4, 5, and 6 displayed critical BC. Although the participants were divided into two groups according to their experience (G1 with <10 years and G2 with >10 years), both groups showed a statistically significant difference in the proposed treatment plans . In case 1, 43.4% of orthodontists proposed anterior tooth vestibularization in questionnaire 1, while in questionnaire 2, 36.5% proposed interproximal reduction. In cases 2, 4, 5 and 6, orthognathic surgery was the most reported treatment plan for both questionnaires (<41.5%). In case 3, tooth extraction was the most reported treatment plan in both questionnaires 1 (52.2%) and 2 (55.3%) . Most participants dismissed the need for CBCT information on case planning in questionnaire 1 in all six cases. In case 6, most orthodontists declared that CBCT information was decisive in treatment planning .
Data from this study revealed that CBCT information regarding BC of anterior teeth significantly impacted the treatment plans proposed by Brazilian orthodontists. Our findings demonstrated that CBCT examinations lead to a treatment plan different than initially proposed by orthodontists. Herein, most participants completed their postgraduate program from the southeastern and northeastern, where accounts the largest number of orthodontic programs in Brazil. Most orthodontists (66.7%) had more than 10 years of experience, which may have influenced the low frequency of CBCT referral (4.4%), since it is a relatively new imaging technique and may imply a previous experience of the examination and software. Unlike 2D image, CBCT image can be used to measure the thickness and height of buccal alveolar plates with high precision and accuracy. , , , In other hand, CBCT might overestimate the actual measurements of periodontal defects, and aspects of image acquisition such as FOV, and voxel size can influence the diagnosis of bone coverage. Nevertheless, voxels smaller than 0.150mm are reliable for identifying periodontal bone defects. Thus, CBCT imaging can improve periodontal diagnosis and determine risk assessment before establishing treatment procedures. Although orthodontists should strive to use a radiation dose “as low as reasonably achievable” (ALARA), CBCT scans of patients with thin dentoalveolar phenotypes and malocclusions requiring tooth movements toward bone boundaries can be invaluable in treatment planning. The diagnostic accuracy and treatment planning efficacy with CBCT information can change the treatment plan. The present study respected ALARA recommendations regarding CBCT indications. The findings indicated that CBCT is requested in cases of tooth ankylosis, surgery, tooth impaction, and root resorption; and, in lower frequencies, while analyzing the periodontal issues and the amount of intermaxillary suture opening. Therefore, in this study it was observed that most orthodontists would overlook the need of CBCT information in the first questionnaire for their treatment plans. The frequency of change in treatment plan after CBCT information was substantial in all six cases, despite the years of practice. Clinical decision-making in orthodontic planning is a complex process, and may be based on personal preferences, training, and experience. Interestingly, significant changes in the treatment plan were observed in all cases. The revelation of significant alveolar defects, such as thin buccal cortical plates, could play an important role in the treatment plan. - CBCT information can significantly affect the orthodontist’s decision while developing the treatment plan, as observed in this study. It was noticed that orthodontists realized the importance of CBCT imaging while assessing buccal BC, once they understood the technology and were trained to request and interpret the CBCT images. , , In this study, there were eight treatment plan options and there was variability in the choice of plans among professionals between the two questionnaires. However, there was consistency in the treatment plan when evaluating the choice individually for each case, after the tomographic information. Generally, most changes in planning after CBCT information were to avoid tooth projection, preserving the buccal bone plate, even when the bone coverage was not considered critical. Treatment of malocclusions requires information regarding the alveolar bone conditions, especially in cases of lack of space management, and the buccolingual direction determination of tooth movement is needed. Conservative approaches, e.g., buccolingual inclination or translation, expansions, and interproximal reductions, can be substituted by extractions or orthognathic surgeries. If adequate knowledge of BC conditions is provided through CBCT information, favorable prognostic can be obtained. In some cases, it might be advisable to consider soft tissue and/or bone augmentation to prevent deleterious sequelae after orthodontic treatment. Thus, the present study analyzed the impact of CBCT information on orthodontic treatment planning of cases with thin or critical anterior BC by Brazilian orthodontists, and highlighted its importance. To the best of our knowledge, this is the first study to analyze the effect of BC assessed through CBCT on orthodontist’s decision-making ability. This study has strengths and limitations. The main strength was that participants had no previous knowledge about the objective of the research, in a prospective design and with a large sample size included. A major concern was the limited consistency in the orthodontist’s treatment plans; therefore, a greater sample than the calculated number was included. Furthermore, the second questionnaire was sent immediately after receiving the response to the first, assuring that the orthodontist would have the previous treatment plan in recent memory, avoiding a recall bias. One limitation of this study was that the participants had no chance to examine the patient. Nevertheless, certified orthodontists were trained in defining treatment plans using patient records. Furthermore, the patient’s chief complaint was described for each case. The evaluation of BC through CBCT images affected orthodontic diagnosis and treatment planning of Brazilian orthodontists. It constitutes an important tool for diagnostic evaluation, and information obtained through CBCT may change the treatment plan, to avoid creating or augmenting favorable conditions for fenestrations and/or dehiscence. Despite the clinical relevance of the results of this study, the potential effects of the radiation dose of CBCT can not be underestimate. - Therefore, the orthodontist should weigh the potential risks and benefits in diagnosis and treatment planning.
In conclusion, significant differences in orthodontic treatment planning using 2D and 3D information were observed, regardless of the professional experience. Information obtained through CBCT regarding alveolar BC influenced the treatment plan; therefore, in selected cases of borderline malocclusion or thin periodontal profile, CBCT can be a valuable tool for orthodontic diagnosis and treatment planning.
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Cervical cancer screening by cotesting method for Vietnamese women 25–55 years old: a cost-effectiveness analysis | 5f9828ab-9de1-4096-97b1-58a6cb8bc5fd | 11758702 | Surgical Procedures, Operative[mh] | According to global statistic on cancer, cervical cancer (CC) poses a considerable disease burden for low-income and middle-income countries (LMICs), but it can be effectively controlled through widespread coverage of HPV vaccine and cervical screening programmes. In Vietnam, CC ranks among the most prevalent cancers affecting women. From 2016, Vietnam Ministry of Health (MOH) approved a national action plan for the prevention and control of CC,covering the period from 2016 to 2025. However, funding for the screening programme has not been incorporated into the national health insurance, relies instead on local budgets and official development aid sources. This unstable budget allocation presents a significant challenge to achieving the objectives outlined in the action plan. According to the guidelines for control and prevention of CC from MOH, screening methods are recommended for women aged 21–65 years and include (1) cervical cytology, (2) cervical observation with axis acetic (VIA, visual inspection with acetic acid) or examination of the cervix with Lugol’s solution (VILI, visual inspection with Lugol’s Iodine) and (3) test to detect human papillomavirus (HPV) types. These tests can be utilised either independently or in combination, with intervals ranging from 2 to 5 years. Specifically, cotesting, which combines HPV testing with cervical cytology, allows for an extended screening interval of up to 5 years, thereby reducing the overall number of screenings in a woman’s lifetime. Among the available screening methods, colposcopy is considered the least effective for early diagnosis of CC and is primary recommended in low-income countries. Cytology and HPV testing are widely used and recommended as primary methods in numerous national programmes. While HPV testing is characterised by lower specificity, the sensitivity of cytology is highly contingent on quality control and assurance measures. A review by Herbert et al indicated that the limitations of HPV testing may be offset by cytology and vice versa. Thus, the combination of these tests (cotesting) represents a promising approach in populations with low HPV vaccination prevalence. Given that Vietnam is a LMIC with low HPV vaccination coverage, there is an imperative to identify a primary cervical screening method that is both effective and cost-efficient. Recent studies have demonstrated that cotesting is more cost-effective than cervical cytology alone, owing to reduced screening cycles and improved sensitivity and specificity. Following Vietnam MOH guidelines, the cotesting method is recommended for women aged 30 years old and older. However, HPV infection can occur at younger ages; the average age of first sexual initiation among Vietnamese youth was reported to be 18.7 years in 2018. Women infected with HPV may develop CC within 10–20 years depending on their immune response, with a more rapid progression likely in individual with compromised immune systems. Consequently, implementing a CC screening programme for younger women is essential for preventing disease progression. Current guidelines in the USA, Europe and Australia advocate for the initiation of CC screening at the age 25. In high-income countries (HICs), women aged 21–65 years undergo routine CC screening at interval of 3–5 years, depending on the primary screening method. However, this screening strategy poses a challenge for LMIC due to financial constraints and scarcity of infrastructure. In LMIC, WHO suggested that women aged 35–54 years with adequate screening history and no abnormal results may safely discontinue screening, given their low risk for HPV infection. An adequate screening history is defined as three consecutive negative cytology results or two consecutive negative cotesting results within the past 10 years, with the most recent test performed within the last 5 years. Accordingly, this study proposes an alternative screening strategy whereby women aged 25–55 years undergo cotesting three times consecutively at 5 year interval. For example, a 25-year-old woman will be screened CC at the age of 25, 30 and 35. Should this approach be found cost-effective, further research will be necessary to explore whether adjustments to the screening age interval might yield even greater cost-effectiveness. To date, only two studies have investigated on the cost-effectiveness of cervical cytology in Vietnam: one conducted by Suba et al in 2001 and another by Kim et al in 2008. Suba et al concluded that cervical cytology was the optimal screening method at that time due to a lack of resources for other screening methods. Kim et al study mainly focused on the cost-effectiveness of HPV vaccination in combination with three times CC screening in a whole woman life or once in every 5 years in Vietnam. However, research assessing the cost-effectiveness of cervical screening methods in Vietnam remains limited, particularly concerning newer approaches such as cotesting. Therefore, this study aims to investigate the cost-effectiveness of CC screening three times consecutively at 5 year interval using the cotesting method compared with five times consecutively at 2 year interval using the cytology method for Vietnamese women aged 25–55 years.
Study design The study applied a cost-effectiveness analysis (CEA) method using Markov modelling to assess the efficiency of the cotesting method in CC screening for Vietnamese women from 25 to 55 years old. The analysis stimulated a cohort of 1000 women at the age of 25 and followed them through yearly cycle until they reached their upper limited screening age. For instance, the group aged 25–29 was modelled and analysed until they turned 55, as was the group aged 30–34, and so forth for the remaining age groups. We applied the provider perspective for the cost-effective analysis. Input parameters for the Markov modelling were obtained through a comprehensive literature review, drawing from common CEA models that simulate the natural progression of HPV infection. These inputs included transition probabilities, efficacy of screening methods, cost for treatment and screening tests and quality-adjusted life years (QALYs) for various health states. Medical and financial experts in the Viet Nam National Cancer Hospital (K hospital) were consulted to validate and adjust the input parameters. Then, a Microsoft Excel template was used to perform the simulation and calculate an incremental cost-effectiveness ratio (ICER). Due to the differing transition probabilities across age groups, we developed six distinct Markov models for cohorts of 1000 women in each age group (25–29, 30–34, 35–39, 40–44, 45–49 and 50–55) to resemble the target women population from 25 to 55 years old. These groups underwent CC screening either three times consecutive using the cotesting method or five times consecutive using the cytology method. For example, the 25–29 age group was screened at the ages 25, 30 and 35 with cotesting and at the ages 25, 27, 29, 31 and 33 with cytology. The Markov model applied the same transition probabilities based on the starting cohort age and simulated the incidence of CC by age 55. Consequently, older cohorts underwent fewer screening rounds. Specifically, women aged 50–55 were screened only once using co-testing and twice using cytology. Patients and public involvement No patients or members of the public were involved in the design, conduct, reporting or dissemination plans of this study. Study population The incidence of CC in Vietnam increased rapidly in women aged 30–34, peaking in the 55–59 age group before gradually decreasing in older populations. Since CC typically develops many years after HPV infection, and the average age of first sexual initiation in Vietnam was 18.7 years, women aged from 25 to 55 years were identified as the target group for this study. This group faces a higher risk of CC development due to lack of HPV vaccination coverage and the absence of national health insurance reimbursement for CC screening in Vietnam. To conduct the evaluation, we constructed a hypothetical cohort of one million women aged from 25 to 55, representing those at high risk of CC. These women were assumed to follow the same natural history of disease, screening, diagnosis, and treatment pathways. Intervention and comparator The cotesting method has been shown to be more cost-effective than cytology or HPV testing alone in routine CC screening up to the upper screening age limit in HICs. However, the LMICs cannot implement a routine CC screening for all target women due to limited resources. WHO and previous studies have recommended at least three consecutive screenings as a feasible strategy for CC screening in LMICs. This study investigates the cost-effectiveness of the cotesting method in Vietnam compared with the cytology method, which has been proven cost-effective for over a decade. The research uses the Markov model to compare two screening strategies for Vietnamese women aged 25–55: Cotesting method: combined cytology and HPV test, performed three times with a 5 year interval. Cytology method: performed five times with a 2 year interval. For example: The 25–29 group was screened at ages of 25, 30 and 35 using the cotesting method, and at ages of 25, 27, 29, 31 and 33 using the cytology method. The 30–34 group was screened at ages of 30, 35 and 40 using the cotesting method, and at ages of 30, 32, 34, 36 and 37 using the cytology method. Markov model The model and time horizon were based on the natural epidemiology of CC progression, reviewed from medical literature and results from randomised control trials and retrospective cohort studies. Most reviewed studies used Markov model with 1 year cycles for analysis, which was also adopted for this study. Additionally, high-risk HPV (HPVhr) natural history models were reviewed, and clinical experts were consulted for adjustment. Since the proposed Markov model shared similar health states with other models, clinical experts did not suggest any changes. The Markov model with seven states was used to simulate the natural disease progression of HPV infection with 1 year cycles . The Markov model was based on following assumptions: A cohort of 1000 undiagnosed women, aged 25–55 and engaging in sexual activity, would be screened using both the cotesting and cytology methods without adding new cohorts. None of the women were vaccinated against HPV. None of them has experienced hysterectomy. Full participation in screening programmes was assumed. All detected cases of HPV positive, cervical intraepithelial neoplasia (CIN) 1, CIN 2, CIN 3 and CC would receive treatment. QALYs were used to measure health outcomes. The Markov model was developed with health states based on history of natural HPV infection which adopted from Felix et al (2016). Input parameters The effectiveness of screening tests, defined by their sensitivity and specificity, is detailed in . These data were obtained from systematic reviews and meta-analyses. Screening costs were sourced from Circulars no 13/2019TT-BYT, which regulates medical service prices in Vietnam ( https://thuvienphapluat.vn/ ), and treatment costs were drawn from Nguyen et al ’s study on the medical costs of treating CC at central hospitals in Vietnam, analysed from a provider perspective . Because treatment cost for CC varied according to severity level and treatment pathways for CIN 1, CIN 2 and CIN 3 were similar; hence, we used average cost of scenarios to generate the treatment cost for these states. The cost inputs were adjusted to 2022 values based on Consumer price index published by Viet Nam General Statistics Office. Currently, there are no Vietnamese data on QALY weights for the different states in the Markov model. Similar cost-effectiveness analyses of CC screening in China, Taiwan and Thailand used utility weights from clinical trials conducted in countries such as the USA, Canada and the UK. In this study, we applied data from Warner et al , which derived QALYs for CIN 1, CIN 2, CIN 3 and CC states from population surveillance in Canada and the USA using the standard gamble method . The QALY for CC state was averaged across treatment phases in accordance with the International Federation of Obstetrics and Gynaecology states to avoid over estimation utility for CC. For transition probabilities related to disease progression and regression , we consulted data from the study ‘Screening for Cervical Cancer: A Decision Analysis for the U.S. Preventive Services Task Force’ and the study ‘The Clinical and Economic Benefits of Co-Testing vs Primary HPV Testing for Cervical Cancer Screening: A Modeling Analysis’. These studies used data from the ATHENA trial (The Addressing the Need for Advanced HPV Diagnostics study) conducted in a population with low HPV vaccination coverage (2%), making the data suitable for the Vietnam context. Cost-effectiveness analysis and sensitivity analysis After adjusting all costs for inflation to 2022 and applying a discount rate of 3%, an ICER was computed to evaluate efficiency of the cotesting method compared with the cytology method. Up to date, there are no specific ICER thresholds for CEA in Vietnam. Therefore, we followed the older recommendations of WHO, which suggested that if the ICER was lower than 1–3 gross domestic product (GDP) per capita, the intervention may be considered cost-effective. According to the GDP data of Vietnam published by the World Bank in 2022, it was US$2785.7/per capita. The exchange rate used for converting VND to USD was 23 060 VND per USD, based on Vietcombank’s rate. Thus, 1 GDP per capita equals 63.68 million VND (US$2786) and 3 GDP per capita equals 191.04 million VND (US$8357). For sensitivity analysis, we conducted both probabilistic sensitivity analysis (PSA) and deterministic sensitivity analysis (DSA). PSA was performed using Monte Carlo simulations (1000 iterations) to present probabilities of cost-effectiveness and the cost-effectiveness acceptancy curve (CEAC) corresponding to the Vietnam’s GDP thresholds, to illustrate the impact of uncertainty on the ICER. In the one-way DSA, all parameters, including the cost of the cotesting and cytology methods, treatment services and QALYs, were analysed. We identified 15 influential parameters and evaluated the impact of changes in the number of cytology screening rounds on the ICER. The results were presented using Tornado diagrams.
The study applied a cost-effectiveness analysis (CEA) method using Markov modelling to assess the efficiency of the cotesting method in CC screening for Vietnamese women from 25 to 55 years old. The analysis stimulated a cohort of 1000 women at the age of 25 and followed them through yearly cycle until they reached their upper limited screening age. For instance, the group aged 25–29 was modelled and analysed until they turned 55, as was the group aged 30–34, and so forth for the remaining age groups. We applied the provider perspective for the cost-effective analysis. Input parameters for the Markov modelling were obtained through a comprehensive literature review, drawing from common CEA models that simulate the natural progression of HPV infection. These inputs included transition probabilities, efficacy of screening methods, cost for treatment and screening tests and quality-adjusted life years (QALYs) for various health states. Medical and financial experts in the Viet Nam National Cancer Hospital (K hospital) were consulted to validate and adjust the input parameters. Then, a Microsoft Excel template was used to perform the simulation and calculate an incremental cost-effectiveness ratio (ICER). Due to the differing transition probabilities across age groups, we developed six distinct Markov models for cohorts of 1000 women in each age group (25–29, 30–34, 35–39, 40–44, 45–49 and 50–55) to resemble the target women population from 25 to 55 years old. These groups underwent CC screening either three times consecutive using the cotesting method or five times consecutive using the cytology method. For example, the 25–29 age group was screened at the ages 25, 30 and 35 with cotesting and at the ages 25, 27, 29, 31 and 33 with cytology. The Markov model applied the same transition probabilities based on the starting cohort age and simulated the incidence of CC by age 55. Consequently, older cohorts underwent fewer screening rounds. Specifically, women aged 50–55 were screened only once using co-testing and twice using cytology.
No patients or members of the public were involved in the design, conduct, reporting or dissemination plans of this study.
The incidence of CC in Vietnam increased rapidly in women aged 30–34, peaking in the 55–59 age group before gradually decreasing in older populations. Since CC typically develops many years after HPV infection, and the average age of first sexual initiation in Vietnam was 18.7 years, women aged from 25 to 55 years were identified as the target group for this study. This group faces a higher risk of CC development due to lack of HPV vaccination coverage and the absence of national health insurance reimbursement for CC screening in Vietnam. To conduct the evaluation, we constructed a hypothetical cohort of one million women aged from 25 to 55, representing those at high risk of CC. These women were assumed to follow the same natural history of disease, screening, diagnosis, and treatment pathways.
The cotesting method has been shown to be more cost-effective than cytology or HPV testing alone in routine CC screening up to the upper screening age limit in HICs. However, the LMICs cannot implement a routine CC screening for all target women due to limited resources. WHO and previous studies have recommended at least three consecutive screenings as a feasible strategy for CC screening in LMICs. This study investigates the cost-effectiveness of the cotesting method in Vietnam compared with the cytology method, which has been proven cost-effective for over a decade. The research uses the Markov model to compare two screening strategies for Vietnamese women aged 25–55: Cotesting method: combined cytology and HPV test, performed three times with a 5 year interval. Cytology method: performed five times with a 2 year interval. For example: The 25–29 group was screened at ages of 25, 30 and 35 using the cotesting method, and at ages of 25, 27, 29, 31 and 33 using the cytology method. The 30–34 group was screened at ages of 30, 35 and 40 using the cotesting method, and at ages of 30, 32, 34, 36 and 37 using the cytology method.
The model and time horizon were based on the natural epidemiology of CC progression, reviewed from medical literature and results from randomised control trials and retrospective cohort studies. Most reviewed studies used Markov model with 1 year cycles for analysis, which was also adopted for this study. Additionally, high-risk HPV (HPVhr) natural history models were reviewed, and clinical experts were consulted for adjustment. Since the proposed Markov model shared similar health states with other models, clinical experts did not suggest any changes. The Markov model with seven states was used to simulate the natural disease progression of HPV infection with 1 year cycles . The Markov model was based on following assumptions: A cohort of 1000 undiagnosed women, aged 25–55 and engaging in sexual activity, would be screened using both the cotesting and cytology methods without adding new cohorts. None of the women were vaccinated against HPV. None of them has experienced hysterectomy. Full participation in screening programmes was assumed. All detected cases of HPV positive, cervical intraepithelial neoplasia (CIN) 1, CIN 2, CIN 3 and CC would receive treatment. QALYs were used to measure health outcomes. The Markov model was developed with health states based on history of natural HPV infection which adopted from Felix et al (2016).
The effectiveness of screening tests, defined by their sensitivity and specificity, is detailed in . These data were obtained from systematic reviews and meta-analyses. Screening costs were sourced from Circulars no 13/2019TT-BYT, which regulates medical service prices in Vietnam ( https://thuvienphapluat.vn/ ), and treatment costs were drawn from Nguyen et al ’s study on the medical costs of treating CC at central hospitals in Vietnam, analysed from a provider perspective . Because treatment cost for CC varied according to severity level and treatment pathways for CIN 1, CIN 2 and CIN 3 were similar; hence, we used average cost of scenarios to generate the treatment cost for these states. The cost inputs were adjusted to 2022 values based on Consumer price index published by Viet Nam General Statistics Office. Currently, there are no Vietnamese data on QALY weights for the different states in the Markov model. Similar cost-effectiveness analyses of CC screening in China, Taiwan and Thailand used utility weights from clinical trials conducted in countries such as the USA, Canada and the UK. In this study, we applied data from Warner et al , which derived QALYs for CIN 1, CIN 2, CIN 3 and CC states from population surveillance in Canada and the USA using the standard gamble method . The QALY for CC state was averaged across treatment phases in accordance with the International Federation of Obstetrics and Gynaecology states to avoid over estimation utility for CC. For transition probabilities related to disease progression and regression , we consulted data from the study ‘Screening for Cervical Cancer: A Decision Analysis for the U.S. Preventive Services Task Force’ and the study ‘The Clinical and Economic Benefits of Co-Testing vs Primary HPV Testing for Cervical Cancer Screening: A Modeling Analysis’. These studies used data from the ATHENA trial (The Addressing the Need for Advanced HPV Diagnostics study) conducted in a population with low HPV vaccination coverage (2%), making the data suitable for the Vietnam context.
After adjusting all costs for inflation to 2022 and applying a discount rate of 3%, an ICER was computed to evaluate efficiency of the cotesting method compared with the cytology method. Up to date, there are no specific ICER thresholds for CEA in Vietnam. Therefore, we followed the older recommendations of WHO, which suggested that if the ICER was lower than 1–3 gross domestic product (GDP) per capita, the intervention may be considered cost-effective. According to the GDP data of Vietnam published by the World Bank in 2022, it was US$2785.7/per capita. The exchange rate used for converting VND to USD was 23 060 VND per USD, based on Vietcombank’s rate. Thus, 1 GDP per capita equals 63.68 million VND (US$2786) and 3 GDP per capita equals 191.04 million VND (US$8357). For sensitivity analysis, we conducted both probabilistic sensitivity analysis (PSA) and deterministic sensitivity analysis (DSA). PSA was performed using Monte Carlo simulations (1000 iterations) to present probabilities of cost-effectiveness and the cost-effectiveness acceptancy curve (CEAC) corresponding to the Vietnam’s GDP thresholds, to illustrate the impact of uncertainty on the ICER. In the one-way DSA, all parameters, including the cost of the cotesting and cytology methods, treatment services and QALYs, were analysed. We identified 15 influential parameters and evaluated the impact of changes in the number of cytology screening rounds on the ICER. The results were presented using Tornado diagrams.
Incremental cost-effectiveness ratio (ICER) The results showed that cotesting was not cost-effective compared with cytology . The total screening costs and QALYs for each age group was summarised in . In general, in five age groups (25–29, 35–39, 40–44, 45–49 and 50–55), cotesting were dominated by cytology, meaning that cotesting had higher cost and lower QALYs. In the 25–29 group, the total cost of the cotesting programme was highest with US$1266 with a total QALYs of 24.72. While the cytology programme cost less at US$1122 and provided higher QALYs at 26.34. For the 30–34 age group, the cost of cotesting was lower than cytology; however, switching from cytology to cotesting would result in a QALY loss. Cotesting could be a cost-effective alternative if the savings per QALY lost exceeded the threshold, but this was not the case here, as Vietnam’s GDP per capita was US$2786, and the saving cost was only US$3. For both screening strategies, the total cost for other age groups decreased significantly. In the cotesting programme, the total cost decreased from US$516 to US$198, while the QALYs dropped from 18.59 to 6.07. Similarly, in the cytology programme, the total cost decreased from US$484 to US$146, with QALYs falling 20.54 to 7.26. Despite higher cost, the cotesting method reduced the incidence of CC and CIN 1/2/3 cases more effectively than cytology. The cotesting prevented 887 CIN 1/2/3 cases and 32 CC cases, compared with 627 CIN 1/2/3 cases and 24 CC cases prevented by cytology, when compared with a no-screening scenario. But the number of false positive cases detected by the cotesting method was double that of the cytology method. The cost for older age groups decreased significantly because the model only stimulated the cohort until they reached 55 years old, resulting in fewer screening cycles for older women and, consequently, lower cost. Sensitivity analysis In the one-way DSA, 23 parameters from cost, transition probabilities and QALYs were analysed to find the most influential parameters. After evaluating the differences in ICER, 15 influential parameters were identified. In general, the top 15 influential factors varied among age groups. The Tornado diagrams revealed that the ICER was most sensitive to the transition probability from HPVhr (−) state to HPVhr state and the prevalence of HPVhr cases in general female population across all age groups . The cost and effectiveness of screening tests has a smaller impact on ICER changes in most age groups, except in the 50–55 age group, where the cost of cotesting ranked third, while the costs of the HPV and cytology tests ranked sixth and seventh, respectively. The treatment cost of CC had a significant impact on the ICER, particularly in the 35–39 age group, where it ranked second, and it ranked third in the 25–29, 30–34 and 40–44 groups and fourth in the 45–49 group. However, in the 50–55 age group, this parameter had little influence on the ICER changes. Cost effectiveness planes for 1000 Monte Carlo simulations and CEAC are shown from . The ICER simulations fluctuated primarily in the north-west and south-west quadrants, indicating that the strategy of three times consecutive screening via cotesting was dominated by the comparator (cytology). The CEACs also confirmed the robustness of the PSA and DSA results, showing that the probability of the ICER remaining below Vietnam’s willingness-to-pay (WTP) threshold (1–3 times GDP per capita) was relatively low, at around 40%–50% across all age groups. The probability of cotesting achieving cost-effectiveness approached 0% as the WTP threshold increased beyond US$1200. In contrast, the cytology method demonstrated a higher probability (40%–60%) of achieving cost-effectiveness, with the CEAC for cytology approaching 100% at the WTP threshold. This suggests that cytology is more likely to be cost-effective, even when the WTP is lower than 1 GDP (US$2786). To analyse the impact of the number of CC screening rounds on the ICER, an additional scenario was conducted to compare cost-effectiveness of three times consecutive cotesting and three times consecutive cytology for Vietnamese women 25–30 years old. In this scenario, the cotesting method (cost: US$1202 and QALYs: 24.18) was superior to the cytology method (cost: US$955 and QALYs: 22.55) with an ICER of 152 USD per QALY gained which is lower than 1 GDP per capita (US$2786). Under uncertainty conditions, 1000 Monte Carlo simulations for this scenario were concentrated in the north-east quadrant, indicating that the number of screening rounds significantly impacts the ICER.
The results showed that cotesting was not cost-effective compared with cytology . The total screening costs and QALYs for each age group was summarised in . In general, in five age groups (25–29, 35–39, 40–44, 45–49 and 50–55), cotesting were dominated by cytology, meaning that cotesting had higher cost and lower QALYs. In the 25–29 group, the total cost of the cotesting programme was highest with US$1266 with a total QALYs of 24.72. While the cytology programme cost less at US$1122 and provided higher QALYs at 26.34. For the 30–34 age group, the cost of cotesting was lower than cytology; however, switching from cytology to cotesting would result in a QALY loss. Cotesting could be a cost-effective alternative if the savings per QALY lost exceeded the threshold, but this was not the case here, as Vietnam’s GDP per capita was US$2786, and the saving cost was only US$3. For both screening strategies, the total cost for other age groups decreased significantly. In the cotesting programme, the total cost decreased from US$516 to US$198, while the QALYs dropped from 18.59 to 6.07. Similarly, in the cytology programme, the total cost decreased from US$484 to US$146, with QALYs falling 20.54 to 7.26. Despite higher cost, the cotesting method reduced the incidence of CC and CIN 1/2/3 cases more effectively than cytology. The cotesting prevented 887 CIN 1/2/3 cases and 32 CC cases, compared with 627 CIN 1/2/3 cases and 24 CC cases prevented by cytology, when compared with a no-screening scenario. But the number of false positive cases detected by the cotesting method was double that of the cytology method. The cost for older age groups decreased significantly because the model only stimulated the cohort until they reached 55 years old, resulting in fewer screening cycles for older women and, consequently, lower cost.
In the one-way DSA, 23 parameters from cost, transition probabilities and QALYs were analysed to find the most influential parameters. After evaluating the differences in ICER, 15 influential parameters were identified. In general, the top 15 influential factors varied among age groups. The Tornado diagrams revealed that the ICER was most sensitive to the transition probability from HPVhr (−) state to HPVhr state and the prevalence of HPVhr cases in general female population across all age groups . The cost and effectiveness of screening tests has a smaller impact on ICER changes in most age groups, except in the 50–55 age group, where the cost of cotesting ranked third, while the costs of the HPV and cytology tests ranked sixth and seventh, respectively. The treatment cost of CC had a significant impact on the ICER, particularly in the 35–39 age group, where it ranked second, and it ranked third in the 25–29, 30–34 and 40–44 groups and fourth in the 45–49 group. However, in the 50–55 age group, this parameter had little influence on the ICER changes. Cost effectiveness planes for 1000 Monte Carlo simulations and CEAC are shown from . The ICER simulations fluctuated primarily in the north-west and south-west quadrants, indicating that the strategy of three times consecutive screening via cotesting was dominated by the comparator (cytology). The CEACs also confirmed the robustness of the PSA and DSA results, showing that the probability of the ICER remaining below Vietnam’s willingness-to-pay (WTP) threshold (1–3 times GDP per capita) was relatively low, at around 40%–50% across all age groups. The probability of cotesting achieving cost-effectiveness approached 0% as the WTP threshold increased beyond US$1200. In contrast, the cytology method demonstrated a higher probability (40%–60%) of achieving cost-effectiveness, with the CEAC for cytology approaching 100% at the WTP threshold. This suggests that cytology is more likely to be cost-effective, even when the WTP is lower than 1 GDP (US$2786). To analyse the impact of the number of CC screening rounds on the ICER, an additional scenario was conducted to compare cost-effectiveness of three times consecutive cotesting and three times consecutive cytology for Vietnamese women 25–30 years old. In this scenario, the cotesting method (cost: US$1202 and QALYs: 24.18) was superior to the cytology method (cost: US$955 and QALYs: 22.55) with an ICER of 152 USD per QALY gained which is lower than 1 GDP per capita (US$2786). Under uncertainty conditions, 1000 Monte Carlo simulations for this scenario were concentrated in the north-east quadrant, indicating that the number of screening rounds significantly impacts the ICER.
Main findings Based on our analysis in the results part, CC screening by three times consecutive cotesting was not cost-effective compared with CC screening by five times cytology. While the cotesting strategy prevented more cases of CIN 1/2/3 and CC than the cytology method, the cost was significantly higher. Additionally, the large number of false positives detected by cotesting raised concerns about its potential impact on patients’ mental health due to overtreatment, and it posed a financial burden on the public health budget. The one-way DSA indicated that the ICER was most sensitive to the transition probability from HPVhr to HPVhr and the prevalence of HPVhr in the general population. Conversely, the cost and effectiveness of screening tests had minimal impacts. The PSA and CEAC further confirmed these findings. At Vietnam’s WTP threshold, the probability of cost-effectiveness for cotesting was close to 0, whereas the cytology method had a 100% probability of being cost-effective at around US$1200, well below 1 GDP per capita (US$2786). Our study proved that the cotesting was dominated by the cytology and this result contrasted with findings from studies of Ian Cromwell et al (2021), Adam Keane et al (2020), Anastasios Skroumpelos et al (2019), Taejong Song et al (2018), Jie-Bin Lew et al (2017) and Thomas Wright et al (2016). Those studies indicated that cotesting was cost-effective in preventing CC incidence and deaths compared with cytology. However, when the comparator was the HPV test, the difference in results was not as significant. Among all screening strategies, cotesting was associated with the lowest CC incidence and high performance in detecting early-stage CC. Although cotesting gained more QALYs, this difference was not significant, and it came with the highest cost per screened woman, a critical consideration for policymakers. Several factors may explain the differing outcomes. First, our study used a 2 year screening interval for cytology, whereas most other studies used a 3 year interval. A shorter screening interval could lead to fewer missed diagnoses in the simulated cohorts, resulting in higher QALY weights. Second, the frequency of CC screening also differed: previous studies considered screening throughout a woman’s lifetime until she reached the upper age limit, whereas our model focused only on three or five consecutive screenings. Consequently, women over the age of 49 received fewer screenings in our model, particularly those in the cotesting group, even though the incidence of CC in Vietnam peaks in women over 45. This reduction in screening for older women resulted in smaller QALY gains for the cotesting group. In the DSA, we found out that the ICER was the most sensitive to the transition probability from HPVhr (−) state to HPVhr state, the prevalence of HPVhr in general women population and the number of CC screening rounds. Although we cannot influence the transition probability, the other factors could be addressed through expanded HPV vaccination programmes and implemented CC screening strategies. Vietnam could increase HPV vaccination coverage by negotiating lower vaccine prices, potentially at US$4.55 per dose (65–67). These factors influence the ICER range, which still falls below Vietnam’s current cost-effectiveness thresholds (1–3 GDP per capita). However, increasing the number of routine cytology screenings could reverse the cost-effectiveness results, making cytology more favourable than cotesting. Future research should explore the optimal number of cytology screenings needed to achieve greater cost-effectiveness compared with cotesting. Our study demonstrated that cotesting was dominated by cytology across all age groups, resulting in lower QALY gains. If we reversed the intervention and comparator in the study, the savings would range from US$16 to US$64 per QALY gained. However, in the second scenario (three rounds of cotesting vs three rounds of cytology), the ICER was US$152 per QALY gained, meaning that women would need to pay US$152 to gain 1 QALY, whereas they could save money with the alternative approach. The findings from both scenarios show that while cotesting can reduce CC incidence, it comes at a much higher cost. Another significant drawback of cotesting is the increase in false positives, which adds to the financial burden due to the need for triage and follow-up tests. These results align with previous studies that highlight the medical benefits of cotesting, although at a substantially higher cost. Strength and limitations The study noted some limitations. First, this research proposed an age interval from 25 to 55 years for CC screening, while national guidelines from other countries targeted women from 21 to 65 or even up to 79 years. Therefore, future research should investigate the appropriate age interval for CC screening in Vietnam. Second, the research did not consider effects from HPV vaccination on the transition probabilities of the Markov model. Although the prevalence of HPV vaccinated women in the target group in Vietnam is unknown, it might decrease sensitivity and positive predicted value of the cytology method. To adjust the input parameters, we used data from studies about efficacy and effectiveness of cytology-based screening method in LMICs. Third, for the 21 transition probabilities that were used in the Markov model, only one assumption about the lower and upper ranges was made. This assumption was based on the study ‘Effectiveness and cost-effectiveness of eliminating cervical cancer through a tailored optimal pathway: a modelling study’ in China which applies ±25% for the range of transition probabilities. We also validated this percentage point and the secondary data with four Vietnamese clinical experts to ensure the best available evidence to the researcher’s knowledge. However, this limitation suggests the need for more localised data, specifically on transition probabilities. Another limitation in our study was the QALY weights which were sourced from the study from a large population in Canada and USA. The weights were elicited by the standard gamble method, which is a valid approach but not the most commonly used in recent studies. Although we found no substantial differences between QALY weights from the EQ-5D method and the standard gamble method. Hence, we decided to use the data on QALY weights from Warner et al (2015). They still contributed to the limitations of our study and future studies should focus on deriving QALY weights more specific to the Vietnamese population. To manage uncertainties, we used PSA. However, the original study about the transition probabilities of the Markov model did not publish the SD, 95% CI, range or full parameter distributions. As a results, we estimated these values based on the transition probabilities and the data from the studies ‘A dynamic Bayesian Markov model for health economic evaluations of interventions in infectious disease’ to generate β distribution for the PSA. Applying the same distribution for all age groups in the PSA might limit the precision of our results. There is a need to conduct systematic reviews and meta-analysis about the transition probabilities of CC development in the general population to address this in future studies.
Based on our analysis in the results part, CC screening by three times consecutive cotesting was not cost-effective compared with CC screening by five times cytology. While the cotesting strategy prevented more cases of CIN 1/2/3 and CC than the cytology method, the cost was significantly higher. Additionally, the large number of false positives detected by cotesting raised concerns about its potential impact on patients’ mental health due to overtreatment, and it posed a financial burden on the public health budget. The one-way DSA indicated that the ICER was most sensitive to the transition probability from HPVhr to HPVhr and the prevalence of HPVhr in the general population. Conversely, the cost and effectiveness of screening tests had minimal impacts. The PSA and CEAC further confirmed these findings. At Vietnam’s WTP threshold, the probability of cost-effectiveness for cotesting was close to 0, whereas the cytology method had a 100% probability of being cost-effective at around US$1200, well below 1 GDP per capita (US$2786). Our study proved that the cotesting was dominated by the cytology and this result contrasted with findings from studies of Ian Cromwell et al (2021), Adam Keane et al (2020), Anastasios Skroumpelos et al (2019), Taejong Song et al (2018), Jie-Bin Lew et al (2017) and Thomas Wright et al (2016). Those studies indicated that cotesting was cost-effective in preventing CC incidence and deaths compared with cytology. However, when the comparator was the HPV test, the difference in results was not as significant. Among all screening strategies, cotesting was associated with the lowest CC incidence and high performance in detecting early-stage CC. Although cotesting gained more QALYs, this difference was not significant, and it came with the highest cost per screened woman, a critical consideration for policymakers. Several factors may explain the differing outcomes. First, our study used a 2 year screening interval for cytology, whereas most other studies used a 3 year interval. A shorter screening interval could lead to fewer missed diagnoses in the simulated cohorts, resulting in higher QALY weights. Second, the frequency of CC screening also differed: previous studies considered screening throughout a woman’s lifetime until she reached the upper age limit, whereas our model focused only on three or five consecutive screenings. Consequently, women over the age of 49 received fewer screenings in our model, particularly those in the cotesting group, even though the incidence of CC in Vietnam peaks in women over 45. This reduction in screening for older women resulted in smaller QALY gains for the cotesting group. In the DSA, we found out that the ICER was the most sensitive to the transition probability from HPVhr (−) state to HPVhr state, the prevalence of HPVhr in general women population and the number of CC screening rounds. Although we cannot influence the transition probability, the other factors could be addressed through expanded HPV vaccination programmes and implemented CC screening strategies. Vietnam could increase HPV vaccination coverage by negotiating lower vaccine prices, potentially at US$4.55 per dose (65–67). These factors influence the ICER range, which still falls below Vietnam’s current cost-effectiveness thresholds (1–3 GDP per capita). However, increasing the number of routine cytology screenings could reverse the cost-effectiveness results, making cytology more favourable than cotesting. Future research should explore the optimal number of cytology screenings needed to achieve greater cost-effectiveness compared with cotesting. Our study demonstrated that cotesting was dominated by cytology across all age groups, resulting in lower QALY gains. If we reversed the intervention and comparator in the study, the savings would range from US$16 to US$64 per QALY gained. However, in the second scenario (three rounds of cotesting vs three rounds of cytology), the ICER was US$152 per QALY gained, meaning that women would need to pay US$152 to gain 1 QALY, whereas they could save money with the alternative approach. The findings from both scenarios show that while cotesting can reduce CC incidence, it comes at a much higher cost. Another significant drawback of cotesting is the increase in false positives, which adds to the financial burden due to the need for triage and follow-up tests. These results align with previous studies that highlight the medical benefits of cotesting, although at a substantially higher cost.
The study noted some limitations. First, this research proposed an age interval from 25 to 55 years for CC screening, while national guidelines from other countries targeted women from 21 to 65 or even up to 79 years. Therefore, future research should investigate the appropriate age interval for CC screening in Vietnam. Second, the research did not consider effects from HPV vaccination on the transition probabilities of the Markov model. Although the prevalence of HPV vaccinated women in the target group in Vietnam is unknown, it might decrease sensitivity and positive predicted value of the cytology method. To adjust the input parameters, we used data from studies about efficacy and effectiveness of cytology-based screening method in LMICs. Third, for the 21 transition probabilities that were used in the Markov model, only one assumption about the lower and upper ranges was made. This assumption was based on the study ‘Effectiveness and cost-effectiveness of eliminating cervical cancer through a tailored optimal pathway: a modelling study’ in China which applies ±25% for the range of transition probabilities. We also validated this percentage point and the secondary data with four Vietnamese clinical experts to ensure the best available evidence to the researcher’s knowledge. However, this limitation suggests the need for more localised data, specifically on transition probabilities. Another limitation in our study was the QALY weights which were sourced from the study from a large population in Canada and USA. The weights were elicited by the standard gamble method, which is a valid approach but not the most commonly used in recent studies. Although we found no substantial differences between QALY weights from the EQ-5D method and the standard gamble method. Hence, we decided to use the data on QALY weights from Warner et al (2015). They still contributed to the limitations of our study and future studies should focus on deriving QALY weights more specific to the Vietnamese population. To manage uncertainties, we used PSA. However, the original study about the transition probabilities of the Markov model did not publish the SD, 95% CI, range or full parameter distributions. As a results, we estimated these values based on the transition probabilities and the data from the studies ‘A dynamic Bayesian Markov model for health economic evaluations of interventions in infectious disease’ to generate β distribution for the PSA. Applying the same distribution for all age groups in the PSA might limit the precision of our results. There is a need to conduct systematic reviews and meta-analysis about the transition probabilities of CC development in the general population to address this in future studies.
The study found that three consecutive CC screenings using cotesting at 5 year intervals were less cost-effective and less beneficial than five screenings using cytology at 2 year intervals. Although the cotesting method was cost-effective in one scenario under DSA, it required women to pay US$152 per QALY gained (ICER=US$152). For the base-case analysis, using cytology as the intervention could lead to savings ranging from US$16 to 64 per QALY gained, which would be appealing for both healthcare decision-makers and users. Additionally, cotesting resulted in a significant increase in referrals and unnecessary treatment of healthy women, posing a financial burden on Vietnam’s healthcare system and creating potential mental health concerns for patients. Given the reasonable clinical benefits and cost savings per QALY gained, the strategy of five consecutive cytology screenings for women aged 25–55 years can be recommended for CC screening in Vietnam.
10.1136/bmjopen-2023-082145 online supplemental file 1 10.1136/bmjopen-2023-082145 online supplemental file 2 10.1136/bmjopen-2023-082145 online supplemental file 3 10.1136/bmjopen-2023-082145 online supplemental file 4 10.1136/bmjopen-2023-082145 online supplemental file 5 10.1136/bmjopen-2023-082145 online supplemental file 6 10.1136/bmjopen-2023-082145 online supplemental file 7 10.1136/bmjopen-2023-082145 online supplemental file 8 10.1136/bmjopen-2023-082145 online supplemental file 9 10.1136/bmjopen-2023-082145 online supplemental file 10
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Feasibility of EBUS-TBNA for histopathological and molecular diagnostics of NSCLC—A retrospective single-center experience | 9fb2f5ed-7a6e-4050-aef6-d0e88a57d89e | 8809531 | Pathology[mh] | Lung cancer, a severe disease with increasing incidence, is the leading cause of cancer-related death globally . Investigation, diagnosis, precise staging, and genomic profiling of lung cancer is a demanding but fundamental process for adequate cancer treatment. For non-small cell lung cancer (NSCLC), representing 85% of all lung cancer cases, several new treatment alternatives have emerged during recent years and may now include, for example, tyrosine kinase inhibitors, targeting tumor-specific mutations/fusion genes, or inhibitors of the immune checkpoint molecules PD1/PD-L1 (programmed death-ligand 1). Today’s therapy decisions thus require thorough molecular analyses to identify clinically relevant alterations. In line with this, the Clinical Practice Guidelines for NSCLC of National Comprehensive Cancer Network (NCCN) 2017 recommend parallel diagnosis, staging, and molecular genetic testing . Being proven as a minimally invasive and effective technique for assessment of the pathology of mediastinal and hilar lymph nodes (LN) along with pulmonary masses proximate to the airway, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has become a preferred investigation method amongst practitioners of pulmonary medicine worldwide. Performed in outpatient settings with high accuracy and minimal complication rate, EBUS-TBNA has been confirmed to be a safe procedure . EBUS-TBNA has now been accepted as a procedure of choice to diagnose and stage locally advanced lung cancer and is recommended by national and international guidelines . Supplementing EBUS-TBNA with transesophageal bronchoscopic ultrasound-guided fine needle aspiration (EUS/EUS-B-FNA) extends the ability to sample multiple intrathoracic LN stations as well as distant metastasis and structures below the diaphragm. Current American College of Chest Physicians (ACCP) lung cancer guidelines recommend EBUS-TBNA and EUS/EUS-B-FNA over invasive mediastinal and surgical staging as the initial staging of NSCLC . In the majority of lung cancer cases, the diagnosis is confirmed by small cytological and/or biopsy specimens, with EBUS-TBNA as a frequent modality of tumor cell acquisition. Studies evaluating EBUS-TBNA in tissue sampling for histopathological diagnostics of lung cancer have demonstrated success rates ranging from 89% to 98% . Molecular analysis can be performed on cytology or biopsy specimens acquired by EBUS-TBNA . The adequacy of EBUS-TBNA samples for molecular testing depends primarily on the absolute number of viable tumor cells, the percentage of tumor cells in the material, and the sensitivity of the particular molecular test . Surveys from different centers demonstrate significant variations of EBUS-TBNA regarding diagnostic yield, sensitivity, negative predictive value, and success rate of genetic testing . Many studies conclude that EBUS-TBNA specimens are sufficient for histological subtyping of NSCLC as well as targeted EGFR mutation and ALK gene fusion analysis . A limited number of studies have evaluated the suitability of EBUS-TBNA specimen for parallel multiple gene alteration analysis . Molecular profiling of lung cancer is today widely performed by massive parallel sequencing (MPS) using targeted gene panels. However, the adequacy of the EBUS-TBNA cytology specimen for analysis of predictive biomarkers using targeted MPS panels is insufficiently explored . This study aims to evaluate the adequacy and sufficiency of EBUS-TBNA cytology specimen for subtyping, molecular genetic profiling, and analysis of PD-L1 in a consecutive series of patients diagnosed with NSCLC. Analytical data A retrospective review of the medical records of all patients examined with flexible and EBUS bronchoscopy as a part of the diagnostic process for suspected or known lung cancer, between January 1, 2017, and April 23, 2018, at the unit for Interventional Pulmonology (IP) at the Department of Respiratory Diseases and Allergology, University Hospital of Skåne, Sweden was performed. The University Hospital of Skåne is providing highly specialized patient care, diagnosis, and treatment for approximately 1.3 million people throughout the southern part of Sweden. Bronchoscopy with EBUS-TBNA was introduced in the IP center in 2004. The IP center is one of Sweden’s largest in volume, with more than 2000 interventions per year: approx. 800 EBUS-TBNA, 300 electromagnetic navigational bronchoscopies, over 100 advanced rigid bronchoscopies with endobronchial therapy and many chest tube insertions, thoracenteses, and medical thoracoscopies. The Regional Ethical Review Board at the University of Lund, Sweden, approved this study (reg nr 2018/730). The Ethical Review Board permitted Marija Karadzovska-Kotevska to extract patients’ data from the medical records. The data set was fully anonymized for all researchers, including Marija Karadzovska-Kotevska, before the initiation of the statistical analysis of the material. Patients’ medical records were accessed between January 2019 and April 2019. Patients included in our study were examined with bronchoscopy with EBUS-TBNA for suspected or known lung cancer at the Unit for Interventional Pulmonology at University Hospital of Skåne in the period between January 2017 and April 2018. The Ethical Review Board did not request participant consent, referring to the study’s retrospective design and that the review of medical records did not imply any risk for injury or discomfort of the participants. All patients had been previously examined with computed tomography of the chest (chest CT), supplemented by positron emission tomography-computed tomography (PET CT) scan when indicated, and had confirmed pulmonary mass and/or mediastinal lymphadenopathy. Subsequently, conventional or advanced flexible bronchoscopies with EBUS-TBNA procedures were performed. We reviewed the outcomes of the pathological assessments of the EBUS-TBNA specimen in all the patients from the study period. To investigate the performance of EBUS-TBNA cytology specimen for histological subtyping of NSCLC and MPS analysis, we included in a consecutive manner all patients where bronchoscopy with EBUS-TBNA revealed metastasis of NSCLC in the examined intrathoracic LN during the study period. The final patient database included: patient’s demographics, clinical characteristics, clinical TNM determined from CT and PET CT scan, the number and location of LN stations sampled, representativity of EBUS-TBNA for every LN position sampled, final histopathological diagnosis, whether mutation/fusion gene status and PD-L1 status were determined on EBUS-TBNA material or other cytology or histology specimen, as well as the portion of bronchoscopies with EBUS-TBNA sampling in which molecular analysis was successful. Procedural data The equipment necessary for the procedure and EBUS-TBNA technique have been fully described by Herth et al. EBUS-TBNA bronchoscopy procedures were performed after topical anesthesia and in monitored moderate conscious sedation with Alfentanil and /or Midazolam commonly in an outpatient setting (Centre for IP in Lund). A convex probe ultrasound flexible video bronchoscope (Olympus BF-UC 180F; Olympus Corp., Tokyo, Japan) was used. Fine needle aspiration was frequently performed with a 22-gauge or sporadically with 21-gauge EBUS-TBNA dedicated needles (Olympus ViziShot, NA-U401SX-4022, and NA-201SX-4022, Olympus Corp.) All the procedures were performed by six operators, four amongst them with many years of experience in the field and two newly trained in the EBUS technique. Each EBUS-TBNA bronchoscopy started with a thorough ultrasound investigation of the intrathoracic LN status. Mediastinal lesions and pathologically enlarged LN, with short-axis larger than 10 mm on CT scan and/or LN with increased metabolic activity on PET CT, were identified and sampled in real-time. Sonographic evaluation of the LN regarding shape, size, margins, echogenicity, presence of hilum, microcalcifications, and necrosis was performed. In concordance with the recommended Guideline for the acquisition and preparation of EBUS-TBNA specimens for diagnostic and molecular testing, 3–5 passes were made per LN station with 10–15 needle revolutions within the lymph node per pass . Rapid on-site evaluation (ROSE) was used during all EBUS-TBNA procedures. Intraprocedural specimen adequacy was defined by a sufficient number of lymphocytes in the smears and determined in all cases by ROSE. In the post-procedural evaluation of smears and ThinPrep ® slides, a representative LN sample exhibited >40 lymphocytes in at least one high-power field, several (5+) clusters of pigmented macrophages, granulomas, or metastasis. Predictive analyses Our department uses a reflex testing approach for lung cancer. Although not mandatory according to guidelines, we aim to perform treatment predictive testing for all advanced NSCLC (also squamous cell carcinomas). The best sample, or samples, from each case, are selected for analysis. Often the cells of a cytological smear are scraped off for MPS while a biopsy is used for diagnostic and predictive IHC, including PD-L1 testing. If no biopsy exists, then a cell block is used for IHC. During Jan 2017 to March 2018, mutations were analyzed with the Ion AmpliSeq ™ Colon and Lung Panel v2 (Thermo Fisher Scientific, Waltham, MA) except in cases with a limited amount of DNA when Therascreen ® EGFR RGQ PCR (Qiagen, Hilden, Germany) was used instead. While ALK and ROS1 fusions were analyzed with IHC for biopsies (clone D5F3, Ventana Medical Systems Inc, Tucson, AZ, and clone D4D6, Cell Signaling Technologies, Leiden, the Netherlands, respectively), FISH was used for cell blocks (Vysis ALK Break Apart FISH Probe and Vysis ROS1 Break Apart FISH Probe, respectively, both Abbott Laboratories, Abbott Park, IL). The reason for this was repeated false-negative ALK IHC with the used preparation method for cytology (fixation with CytoLyt ® and cell block preparation with Cellient ™ ). FISH was also used when IHC staining was inconclusive and to confirm a positive ROS1 staining, while FISH confirmation was not mandatory (and hence not always performed) for a positive IHC staining for ALK during the study period. From March 2018, mutations and fusions were both analyzed with the Oncomine ™ Focus Assay (Thermo Fisher Scientific, Waltham, MA), with PCR for EGFR mutations and IHC and FISH for ALK and ROS1 fusions (as prior to March 2018) as backup methods. PD-L1 was assessed with the 28–8 clone (Abcam, Cambridge, UK) until the end of 2017 and with the 22C3 assay (Agilent/pharmDx, Santa Clara, CA) since the beginning of 2018. For both assays, staining was performed on a Ventana Benchmark Ultra (Ventana Medical Systems Inc, Tucson, AZ) using the OptiView visualization system. Routine mutation, fusion, and PD-L1 analysis at different times during the study period are shown in the . A retrospective review of the medical records of all patients examined with flexible and EBUS bronchoscopy as a part of the diagnostic process for suspected or known lung cancer, between January 1, 2017, and April 23, 2018, at the unit for Interventional Pulmonology (IP) at the Department of Respiratory Diseases and Allergology, University Hospital of Skåne, Sweden was performed. The University Hospital of Skåne is providing highly specialized patient care, diagnosis, and treatment for approximately 1.3 million people throughout the southern part of Sweden. Bronchoscopy with EBUS-TBNA was introduced in the IP center in 2004. The IP center is one of Sweden’s largest in volume, with more than 2000 interventions per year: approx. 800 EBUS-TBNA, 300 electromagnetic navigational bronchoscopies, over 100 advanced rigid bronchoscopies with endobronchial therapy and many chest tube insertions, thoracenteses, and medical thoracoscopies. The Regional Ethical Review Board at the University of Lund, Sweden, approved this study (reg nr 2018/730). The Ethical Review Board permitted Marija Karadzovska-Kotevska to extract patients’ data from the medical records. The data set was fully anonymized for all researchers, including Marija Karadzovska-Kotevska, before the initiation of the statistical analysis of the material. Patients’ medical records were accessed between January 2019 and April 2019. Patients included in our study were examined with bronchoscopy with EBUS-TBNA for suspected or known lung cancer at the Unit for Interventional Pulmonology at University Hospital of Skåne in the period between January 2017 and April 2018. The Ethical Review Board did not request participant consent, referring to the study’s retrospective design and that the review of medical records did not imply any risk for injury or discomfort of the participants. All patients had been previously examined with computed tomography of the chest (chest CT), supplemented by positron emission tomography-computed tomography (PET CT) scan when indicated, and had confirmed pulmonary mass and/or mediastinal lymphadenopathy. Subsequently, conventional or advanced flexible bronchoscopies with EBUS-TBNA procedures were performed. We reviewed the outcomes of the pathological assessments of the EBUS-TBNA specimen in all the patients from the study period. To investigate the performance of EBUS-TBNA cytology specimen for histological subtyping of NSCLC and MPS analysis, we included in a consecutive manner all patients where bronchoscopy with EBUS-TBNA revealed metastasis of NSCLC in the examined intrathoracic LN during the study period. The final patient database included: patient’s demographics, clinical characteristics, clinical TNM determined from CT and PET CT scan, the number and location of LN stations sampled, representativity of EBUS-TBNA for every LN position sampled, final histopathological diagnosis, whether mutation/fusion gene status and PD-L1 status were determined on EBUS-TBNA material or other cytology or histology specimen, as well as the portion of bronchoscopies with EBUS-TBNA sampling in which molecular analysis was successful. The equipment necessary for the procedure and EBUS-TBNA technique have been fully described by Herth et al. EBUS-TBNA bronchoscopy procedures were performed after topical anesthesia and in monitored moderate conscious sedation with Alfentanil and /or Midazolam commonly in an outpatient setting (Centre for IP in Lund). A convex probe ultrasound flexible video bronchoscope (Olympus BF-UC 180F; Olympus Corp., Tokyo, Japan) was used. Fine needle aspiration was frequently performed with a 22-gauge or sporadically with 21-gauge EBUS-TBNA dedicated needles (Olympus ViziShot, NA-U401SX-4022, and NA-201SX-4022, Olympus Corp.) All the procedures were performed by six operators, four amongst them with many years of experience in the field and two newly trained in the EBUS technique. Each EBUS-TBNA bronchoscopy started with a thorough ultrasound investigation of the intrathoracic LN status. Mediastinal lesions and pathologically enlarged LN, with short-axis larger than 10 mm on CT scan and/or LN with increased metabolic activity on PET CT, were identified and sampled in real-time. Sonographic evaluation of the LN regarding shape, size, margins, echogenicity, presence of hilum, microcalcifications, and necrosis was performed. In concordance with the recommended Guideline for the acquisition and preparation of EBUS-TBNA specimens for diagnostic and molecular testing, 3–5 passes were made per LN station with 10–15 needle revolutions within the lymph node per pass . Rapid on-site evaluation (ROSE) was used during all EBUS-TBNA procedures. Intraprocedural specimen adequacy was defined by a sufficient number of lymphocytes in the smears and determined in all cases by ROSE. In the post-procedural evaluation of smears and ThinPrep ® slides, a representative LN sample exhibited >40 lymphocytes in at least one high-power field, several (5+) clusters of pigmented macrophages, granulomas, or metastasis. Our department uses a reflex testing approach for lung cancer. Although not mandatory according to guidelines, we aim to perform treatment predictive testing for all advanced NSCLC (also squamous cell carcinomas). The best sample, or samples, from each case, are selected for analysis. Often the cells of a cytological smear are scraped off for MPS while a biopsy is used for diagnostic and predictive IHC, including PD-L1 testing. If no biopsy exists, then a cell block is used for IHC. During Jan 2017 to March 2018, mutations were analyzed with the Ion AmpliSeq ™ Colon and Lung Panel v2 (Thermo Fisher Scientific, Waltham, MA) except in cases with a limited amount of DNA when Therascreen ® EGFR RGQ PCR (Qiagen, Hilden, Germany) was used instead. While ALK and ROS1 fusions were analyzed with IHC for biopsies (clone D5F3, Ventana Medical Systems Inc, Tucson, AZ, and clone D4D6, Cell Signaling Technologies, Leiden, the Netherlands, respectively), FISH was used for cell blocks (Vysis ALK Break Apart FISH Probe and Vysis ROS1 Break Apart FISH Probe, respectively, both Abbott Laboratories, Abbott Park, IL). The reason for this was repeated false-negative ALK IHC with the used preparation method for cytology (fixation with CytoLyt ® and cell block preparation with Cellient ™ ). FISH was also used when IHC staining was inconclusive and to confirm a positive ROS1 staining, while FISH confirmation was not mandatory (and hence not always performed) for a positive IHC staining for ALK during the study period. From March 2018, mutations and fusions were both analyzed with the Oncomine ™ Focus Assay (Thermo Fisher Scientific, Waltham, MA), with PCR for EGFR mutations and IHC and FISH for ALK and ROS1 fusions (as prior to March 2018) as backup methods. PD-L1 was assessed with the 28–8 clone (Abcam, Cambridge, UK) until the end of 2017 and with the 22C3 assay (Agilent/pharmDx, Santa Clara, CA) since the beginning of 2018. For both assays, staining was performed on a Ventana Benchmark Ultra (Ventana Medical Systems Inc, Tucson, AZ) using the OptiView visualization system. Routine mutation, fusion, and PD-L1 analysis at different times during the study period are shown in the . During the study period (January 1, 2017, to April 23, 2018), there were 806 EBUS procedures performed on 765 patients, aiming to diagnose and/or stage suspect or known lung cancer. 296 diagnostic EBUS-TBNA, 260 staging EBUS-TBNA, and 250 radial EBUS procedures were conducted. In patients with concomitant mediastinal lymphadenopathy and lung lesions reachable by radial EBUS, linear EBUS was performed immediately after the radial EBUS, during the same bronchoscopy procedure. The outcomes of the bronchoscopy with radial and/or linear EBUS procedures resulted in diagnosing lung cancer in 209/765 (27%), 174 with NSCLC, 26 with small cell lung cancer (SCLC), and 9 with carcinoids. (Shown in ). In 132 of 174 NSCLC patients (76%), we detected cancer cells in the fine needle aspirate from EBUS-TBNA. These patients thus fulfilled the criteria for being included in a consecutive manner in our cohort. Baseline characteristics are shown in . The majority (95%) of patients were newly diagnosed, whereas relapse or progression of cancer disease and the need for accurate histopathology and molecular status was the reason for EBUS-TBNA in six patients (5%). Initial examination with chest CT and PET CT had classified 63 of 132 patients (48%) in clinical TNM stage III and 61 patients (46%) in clinical TNM stage IV according to the current eighth edition of TNM. The tumors were localized predominantly in the right (35%) or left (17%) upper lobes. In all, 140 bronchoscopies with EBUS were performed in the 132 patients (Diagnostic EBUS bronchoscopy was reattempted in four patients, and two more attempts were made in two patients to establish a diagnosis). The distribution of the tumor lesions according to chest CT and PET CT and sampled LN are shown in . We have analyzed all histological/cytological slides for the adenocarcinoma cases for subtyping. There was a surgical specimen for 15 of the cases, and for the remaining, there was a biopsy (with/without cytology) in 39 and only cytology in 23 cases. Three cases were mixed mucinous/non-mucinous (one surgical resection and two biopsies), while the other cases were non-mucinous. All three with mixed growth pattern were EGFR and ROS 1 negative, one was ALK positive, and one PD-L1 positive (1–4%). Histologic subtypes are reported in . In total, 376 LN stations were sampled in 129 of the 132 patients during 140 EBUS-TBNA procedures (in three patients, EBUS-TBNA specimens were collected only from tumor lesions adjacent to the main bronchi). In 32 patients, only one LN was sampled, whereas 2 LN stations were sampled in 28 patients (22%), 3 LN stations in 32 (24%), 4 LN stations in 33 (25%), and 5 LN stations were sampled in 12 patients (9%). The most frequently sampled position was station 7, followed by 4R and 4L. In 126 out of 140 procedures, all sampled LN stations were representative of lymphoid tissue. In 11 procedures (8%), the EBUS-TBNA aspirate was representative from all but 1 LN station. NSCLC subtyping and molecular analysis from EBUS-TBNA specimen In our statistical analysis for the adequacy of the EBUS-TBNA specimen for molecular analysis, we included in a consecutive manner all patients in which the EBUS-TBNA specimen showed cancer cells from NSCLC. We analyzed the outcome of 96 EBUS specimens (out of 140 EBUS bronchoscopies performed in the cohort). We omitted the ones that were performed with the purpose of mediastinal staging and had diagnosis established previously (13 cases), the ones where molecular analysis was attempted on other specimens from the bronchoscopy as bronchial brushes and washes and biopsies (23 cases), and finally those with cellblock from pleural fluid, liver biopsy as well as previous surgery (8 cases). The choice of the tumor specimen (EBUS cytology, bronchial brush/wash or biopsy) for tumor subtyping and molecular analysis was made at the pathologist’s discretion. Final histologic diagnosis and subtyping of NSCLC by IHC on EBUS-TBNA specimen was obtained in 66 of 80 executed analyses (83%). Molecular analysis by a panel for MPS was attempted on aspirate from EBUS-TBNA in 96 cases and proved sufficient in 51 (53%). Furthermore, partial genetic analyses ( EGFR PCR, ALK IHC and/or FISH, ROS1 IHC and/or FISH) were accomplished in an additional 23 out of 96 cases (24%), thus making the EBUS-TBNA aspirate sufficient for analysis of treatment predictive biomarkers in 74/96 cases (77%) of all tested EBUS-TBNA specimens in the cohort. EBUS-TBNA specimen was insufficient for genetic testing in 22 patients (23%). The EBUS-TBNA specimen showed to be adequate for analysis of EGFR gene mutation, ALK fusion, ROS1 rearrangement, and PD-L1 in 69%, 49%, 36%, and 33%, respectively, from the total number of tested EBUS-TBNA samples, as shown in . The prevalence of EGFR gene mutation in the study group was 11% (14/132). We detected exon 19 deletions in five and L858R mutations in seven patients. Two cases harbored EGFR exon 20 mutations. ALK fusions were identified in four patients (3%), all with lung adenocarcinoma. ROS1 rearrangement was found in one patient (1%). When molecular analysis on air-dried smears from bronchial brushes or tissue from bronchial biopsies sampled during the same bronchoscopy session as EBUS-TBNA was taken into account, an additional 23 cases had complete molecular analysis. As a result, the cumulative adequacy ratio for complete molecular tumor profiling by MPS from the two procedures performed at the same diagnostic session (flexible bronchoscopy and EBUS-TBNA) was 77%. provides a comprehensive overview of the performed IHC, MPS, and targeted driver oncogenes analyses in the cohort group. It demonstrates the adequacy of the EBUS-TBNA cytology sample for subtyping and genetic testing of NSCLC compared to other cytology and biopsy samples collected in the cohort group. NSCLC subtyping and molecular analysis from other tumor specimens in the cohort group Besides the focus on EBUS-TBNA specimens, we reviewed patient records regarding all other tumor specimens, cytology, and biopsies used for molecular testing to demonstrate the proportion of patients in our cohort group with complete genetic tumor profiling before the treatment start. A total number of 493 analyses (MPS, EGFR PCR, ALK IHC and/or FISH, ROS1 IHC and/or FISH, PD-L1 IHC) of predictive biomarkers were performed on air-dried smears, cellblocks, and biopsy tissue in the study group (all procedures included: EBUS cytology, bronchial brushes and forceps biopsies from bronchoscopy, transthoracic tumor biopsy or tissue from previous or later operation). In 102 and 17 patients out of 132 in our cohort group, a complete or partial molecular analysis was performed, respectively (all procedures included). Molecular analyses were not successful in 11 patients. For two patients with squamous cell lung cancer, the cytology material was not sent to molecular analysis. Additionally, we examined patient records concerning given antitumoral treatment after diagnosis. In 12 out of 14 patients with detected EGFR mutation and three out of four patients with detected ALK fusion that were classified as stage IV lung cancer disease after diagnosis, has the analysis of treatment predictive biomarkers led to antitumoral treatment with tyrosine kinase inhibitor in the first-line setting. Two patients with locally advanced cancer disease, harboring EGFR mutation received concurrent chemoradiation therapy according to the current standard of care. One patient harboring ALK fusion was treated with neoadjuvant chemoradiation therapy in a preoperative setting. The cumulative adequacy and outcome of all tested tumor samples in the cohort for the treatment predictive biomarkers and PD-L1 of two of the cohort subgroups, NSCLC adenocarcinoma, respectively NSCLC NOS, are shown in . Complications occurred in 3.6% (5/140) of performed EBUS-TBNA procedures. Postprocedural minor hemoptysis was described in one patient. Two patients reported mild fever a few days after the procedure that resolved spontaneously without any medical intervention. Two patients were hospitalized, one due to sepsis and the other one for mediastinitis, both recovered fully after antibiotic treatment. One patient died 3 days after EBUS-TBNA. The patient was a 77-year-old male with COPD who presented with stage IV lung adenocarcinoma and was admitted to the hospital prior to EBUS-TBNA due to severe dyspnea. The EBUS-TBNA procedure was uncomplicated, but the patient became disoriented the next day. The patient rapidly deteriorated despite the administration of broad-spectrum antibiotics, became unconscious, and died within 72 hours of EBUS-TBNA. The scenario was attributed to sepsis even though the blood culture was negative. A possible differential diagnosis could have been paraneoplastic encephalitis. However, the family strongly opposed an autopsy, and the procedure was not performed. In our statistical analysis for the adequacy of the EBUS-TBNA specimen for molecular analysis, we included in a consecutive manner all patients in which the EBUS-TBNA specimen showed cancer cells from NSCLC. We analyzed the outcome of 96 EBUS specimens (out of 140 EBUS bronchoscopies performed in the cohort). We omitted the ones that were performed with the purpose of mediastinal staging and had diagnosis established previously (13 cases), the ones where molecular analysis was attempted on other specimens from the bronchoscopy as bronchial brushes and washes and biopsies (23 cases), and finally those with cellblock from pleural fluid, liver biopsy as well as previous surgery (8 cases). The choice of the tumor specimen (EBUS cytology, bronchial brush/wash or biopsy) for tumor subtyping and molecular analysis was made at the pathologist’s discretion. Final histologic diagnosis and subtyping of NSCLC by IHC on EBUS-TBNA specimen was obtained in 66 of 80 executed analyses (83%). Molecular analysis by a panel for MPS was attempted on aspirate from EBUS-TBNA in 96 cases and proved sufficient in 51 (53%). Furthermore, partial genetic analyses ( EGFR PCR, ALK IHC and/or FISH, ROS1 IHC and/or FISH) were accomplished in an additional 23 out of 96 cases (24%), thus making the EBUS-TBNA aspirate sufficient for analysis of treatment predictive biomarkers in 74/96 cases (77%) of all tested EBUS-TBNA specimens in the cohort. EBUS-TBNA specimen was insufficient for genetic testing in 22 patients (23%). The EBUS-TBNA specimen showed to be adequate for analysis of EGFR gene mutation, ALK fusion, ROS1 rearrangement, and PD-L1 in 69%, 49%, 36%, and 33%, respectively, from the total number of tested EBUS-TBNA samples, as shown in . The prevalence of EGFR gene mutation in the study group was 11% (14/132). We detected exon 19 deletions in five and L858R mutations in seven patients. Two cases harbored EGFR exon 20 mutations. ALK fusions were identified in four patients (3%), all with lung adenocarcinoma. ROS1 rearrangement was found in one patient (1%). When molecular analysis on air-dried smears from bronchial brushes or tissue from bronchial biopsies sampled during the same bronchoscopy session as EBUS-TBNA was taken into account, an additional 23 cases had complete molecular analysis. As a result, the cumulative adequacy ratio for complete molecular tumor profiling by MPS from the two procedures performed at the same diagnostic session (flexible bronchoscopy and EBUS-TBNA) was 77%. provides a comprehensive overview of the performed IHC, MPS, and targeted driver oncogenes analyses in the cohort group. It demonstrates the adequacy of the EBUS-TBNA cytology sample for subtyping and genetic testing of NSCLC compared to other cytology and biopsy samples collected in the cohort group. Besides the focus on EBUS-TBNA specimens, we reviewed patient records regarding all other tumor specimens, cytology, and biopsies used for molecular testing to demonstrate the proportion of patients in our cohort group with complete genetic tumor profiling before the treatment start. A total number of 493 analyses (MPS, EGFR PCR, ALK IHC and/or FISH, ROS1 IHC and/or FISH, PD-L1 IHC) of predictive biomarkers were performed on air-dried smears, cellblocks, and biopsy tissue in the study group (all procedures included: EBUS cytology, bronchial brushes and forceps biopsies from bronchoscopy, transthoracic tumor biopsy or tissue from previous or later operation). In 102 and 17 patients out of 132 in our cohort group, a complete or partial molecular analysis was performed, respectively (all procedures included). Molecular analyses were not successful in 11 patients. For two patients with squamous cell lung cancer, the cytology material was not sent to molecular analysis. Additionally, we examined patient records concerning given antitumoral treatment after diagnosis. In 12 out of 14 patients with detected EGFR mutation and three out of four patients with detected ALK fusion that were classified as stage IV lung cancer disease after diagnosis, has the analysis of treatment predictive biomarkers led to antitumoral treatment with tyrosine kinase inhibitor in the first-line setting. Two patients with locally advanced cancer disease, harboring EGFR mutation received concurrent chemoradiation therapy according to the current standard of care. One patient harboring ALK fusion was treated with neoadjuvant chemoradiation therapy in a preoperative setting. The cumulative adequacy and outcome of all tested tumor samples in the cohort for the treatment predictive biomarkers and PD-L1 of two of the cohort subgroups, NSCLC adenocarcinoma, respectively NSCLC NOS, are shown in . Complications occurred in 3.6% (5/140) of performed EBUS-TBNA procedures. Postprocedural minor hemoptysis was described in one patient. Two patients reported mild fever a few days after the procedure that resolved spontaneously without any medical intervention. Two patients were hospitalized, one due to sepsis and the other one for mediastinitis, both recovered fully after antibiotic treatment. One patient died 3 days after EBUS-TBNA. The patient was a 77-year-old male with COPD who presented with stage IV lung adenocarcinoma and was admitted to the hospital prior to EBUS-TBNA due to severe dyspnea. The EBUS-TBNA procedure was uncomplicated, but the patient became disoriented the next day. The patient rapidly deteriorated despite the administration of broad-spectrum antibiotics, became unconscious, and died within 72 hours of EBUS-TBNA. The scenario was attributed to sepsis even though the blood culture was negative. A possible differential diagnosis could have been paraneoplastic encephalitis. However, the family strongly opposed an autopsy, and the procedure was not performed. Present and future novel therapeutic possibilities for lung cancer are persistently challenging the process of genomic profiling of NSCLC, making adequate tissue material for molecular analysis essential. Growing international literature supports the hypothesis that minimally invasive procedures as EBUS-TBNA are sufficient for both diagnosis and genetic testing of NSCLC. However, study results have also shown discrepancies in the diagnostic yield and accuracy of EBUS-TBNA for diagnosis and molecular analysis of NSCLC, thus raising the question of generalizability . In our single-center retrospective study, EBUS bronchoscopy as a part of the initial investigational procedure for suspected lung cancer led to a diagnosis and NSCLC subtyping in 83%, and successful analysis of treatment predictive biomarkers in 77% of all analyzed EBUS samples. Importantly, we also confirm that the majority of EBUS-TBNA cytology specimens can support parallel diagnostics, subtyping and molecular characterization of NSCLC. EBUS-TBNA cytology for molecular diagnostics The EBUS-TBNA cytology samples allowed histological subtyping and successful genomic profiling of NSCLC by MPS concurrently in 53% of all analyzed samples. Partial genomic profiling (PCR EGFR and/or FISH/IHC ALK and/or ROS1 ) was obtained in an additional 23 cases (24%), seven of which were squamous cell carcinomas where EGFR status alone was considered sufficient for treatment decision. Rooper et al. presented similar results where simultaneous subtyping and molecular diagnostics were possible in 57.9% of the patients . EBUS-TBNA specimen was insufficient for genetic testing (by MPS or PCR EGFR and/or FISH/IHC ALK and/or ROS1) in 22 of 96 cases (23%) in our study. When reviewing the medical records of those 22 patients aiming to assess if technical issues might have led to poor specimen quality, we could not find anything remarkable other than NSCLC subtyping by IHC has been successfully conducted previously in 8 of total 22 cases (36%) where the EBUS specimen proved insufficient for genetic testing. Several studies have earlier advised moderation in immunohistochemical analysis to preserve tumor specimens to test predictive tumor biomarkers . We further analyzed if there was a correlation between the outcome of the EBUS-TBNA procedure regarding the sufficiency of the EBUS specimen for MPS analysis and the experience of the bronchoscopist. 88 out of 96 EBUS-TBNA procedures were performed by experienced bronchoscopists and 8 by bronchoscopists in training. 47/88 samples obtained by experienced and 4/8 by bronchoscopist in training resulted in an adequate sample for complete MPS analysis. In 21/88 and 2/8, a partial analysis of predictive biomarkers could be performed. 20/88 samples acquired by experienced vs. 2/8 by bronchoscopist in training were insufficient for MPS analysis. The proportion of inadequate EBUS specimens for MPS analysis did not differ significantly between those two subgroups. However, the ratio of procedures performed by inexperienced EBUS bronchoscopists was substantially lower, thus restricting from definite conclusions and requiring further investigation. Treatment predictive markers Fine needle aspirate obtained from LN and lesions adjacent to the main bronchi via EBUS-TBNA was found suitable for the evaluation of EGFR (MPS or PCR) in 69% (66/96) against overall adequacy of 80% for all diagnostic procedures in the cohort group. Success rates for EGFR analysis comparable to our study were demonstrated by both Schuurbiers et al. with 77% and by Garcia-Olivé et al. with 72.2% of EBUS cytology samples sufficient for EGFR evaluation . Navani et al. presented much higher adequacy, with 90% of EBUS specimen sufficient for EGFR analysis . The EGFR mutation prevalence of 11% in our cohort is consistent with the findings in several studies analyzed in a systematic review and meta-analysis presented by Labarca et al. . Our results are coherent with the findings by Isaksson et al. who reported an EGFR prevalence of 10% in the 519 NSCLC patients subjected to the first 1.5 years of treatment predictive MPS testing in our health care region (2015 to mid- 2016) . The suitability of the EBUS-TBNA cytology specimen in our study for ALK analysis by MPS or FISH was 49% (38/77 samples) compared to a 69% adequacy when considering all specimens from every diagnostic procedure conducted in the cohort. The prevalence of ALK fusions was 3%, as could be expected from previous studies presented in a systematic review and meta-analysis presented by Labarca et al. . In our study, ROS1 analysis by IHC/FISH was attempted on 65 EBUS fine needle aspirate specimens. Reliable analysis could be performed in 36% (22/61 samples) presenting ROS1 positivity in only one case (1%). At present, there are only a few reports on the adequacy ratio of EBUS specimen for ROS1 and PD-L1 analysis . Fernandes-Bussy et al. conducted a study with 86 cases and found an 83% adequacy ratio for ROS1 (10/12 samples) . Cicek et al presented a study cohort of 114 patients where ROS1 analysis could be attempted in 98 cases with a 91% adequacy of EBUS specimens for ROS1 analysis . Evaluation of PD-L1 expression in lung cancer has mainly been performed on biopsy tissue specimens. The role of EBUS-TBNA in the analysis of PD-L1 is unclear and present evidence insufficient . A very limited number of studies has been conducted, cautiously indicating that an EBUS-TBNA cytology specimen can support PD-L1 testing and quantification and demonstrating satisfactory PD-L1 outcome concordance between cytology and biopsy specimens . In our cohort, 128 attempts for PD-L1 evaluation were made on various specimens (cytology, biopsies), showing positive findings (PD-L1 staining in 2–99% of tumor cells) in 44 attempts (35%). However, EBUS-TBNA cytology specimens were used for PD-L1 analysis in only 69 cases, with positive PD-L1 findings in 13 cases. These numbers are lower than seen in large consecutive series and merit further investigation . Clinical relevance of the study Our study confirms that diagnostic EBUS-TBNA can be an appropriate procedure for adequate diagnosis, subtyping and genetic testing of NSCLC by MPS. However, the adequacy of the EBUS specimen in our study was lower compared to the outcomes of a few other reported studies . Interestingly, in a recent review article, 33 individual studies were analyzed, and a quality assessment found a high risk of bias for selectively reported results in 17/33 studies . According to this report, only two studies were dedicated to the utility of EBUS-TBNA specimen for MPS analysis, both, however, with data insufficient to be included in the meta-analysis . Our approach with unselective inclusion of all assessed EBUS specimens aimed to reflect the everyday reality in most hospitals. We also decided to take an entire consecutive cohort of patients and procedures performed by all our six bronchoscopists with varying levels of expertise, 4/6 with many years of experience in interventional pulmonology whereas 2/6 under training. We believe that this approach of a mixture of expertise levels could allow generalization of our results to the real-world context, which in particular is referring to community hospitals worldwide with a lower volume of EBUS procedures where a considerable quantity of training period is involved . Several previous studies have shown successful MPS on cell block samples collected by EBUS bronchoscopy. A recently published study with a similar cohort size to our study demonstrated that EBUS-TBNA specimen is adequate for genetic profiling of thoracic malignancies by a panel for MPS performed on paraffin-embedded cellblocks in 93% of the cases. The adequacy in the study period improved from 76.3% in the first third of cases to 92.3% for the final one-third of the cases . However, according to our knowledge, our study is one of very few that has in a novel manner (large consecutive cohort) focused on the assessment of large patient series regarding the feasibility of the cytology smears obtained by EBUS-TBNA for molecular cancer profiling by panels for MPS. Our patient material represents the first evaluation of the outcomes from EBUS-TBNA procedures for diagnostics and molecular profiling of NSCLC in a single high-volume academic IP center in Sweden and consists of a genuinely unselective and consecutive series of all patients referred to our IP center for advanced bronchoscopy procedure with EBUS. Importantly, we included in the study all EBUS specimens that showed cancer cells from NSCLC in an indiscriminating manner, aiming to minimize the selection bias. Our study findings support the benefit of conventional and advanced bronchoscopy combined with EBUS-TBNA performed in one session as a fist-line procedure for prompt and simultaneous diagnosis, precise staging, and genomic profiling of lung cancer. We can also confirm that the analysis of predictive biomarkers is fundamental in process of cancer treatment decision-making. In our patient cohort, the detection of targetable tumor alterations ( EGFR mutation and ALK fusion) has led to treatment with EGFR/ALK specific tyrosine kinase inhibitor in all stage IV patients according to the present-day recommendations. Certain limitations apply to this study. First, we acknowledge the restrictions applicable to retrospective studies. Furthermore, in our real-world approach, we did not re-assess all slides from the EBUS procedures to see if molecular analysis could have also been performed in cases where the clinical pathologist had selected a biopsy for the analysis, nor did we re-evaluate the slides of the group of insufficient ones. In summary, we believe that the results of our study strengthen the evidence that cytology smears provide a reliable sample with good DNA quality for MPS testing and highlight the need for future studies assessing the suitability of EBUS-TBNA specimen for MPS in a prospective setting. The EBUS-TBNA cytology samples allowed histological subtyping and successful genomic profiling of NSCLC by MPS concurrently in 53% of all analyzed samples. Partial genomic profiling (PCR EGFR and/or FISH/IHC ALK and/or ROS1 ) was obtained in an additional 23 cases (24%), seven of which were squamous cell carcinomas where EGFR status alone was considered sufficient for treatment decision. Rooper et al. presented similar results where simultaneous subtyping and molecular diagnostics were possible in 57.9% of the patients . EBUS-TBNA specimen was insufficient for genetic testing (by MPS or PCR EGFR and/or FISH/IHC ALK and/or ROS1) in 22 of 96 cases (23%) in our study. When reviewing the medical records of those 22 patients aiming to assess if technical issues might have led to poor specimen quality, we could not find anything remarkable other than NSCLC subtyping by IHC has been successfully conducted previously in 8 of total 22 cases (36%) where the EBUS specimen proved insufficient for genetic testing. Several studies have earlier advised moderation in immunohistochemical analysis to preserve tumor specimens to test predictive tumor biomarkers . We further analyzed if there was a correlation between the outcome of the EBUS-TBNA procedure regarding the sufficiency of the EBUS specimen for MPS analysis and the experience of the bronchoscopist. 88 out of 96 EBUS-TBNA procedures were performed by experienced bronchoscopists and 8 by bronchoscopists in training. 47/88 samples obtained by experienced and 4/8 by bronchoscopist in training resulted in an adequate sample for complete MPS analysis. In 21/88 and 2/8, a partial analysis of predictive biomarkers could be performed. 20/88 samples acquired by experienced vs. 2/8 by bronchoscopist in training were insufficient for MPS analysis. The proportion of inadequate EBUS specimens for MPS analysis did not differ significantly between those two subgroups. However, the ratio of procedures performed by inexperienced EBUS bronchoscopists was substantially lower, thus restricting from definite conclusions and requiring further investigation. Fine needle aspirate obtained from LN and lesions adjacent to the main bronchi via EBUS-TBNA was found suitable for the evaluation of EGFR (MPS or PCR) in 69% (66/96) against overall adequacy of 80% for all diagnostic procedures in the cohort group. Success rates for EGFR analysis comparable to our study were demonstrated by both Schuurbiers et al. with 77% and by Garcia-Olivé et al. with 72.2% of EBUS cytology samples sufficient for EGFR evaluation . Navani et al. presented much higher adequacy, with 90% of EBUS specimen sufficient for EGFR analysis . The EGFR mutation prevalence of 11% in our cohort is consistent with the findings in several studies analyzed in a systematic review and meta-analysis presented by Labarca et al. . Our results are coherent with the findings by Isaksson et al. who reported an EGFR prevalence of 10% in the 519 NSCLC patients subjected to the first 1.5 years of treatment predictive MPS testing in our health care region (2015 to mid- 2016) . The suitability of the EBUS-TBNA cytology specimen in our study for ALK analysis by MPS or FISH was 49% (38/77 samples) compared to a 69% adequacy when considering all specimens from every diagnostic procedure conducted in the cohort. The prevalence of ALK fusions was 3%, as could be expected from previous studies presented in a systematic review and meta-analysis presented by Labarca et al. . In our study, ROS1 analysis by IHC/FISH was attempted on 65 EBUS fine needle aspirate specimens. Reliable analysis could be performed in 36% (22/61 samples) presenting ROS1 positivity in only one case (1%). At present, there are only a few reports on the adequacy ratio of EBUS specimen for ROS1 and PD-L1 analysis . Fernandes-Bussy et al. conducted a study with 86 cases and found an 83% adequacy ratio for ROS1 (10/12 samples) . Cicek et al presented a study cohort of 114 patients where ROS1 analysis could be attempted in 98 cases with a 91% adequacy of EBUS specimens for ROS1 analysis . Evaluation of PD-L1 expression in lung cancer has mainly been performed on biopsy tissue specimens. The role of EBUS-TBNA in the analysis of PD-L1 is unclear and present evidence insufficient . A very limited number of studies has been conducted, cautiously indicating that an EBUS-TBNA cytology specimen can support PD-L1 testing and quantification and demonstrating satisfactory PD-L1 outcome concordance between cytology and biopsy specimens . In our cohort, 128 attempts for PD-L1 evaluation were made on various specimens (cytology, biopsies), showing positive findings (PD-L1 staining in 2–99% of tumor cells) in 44 attempts (35%). However, EBUS-TBNA cytology specimens were used for PD-L1 analysis in only 69 cases, with positive PD-L1 findings in 13 cases. These numbers are lower than seen in large consecutive series and merit further investigation . Our study confirms that diagnostic EBUS-TBNA can be an appropriate procedure for adequate diagnosis, subtyping and genetic testing of NSCLC by MPS. However, the adequacy of the EBUS specimen in our study was lower compared to the outcomes of a few other reported studies . Interestingly, in a recent review article, 33 individual studies were analyzed, and a quality assessment found a high risk of bias for selectively reported results in 17/33 studies . According to this report, only two studies were dedicated to the utility of EBUS-TBNA specimen for MPS analysis, both, however, with data insufficient to be included in the meta-analysis . Our approach with unselective inclusion of all assessed EBUS specimens aimed to reflect the everyday reality in most hospitals. We also decided to take an entire consecutive cohort of patients and procedures performed by all our six bronchoscopists with varying levels of expertise, 4/6 with many years of experience in interventional pulmonology whereas 2/6 under training. We believe that this approach of a mixture of expertise levels could allow generalization of our results to the real-world context, which in particular is referring to community hospitals worldwide with a lower volume of EBUS procedures where a considerable quantity of training period is involved . Several previous studies have shown successful MPS on cell block samples collected by EBUS bronchoscopy. A recently published study with a similar cohort size to our study demonstrated that EBUS-TBNA specimen is adequate for genetic profiling of thoracic malignancies by a panel for MPS performed on paraffin-embedded cellblocks in 93% of the cases. The adequacy in the study period improved from 76.3% in the first third of cases to 92.3% for the final one-third of the cases . However, according to our knowledge, our study is one of very few that has in a novel manner (large consecutive cohort) focused on the assessment of large patient series regarding the feasibility of the cytology smears obtained by EBUS-TBNA for molecular cancer profiling by panels for MPS. Our patient material represents the first evaluation of the outcomes from EBUS-TBNA procedures for diagnostics and molecular profiling of NSCLC in a single high-volume academic IP center in Sweden and consists of a genuinely unselective and consecutive series of all patients referred to our IP center for advanced bronchoscopy procedure with EBUS. Importantly, we included in the study all EBUS specimens that showed cancer cells from NSCLC in an indiscriminating manner, aiming to minimize the selection bias. Our study findings support the benefit of conventional and advanced bronchoscopy combined with EBUS-TBNA performed in one session as a fist-line procedure for prompt and simultaneous diagnosis, precise staging, and genomic profiling of lung cancer. We can also confirm that the analysis of predictive biomarkers is fundamental in process of cancer treatment decision-making. In our patient cohort, the detection of targetable tumor alterations ( EGFR mutation and ALK fusion) has led to treatment with EGFR/ALK specific tyrosine kinase inhibitor in all stage IV patients according to the present-day recommendations. Certain limitations apply to this study. First, we acknowledge the restrictions applicable to retrospective studies. Furthermore, in our real-world approach, we did not re-assess all slides from the EBUS procedures to see if molecular analysis could have also been performed in cases where the clinical pathologist had selected a biopsy for the analysis, nor did we re-evaluate the slides of the group of insufficient ones. In summary, we believe that the results of our study strengthen the evidence that cytology smears provide a reliable sample with good DNA quality for MPS testing and highlight the need for future studies assessing the suitability of EBUS-TBNA specimen for MPS in a prospective setting. Our results confirm bronchoscopy with EBUS-TBNA as a safe, and minimally invasive procedure of first choice with a central role in the demanding diagnostic and staging workout for lung cancer. Although more data is needed on the utility of EBUS-TBNA cytology specimen for MPS and PD-L1 analysis, EBUS cytology aspirates appear to be reliable for diagnosing and subtyping NSCLC and abundantly for treatment predictive molecular testing. S1 File Routine mutation, fusion, and PD-L1 analysis at different times (the years of inclusion in the study were 01/01/2017-23/04/2018). Note that for NGS and PCR the best material based on tumor cell content and fraction was selected regardless of specimen type. (DOCX) Click here for additional data file. S1 Data Anonymized study dataset. Feasibility of EBUS-TBNA for histopathological and molecular diagnostics of NSCLC—a retrospective single-center experience. (XLSX) Click here for additional data file. |
Dysfunction in mitochondrial electron transport chain drives the pathogenesis of pulmonary arterial hypertension: insights from a multi-omics investigation | ef3d1f6d-8616-417b-b9ef-0e313a8006ff | 11749457 | Biochemistry[mh] | Pulmonary arterial hypertension (PAH) is a progressive disorder with vascular remodeling of pulmonary arteries and elevated pulmonary vascular resistance, posing a significant risk of right ventricular failure and potential mortality . The estimated global prevalence of PAH is 15–50 cases per million individuals . Despite the existence of therapeutic medications targeting prostacyclin and endothelin pathways for PAH, the prognosis of newly diagnosed PAH patients remains suboptimal, with a 5-year survival rate of 61.2% reported by the REVEAL registry, underscoring the need for improved outcomes in this population . Therefore, further elucidation of the molecular mechanisms underlying PAH pathophysiology could potentially advance therapeutic development and improve the prognosis of PAH patients. As a multifactorial disease, the pathogenesis of PAH has been extensively explored. Endothelial dysfunction is hypothesized to play a key role in PAH, as long-term overexpression of vasoconstrictors such as endothelin-1 could exert effects on vascular tone and accelerate vascular remodeling . Inflammation and immune dysregulation are critical factors in the pathogenic mechanisms of PAH as well, indicated by the growing evidence of accumulated inflammatory and immune cells in PAH lungs and walls of remodeled pulmonary vessels . The occurrence and development of PAH is also closely linked with mitochondrial dysfunction. In PAH, altered mitochondrial dynamics have been implicated in promoting cell proliferation/resistance to apoptosis phenotype in pulmonary arterial cells . Furthermore, abnormal expression of reactive oxygen species (ROS), which are mainly produced by mitochondria, have been observed in PAH patients . Mitochondrial dysfunction is known to be associated with dysregulated metabolism, specifically characterized by a metabolic shift from oxidative phosphorylation to glycolysis . However, the specific mitochondrial abnormalities that exert greater impact on the etiology of PAH have yet to be fully comprehended. The elucidation of the pivotal pathogenic mechanisms is crucial for developing more effective treatments for PAH. The primary objectives of this study were to explore the key alterations in mitochondrial function and metabolic profiles in PAH, and to help identify potential diagnostic markers and therapeutic targets for PAH. We integrated three PAH microarray datasets from the Gene Expression Omnibus (GEO) and found significant enrichment of the electron transport chain (ETC)-related signaling pathways in mitochondrial oxidative phosphorylation system in PAH samples. Notably, the electron transport from cytochrome c to oxygen in ETC, which was down-regulated in PAH samples, was identified as the most crucial mitochondria-related pathway via machine learning. By further transcriptomic and metabolomic analyses, we identified 14 mitochondria-related genes and three differential metabolites (citric acid, 3-phenyllactic-acid, and ADP), suggesting that dysregulation of mitochondrial ETC and citric acid cycle might contribute to the development of PAH. These findings offer insights for the development of novel PAH diagnostics and therapies. GEO datasets analysis The selection criteria of GEO datasets were as follows: (1) large sample size; (2) including PAH group and control group; (3) data interpretable. Three PAH datasets (GSE24988, GSE117261, and GSE53408) were retrieved from the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ) for analysis. The basic information of these datasets is given in Tables and . Samples in the GSE24988 dataset were obtained from the recipient organs of pulmonary fibrosis patients undergoing lung transplant, including 94 PAH samples and 22 controls without PAH. The GSE117261 dataset comprises 58 samples from PAH patients undergoing lung transplant and 25 control samples from failed donors (without matching recipients). The GSE53408 dataset includes 12 samples from end stage PAH patients who went through lung transplantation, and 11 control samples from normal tissue of cancer patients undergoing surgery (lobectomy). In total, microarray gene expression data of 164 PAH and 58 control lung samples were obtained. The microarray probes were converted to corresponding gene symbols with the annotation file from the GPL6244 platform (Affymetrix Human Gene 1.0 ST Array) using R software (version 4.2.2, https://www.r-project.org/ ). The Robust Multichip Average (RMA) algorithm was utilized for gene expression normalization. Principal component analysis (PCA) was conducted after merging the gene expression profiles of the three datasets. The batch effects among them were assessed by the “sva” R package and removed by the “combat” function . The three-dimensional (3D) PCA scatter plots before and after removing the batch effects were drawn to show the heterogeneity among the three datasets by the “scatterplot3d” R package. The difference in gene expression levels between PAH and control samples was estimated via “limma” package . Genes with |log 2 fold-change (FC)| ≥ 0.7 and Benjamini–Hochberg (B–H) false discovery rate (FDR) < 0.05 were identified as differentially expressed genes (DEGs) . Functional annotation and enrichment analysis Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed with the expression profiles of DEGs by the “clusterProfiler” R package . The GO gene sets are comprised of biological process (BP), cellular component (CC), and molecular function (MF) subsets. Gene set enrichment analysis (GSEA) was conducted with the “clusterProfiler” and “msigdbr” R packages . Gene set variation analysis (GSVA) was used to calculate the enrichment scores of mitochondria-related pathways in each sample based on the expression profiles of samples by the “GSVA” R package . The mitochondria-related pathway terms and genes were obtained from the Molecular Signatures Database (MSigDB; https://www.gsea-msigdb.org/gsea/index.jsp , v2023.1.Hs). The normalized enrichment score (NES) reflected the degree to which a gene set was overrepresented in the groups. A B–H adjusted P value <0.05 was considered statistically significant. Screening crucial mitochondria-related pathways by machine learning Machine learning algorithms including Naive Bayes, logistic regression, Gradient Boosting Machine (GBM), Adaptive Boosting (Adaboost), and eXtreme Gradient Boosting (XGBoost) were used to screen the most significant mitochondria-related pathways based on the GSVA scores by R . Naive Bayes is based on Bayes’ theorem with the “naive” assumption that features are independent of each other, which simplifies calculations . Logistic regression models the probability that a given input belongs to a particular class using the logistic function . GBM works by building a series of decision trees sequentially, where each tree corrects the errors made by the previous ones . Adaboost operates by combining multiple weak learners to create a strong classifier . XGBoost is an advanced implementation of the gradient boosting algorithm, incorporating several features such as gradient boosting framework, regularization, sparsity awareness, and cross validation . Different algorithms were used for modeling to make the analysis more robust. The risk of overfitting was mitigated by a tenfold cross-validation approach and the control of model complexity. A total of 222 samples (164 PAH and 58 controls) were randomly divided into training (75%) and validation (25%) sets. The SHapley Additive exPlanations (SHAP) algorithm was used to interpret the results of the XGBoost model by quantifying the contribution of each feature to the prediction outcome for individual data points . Receiver operating characteristic (ROC) and precision-recall (PR) curves were plotted to evaluate the performance of the machine learning models in the validation set by the “pROC” and “PRROC” packages. The area under curve (AUC) values on ROC and PR curves represented the accuracy of the models. A decision tree was drawn to show the contributions of each mitochondrial pathway in the model by the “rpart” package. The decision tree models decisions based on a hierarchical structure resembling a tree, where each internal node represents a feature, each branch represents a decision based on that feature, and each leaf node represents the outcome or prediction. Participant inclusion The study was approved by the Ethics Committee of The First Affiliated Hospital, Sun Yat-sen University (No. [2023] 553). Lung tissue specimens were collected from eight adult patients with PAH and eight non-PAH adult patients as the control group undergoing pulmonary lobectomy or partial pulmonary lobectomy at the First Affiliated Hospital, Sun Yat-sen University between February 2023 and April 2023. The lung tissue samples in the control group were collected at a site remote from tumor foci of early-stage lung cancer patients. The diagnosis of PAH was defined as a pulmonary artery systolic pressure (PASP) > 35 mmHg detected by noninvasive echocardiography . Exclusion criteria were as follows: (1) severe cerebrovascular diseases; (2) chronic progressive nephropathy. The baseline information of the PAH and control patients, including body mass index (BMI), pulmonary artery systolic pressure (PASP), forced vital capacity (FVC), and total lung capacity (TLC), were recorded. All subjects provided informed consent for the study. Transcriptomic analysis Total RNA was extracted from the lung tissue samples of PAH and non-PAH patients recruited in our hospital using the Direct-zol RNA MiniPrep Plus Kit (Zymo Research) for RNA sequencing. RNA was quantified by a NanoDrop 8000 UV–Vis Spectrophotometer (Thermo Scientific) and quality was assessed with the Bioanalyzer RNA 6000 Nano Kit (Agilent). Samples with sufficient quantity and quality (RNA integrity number ≥ 6) were retained for sequencing. The purified RNA was used to prepare directional cDNA libraries for sequencing using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina. Quality control (before and after library preparation) was performed with the Bioanalyzer HighSens DNA Kit (Agilent) and the molarity was examined by the Kapa qPCR quantification kit. Paired-end, 75 base pair sequence was generated using an Illumina Novaseq 6000, with the version 2 chemistry. After de-multiplexing and adapter removal, sequence quality was assessed with FastQC. Samples with sufficient quality were aligned to the human genome (version GRCh38) using STAR, which also produced transcript counts. Samples with aberrant alignment characteristics (e.g. low number of mapped reads, high number of reads mapped to multiple locations) were excluded from analysis. Genes with |log 2 fold-change (FC)| ≥ 1 and B–H adjusted P value <0.05 were identified as differentially expressed genes (DEGs). Mass spectrometry (MS)-based metabolomic profiling The liquid chromatography-electrospray ionization-tandem mass spectrometry (LC–ESI–MS/MS, QTRAP ® 6500+) system was used for non-targeted metabolomic profiling. The positive and negative ion modes were analyzed by Analyst 1.6.3 software (Sciex). A mixture of the sample extracts was used for quality control and was inserted into analytical samples. We subsequently obtained the total ion current (TIC) chromatogram and multiple reaction monitoring (MRM) multi-peak chromatogram. A triple quadrupole mass spectrometer was used to select the characteristic ion of each substance. The signal intensity (counts per second) of the characteristic ion was acquired in the detector. MultiQuant software was used to integrate and calibrate the chromatographic peaks in the sample output files by the mass spectrometer. The chromatographic peak area represents the relative content of the corresponding substance, and the chromatographic peak of each metabolite in different samples was corrected based on the retention time and peak type. Orthogonal partial least squares‑discriminant analysis (OPLS-DA) Differential metabolites between PAH and control samples were identified by OPLS-DA, a method that combines orthogonal signal correction and partial least squares‑discriminant analysis to decompose the matrix information of independent variables into related- and unrelated-information of dependent variables . Differential metabolites between groups were selected using variable importance in projection (VIP) of the OPLS-DA model by the “MetaboAnalystR” R package . Metabolites with VIP ≥ 1.0 and |log 2 fold-change (FC)| ≥ 1 were defined as significantly differential metabolites. The heatmap of differential metabolites was drawn by the “pheatmap” R package. KEGG analysis of differential metabolites Metabolites were annotated and mapped to potential metabolic pathways based on the KEGG compound and pathway database ( http://www.kegg.jp/kegg/compound/ ), including carbohydrate, nucleotide, amino acid metabolism and organic substance biodegradation . The hypergeometric test’s P value <0.05 was considered statistically significant for pathway enrichment. Clustering and correlation analyses of DEGs and differential metabolites DEGs were assigned into subgroups by k-means clustering. Correlations between gene expression were assessed by Pearson’s correlation coefficients. Relationships between DEGs and differential metabolites were analyzed by Pearson’s correlation analysis and canonical correlation analysis (CCA). A Pearson’s correlation coefficient >0.7 was considered as strong correlation. The selection criteria of GEO datasets were as follows: (1) large sample size; (2) including PAH group and control group; (3) data interpretable. Three PAH datasets (GSE24988, GSE117261, and GSE53408) were retrieved from the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ) for analysis. The basic information of these datasets is given in Tables and . Samples in the GSE24988 dataset were obtained from the recipient organs of pulmonary fibrosis patients undergoing lung transplant, including 94 PAH samples and 22 controls without PAH. The GSE117261 dataset comprises 58 samples from PAH patients undergoing lung transplant and 25 control samples from failed donors (without matching recipients). The GSE53408 dataset includes 12 samples from end stage PAH patients who went through lung transplantation, and 11 control samples from normal tissue of cancer patients undergoing surgery (lobectomy). In total, microarray gene expression data of 164 PAH and 58 control lung samples were obtained. The microarray probes were converted to corresponding gene symbols with the annotation file from the GPL6244 platform (Affymetrix Human Gene 1.0 ST Array) using R software (version 4.2.2, https://www.r-project.org/ ). The Robust Multichip Average (RMA) algorithm was utilized for gene expression normalization. Principal component analysis (PCA) was conducted after merging the gene expression profiles of the three datasets. The batch effects among them were assessed by the “sva” R package and removed by the “combat” function . The three-dimensional (3D) PCA scatter plots before and after removing the batch effects were drawn to show the heterogeneity among the three datasets by the “scatterplot3d” R package. The difference in gene expression levels between PAH and control samples was estimated via “limma” package . Genes with |log 2 fold-change (FC)| ≥ 0.7 and Benjamini–Hochberg (B–H) false discovery rate (FDR) < 0.05 were identified as differentially expressed genes (DEGs) . Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed with the expression profiles of DEGs by the “clusterProfiler” R package . The GO gene sets are comprised of biological process (BP), cellular component (CC), and molecular function (MF) subsets. Gene set enrichment analysis (GSEA) was conducted with the “clusterProfiler” and “msigdbr” R packages . Gene set variation analysis (GSVA) was used to calculate the enrichment scores of mitochondria-related pathways in each sample based on the expression profiles of samples by the “GSVA” R package . The mitochondria-related pathway terms and genes were obtained from the Molecular Signatures Database (MSigDB; https://www.gsea-msigdb.org/gsea/index.jsp , v2023.1.Hs). The normalized enrichment score (NES) reflected the degree to which a gene set was overrepresented in the groups. A B–H adjusted P value <0.05 was considered statistically significant. Machine learning algorithms including Naive Bayes, logistic regression, Gradient Boosting Machine (GBM), Adaptive Boosting (Adaboost), and eXtreme Gradient Boosting (XGBoost) were used to screen the most significant mitochondria-related pathways based on the GSVA scores by R . Naive Bayes is based on Bayes’ theorem with the “naive” assumption that features are independent of each other, which simplifies calculations . Logistic regression models the probability that a given input belongs to a particular class using the logistic function . GBM works by building a series of decision trees sequentially, where each tree corrects the errors made by the previous ones . Adaboost operates by combining multiple weak learners to create a strong classifier . XGBoost is an advanced implementation of the gradient boosting algorithm, incorporating several features such as gradient boosting framework, regularization, sparsity awareness, and cross validation . Different algorithms were used for modeling to make the analysis more robust. The risk of overfitting was mitigated by a tenfold cross-validation approach and the control of model complexity. A total of 222 samples (164 PAH and 58 controls) were randomly divided into training (75%) and validation (25%) sets. The SHapley Additive exPlanations (SHAP) algorithm was used to interpret the results of the XGBoost model by quantifying the contribution of each feature to the prediction outcome for individual data points . Receiver operating characteristic (ROC) and precision-recall (PR) curves were plotted to evaluate the performance of the machine learning models in the validation set by the “pROC” and “PRROC” packages. The area under curve (AUC) values on ROC and PR curves represented the accuracy of the models. A decision tree was drawn to show the contributions of each mitochondrial pathway in the model by the “rpart” package. The decision tree models decisions based on a hierarchical structure resembling a tree, where each internal node represents a feature, each branch represents a decision based on that feature, and each leaf node represents the outcome or prediction. The study was approved by the Ethics Committee of The First Affiliated Hospital, Sun Yat-sen University (No. [2023] 553). Lung tissue specimens were collected from eight adult patients with PAH and eight non-PAH adult patients as the control group undergoing pulmonary lobectomy or partial pulmonary lobectomy at the First Affiliated Hospital, Sun Yat-sen University between February 2023 and April 2023. The lung tissue samples in the control group were collected at a site remote from tumor foci of early-stage lung cancer patients. The diagnosis of PAH was defined as a pulmonary artery systolic pressure (PASP) > 35 mmHg detected by noninvasive echocardiography . Exclusion criteria were as follows: (1) severe cerebrovascular diseases; (2) chronic progressive nephropathy. The baseline information of the PAH and control patients, including body mass index (BMI), pulmonary artery systolic pressure (PASP), forced vital capacity (FVC), and total lung capacity (TLC), were recorded. All subjects provided informed consent for the study. Total RNA was extracted from the lung tissue samples of PAH and non-PAH patients recruited in our hospital using the Direct-zol RNA MiniPrep Plus Kit (Zymo Research) for RNA sequencing. RNA was quantified by a NanoDrop 8000 UV–Vis Spectrophotometer (Thermo Scientific) and quality was assessed with the Bioanalyzer RNA 6000 Nano Kit (Agilent). Samples with sufficient quantity and quality (RNA integrity number ≥ 6) were retained for sequencing. The purified RNA was used to prepare directional cDNA libraries for sequencing using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina. Quality control (before and after library preparation) was performed with the Bioanalyzer HighSens DNA Kit (Agilent) and the molarity was examined by the Kapa qPCR quantification kit. Paired-end, 75 base pair sequence was generated using an Illumina Novaseq 6000, with the version 2 chemistry. After de-multiplexing and adapter removal, sequence quality was assessed with FastQC. Samples with sufficient quality were aligned to the human genome (version GRCh38) using STAR, which also produced transcript counts. Samples with aberrant alignment characteristics (e.g. low number of mapped reads, high number of reads mapped to multiple locations) were excluded from analysis. Genes with |log 2 fold-change (FC)| ≥ 1 and B–H adjusted P value <0.05 were identified as differentially expressed genes (DEGs). The liquid chromatography-electrospray ionization-tandem mass spectrometry (LC–ESI–MS/MS, QTRAP ® 6500+) system was used for non-targeted metabolomic profiling. The positive and negative ion modes were analyzed by Analyst 1.6.3 software (Sciex). A mixture of the sample extracts was used for quality control and was inserted into analytical samples. We subsequently obtained the total ion current (TIC) chromatogram and multiple reaction monitoring (MRM) multi-peak chromatogram. A triple quadrupole mass spectrometer was used to select the characteristic ion of each substance. The signal intensity (counts per second) of the characteristic ion was acquired in the detector. MultiQuant software was used to integrate and calibrate the chromatographic peaks in the sample output files by the mass spectrometer. The chromatographic peak area represents the relative content of the corresponding substance, and the chromatographic peak of each metabolite in different samples was corrected based on the retention time and peak type. Differential metabolites between PAH and control samples were identified by OPLS-DA, a method that combines orthogonal signal correction and partial least squares‑discriminant analysis to decompose the matrix information of independent variables into related- and unrelated-information of dependent variables . Differential metabolites between groups were selected using variable importance in projection (VIP) of the OPLS-DA model by the “MetaboAnalystR” R package . Metabolites with VIP ≥ 1.0 and |log 2 fold-change (FC)| ≥ 1 were defined as significantly differential metabolites. The heatmap of differential metabolites was drawn by the “pheatmap” R package. Metabolites were annotated and mapped to potential metabolic pathways based on the KEGG compound and pathway database ( http://www.kegg.jp/kegg/compound/ ), including carbohydrate, nucleotide, amino acid metabolism and organic substance biodegradation . The hypergeometric test’s P value <0.05 was considered statistically significant for pathway enrichment. DEGs were assigned into subgroups by k-means clustering. Correlations between gene expression were assessed by Pearson’s correlation coefficients. Relationships between DEGs and differential metabolites were analyzed by Pearson’s correlation analysis and canonical correlation analysis (CCA). A Pearson’s correlation coefficient >0.7 was considered as strong correlation. Identification of DEGs in the PAH datasets from GEO Three microarray datasets from GEO with a total of 222 lung samples (164 PAH and 58 controls) were integrated for the analysis. Certain heterogeneity existed among the three datasets in the initial PCA graph (Fig. A). After removing batch effects, the gene expression data in the three datasets exhibit similar distributions, suitable for further analysis (Fig. B). The differential expression analysis identified 36 DEGs between PAH and control samples (Fig. C; Additional file ). The genes displaying the most significantly up-regulation in PAH included peptidase inhibitor 15 ( PI15 ), asporin ( ASPN ), collagen type xiv alpha 1 chain ( COL14A1 ), and secreted frizzled related protein 2 ( SFRP2 ), whereas the genes with the most remarkable down-regulation in PAH were S100 calcium binding protein A8 ( S100A8 ), S100 calcium binding protein A12 ( S100A12 ), and ficolin 3 ( FCN3 ). Significant alterations of mitochondria-related pathways in PAH Following the identification of DEGs in the PAH datasets, KEGG enrichment analysis was performed and six significantly enriched pathways were identified, including mitochondria-related pathways (calcium and cAMP signaling pathways) as well as cardiac disease-related pathways (arrhythmogenic right ventricular cardiomyopathy and hypertrophic cardiomyopathy) (Fig. D). The GO enrichment analysis demonstrated notable enrichment of pathways related to transmembrane transport, ion gated channel activity, and metabolic processes (Fig. A). As mitochondrial dysfunction plays an important role in PAH, we subsequently evaluated the mitochondria-related pathways using the GSVA scoring method. A total of 30 mitochondria-related pathways showed significant differences between PAH and control samples (Fig. B). The GSEA revealed substantial up-regulation of ATP dependent activity (NES = 1.896, adjusted P value = 6.85E−08) and down-regulation of the ETC in mitochondrial oxidative phosphorylation system (NES = −2.158, adjusted P value = 0.001) in PAH (Fig. C, D). Collectively, the development of PAH is linked with a variety of cell signaling pathways, particularly those that involve disruption in mitochondrial functionality. Electron transport from cytochrome c to oxygen in ETC as the most critical mitochondria-related pathway in PAH To determine the most crucial mitochondria-related pathway in PAH, machine learning algorithms including Naïve Bayes, logistic regression, GBM, Adaboost, XGBoost, and decision tree were employed. The AUCs on ROC and PR curves of the Naïve Bayes model were 0.631 and 0.819, respectively (Fig. A). The models of logistic regression (ROC-AUC: 0.635, PR-AUC: 0.838; Fig. A), GBM (ROC-AUC: 0.631, PR-AUC: 0.803; Fig. B), and Adaboost (ROC-AUC: 0.666, PR-AUC: 0.830; Fig. B) exhibited similar accuracy. In the decision tree model, the pathway of mitochondrial electron transport (cytochrome c to oxygen) accounted for the largest part, followed by the regulation of mitophagy and the activation of NOXA and translocation to mitochondria (Fig. C). The SHAP algorithm for XGBoost model interpretation identified five mitochondria-related pathways with the greatest influence on the model (Fig. A). The SHAP force plot and summary plot indicated that the pathway of mitochondrial electron transport (cytochrome c to oxygen) was of the greatest impact on the model (Fig. B, C), implying its optimal performance in distinguishing PAH from control samples. The GSEA showed the pathway of mitochondrial electron transport from cytochrome c to oxygen was significantly down-regulated in the PAH group (NES = −1.851, adjusted P value = 0.015; Fig. D). Taken together, the electron transport from cytochrome c to oxygen in mitochondria is postulated to be the most significant mitochondria-related pathway implicated in the pathogenesis of PAH. Transcriptomic analysis of clinical PAH lung tissue samples To further investigate the findings, lung tissue samples from eight PAH and eight non-PAH patients were obtained for transcriptomic profiling. Both the PAH and control groups were Han Chinese. The PAH group included 4 males and 4 females, aged 65.50 ± 6.41 years old, with BMI 22.03 ± 2.48 kg/m 2 , PASP 43.13 ± 3.14 mmHg, FVC 95.63 ± 14.90, and TLC 92.50 ± 5.25. The control group included 5 males and 3 females, aged 60.63 ± 10.39 years old, with BMI 23.02 ± 3.01 kg/m 2 , PASP 20.38 ± 2.83 mmHg, FVC 97.38 ± 6.52, and TLC 97.50 ± 18.25. Through the transcriptomic analysis, a total of 361 DEGs were classified into three subgroups by K-means clustering (Fig. A; Additional file ). Through KEGG analysis of the DEGs, six major categories of pathways were significantly enriched, including the metabolism pathway (Fig. B). Among the 361 DEGs, 204 genes displayed up-regulated expression patterns and 157 genes were down-regulated in the PAH group compared with the controls (Fig. C). Subsequently, a total of 630 mitochondria-related genes (mito-genes) were acquired from the MSigDB (Additional file ). Analysis of the Venn diagram revealed a shared set of 14 genes between the DEGs and the mito-genes, the majority of which exhibited elevated levels of expression in PAH (Fig. D, E). The interrelationships of the 14 genes were estimated by Pearson’s correlation coefficients, which demonstrated a close association between mitochondrial-related genes, including mitochondrially encoded ATP synthase membrane subunit 6 ( MT-ATP6 ), MT-ATP8 , mitochondrially encoded NADH dehydrogenase 4L ( MT-ND4L ), and mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 5 ( MT-ND5 ) (Fig. F). Strong correlations were also observed between metallothionein 1E ( MT1E ), MT1F , and MT1G (Fig. F). These results indicate the involvement of altered metabolism and mitochondrial function in PAH at the transcriptional level. Metabolomic profiling of clinical PAH and control lung tissue samples Metabolomic analysis was performed to investigate the metabolic alterations in PAH. The levels of 63 metabolites were detected in the lung tissue samples, and three of them (citric acid, 3-phenyllactic-acid, and ADP) exhibited significant alterations in PAH samples compared with controls (Fig. A; Additional file ). Specifically, the PAH samples appeared to have decreased levels of citric acid and elevated levels of 3-phenyllactic-acid as well as ADP. To further identify key genes related to the differential metabolites, Pearson’s correlation analysis was performed between the expression levels of the DEGs and the three metabolites, and a correlation clustering heatmap was drawn based on the Pearson’s correlation coefficients between the DEGs and metabolites (Fig. B). KEGG pathway analysis was performed on the identified differential metabolites, and the results were integrated with the enriched KEGG pathways of DEGs identified through transcriptomic analysis to obtain the shared pathways. CCA analysis was performed between the DEGs and differential metabolites in the shared KEGG pathways, and DEGs were closely linked with citric acid (Fig. C). Four mito-genes were highly correlated with the metabolites (citric acid and ADP) as well, including cholinergic receptor nicotinic alpha 1 subunit ( CHRNA1 ), ornithine transcarbamylase ( OTC ), kit proto-oncogene ( KIT ), and calcium/calmodulin dependent protein kinase II alpha ( CAMK2A ) (Fig. D). Through transcriptomic and metabolomic analyses, we identified three key metabolites (citric acid, 3-phenyllactic-acid, and ADP) and four related mito-genes ( CHRNA1 , OTC , KIT , and CAMK2A ) in PAH pathogenesis. Three microarray datasets from GEO with a total of 222 lung samples (164 PAH and 58 controls) were integrated for the analysis. Certain heterogeneity existed among the three datasets in the initial PCA graph (Fig. A). After removing batch effects, the gene expression data in the three datasets exhibit similar distributions, suitable for further analysis (Fig. B). The differential expression analysis identified 36 DEGs between PAH and control samples (Fig. C; Additional file ). The genes displaying the most significantly up-regulation in PAH included peptidase inhibitor 15 ( PI15 ), asporin ( ASPN ), collagen type xiv alpha 1 chain ( COL14A1 ), and secreted frizzled related protein 2 ( SFRP2 ), whereas the genes with the most remarkable down-regulation in PAH were S100 calcium binding protein A8 ( S100A8 ), S100 calcium binding protein A12 ( S100A12 ), and ficolin 3 ( FCN3 ). Following the identification of DEGs in the PAH datasets, KEGG enrichment analysis was performed and six significantly enriched pathways were identified, including mitochondria-related pathways (calcium and cAMP signaling pathways) as well as cardiac disease-related pathways (arrhythmogenic right ventricular cardiomyopathy and hypertrophic cardiomyopathy) (Fig. D). The GO enrichment analysis demonstrated notable enrichment of pathways related to transmembrane transport, ion gated channel activity, and metabolic processes (Fig. A). As mitochondrial dysfunction plays an important role in PAH, we subsequently evaluated the mitochondria-related pathways using the GSVA scoring method. A total of 30 mitochondria-related pathways showed significant differences between PAH and control samples (Fig. B). The GSEA revealed substantial up-regulation of ATP dependent activity (NES = 1.896, adjusted P value = 6.85E−08) and down-regulation of the ETC in mitochondrial oxidative phosphorylation system (NES = −2.158, adjusted P value = 0.001) in PAH (Fig. C, D). Collectively, the development of PAH is linked with a variety of cell signaling pathways, particularly those that involve disruption in mitochondrial functionality. To determine the most crucial mitochondria-related pathway in PAH, machine learning algorithms including Naïve Bayes, logistic regression, GBM, Adaboost, XGBoost, and decision tree were employed. The AUCs on ROC and PR curves of the Naïve Bayes model were 0.631 and 0.819, respectively (Fig. A). The models of logistic regression (ROC-AUC: 0.635, PR-AUC: 0.838; Fig. A), GBM (ROC-AUC: 0.631, PR-AUC: 0.803; Fig. B), and Adaboost (ROC-AUC: 0.666, PR-AUC: 0.830; Fig. B) exhibited similar accuracy. In the decision tree model, the pathway of mitochondrial electron transport (cytochrome c to oxygen) accounted for the largest part, followed by the regulation of mitophagy and the activation of NOXA and translocation to mitochondria (Fig. C). The SHAP algorithm for XGBoost model interpretation identified five mitochondria-related pathways with the greatest influence on the model (Fig. A). The SHAP force plot and summary plot indicated that the pathway of mitochondrial electron transport (cytochrome c to oxygen) was of the greatest impact on the model (Fig. B, C), implying its optimal performance in distinguishing PAH from control samples. The GSEA showed the pathway of mitochondrial electron transport from cytochrome c to oxygen was significantly down-regulated in the PAH group (NES = −1.851, adjusted P value = 0.015; Fig. D). Taken together, the electron transport from cytochrome c to oxygen in mitochondria is postulated to be the most significant mitochondria-related pathway implicated in the pathogenesis of PAH. To further investigate the findings, lung tissue samples from eight PAH and eight non-PAH patients were obtained for transcriptomic profiling. Both the PAH and control groups were Han Chinese. The PAH group included 4 males and 4 females, aged 65.50 ± 6.41 years old, with BMI 22.03 ± 2.48 kg/m 2 , PASP 43.13 ± 3.14 mmHg, FVC 95.63 ± 14.90, and TLC 92.50 ± 5.25. The control group included 5 males and 3 females, aged 60.63 ± 10.39 years old, with BMI 23.02 ± 3.01 kg/m 2 , PASP 20.38 ± 2.83 mmHg, FVC 97.38 ± 6.52, and TLC 97.50 ± 18.25. Through the transcriptomic analysis, a total of 361 DEGs were classified into three subgroups by K-means clustering (Fig. A; Additional file ). Through KEGG analysis of the DEGs, six major categories of pathways were significantly enriched, including the metabolism pathway (Fig. B). Among the 361 DEGs, 204 genes displayed up-regulated expression patterns and 157 genes were down-regulated in the PAH group compared with the controls (Fig. C). Subsequently, a total of 630 mitochondria-related genes (mito-genes) were acquired from the MSigDB (Additional file ). Analysis of the Venn diagram revealed a shared set of 14 genes between the DEGs and the mito-genes, the majority of which exhibited elevated levels of expression in PAH (Fig. D, E). The interrelationships of the 14 genes were estimated by Pearson’s correlation coefficients, which demonstrated a close association between mitochondrial-related genes, including mitochondrially encoded ATP synthase membrane subunit 6 ( MT-ATP6 ), MT-ATP8 , mitochondrially encoded NADH dehydrogenase 4L ( MT-ND4L ), and mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 5 ( MT-ND5 ) (Fig. F). Strong correlations were also observed between metallothionein 1E ( MT1E ), MT1F , and MT1G (Fig. F). These results indicate the involvement of altered metabolism and mitochondrial function in PAH at the transcriptional level. Metabolomic analysis was performed to investigate the metabolic alterations in PAH. The levels of 63 metabolites were detected in the lung tissue samples, and three of them (citric acid, 3-phenyllactic-acid, and ADP) exhibited significant alterations in PAH samples compared with controls (Fig. A; Additional file ). Specifically, the PAH samples appeared to have decreased levels of citric acid and elevated levels of 3-phenyllactic-acid as well as ADP. To further identify key genes related to the differential metabolites, Pearson’s correlation analysis was performed between the expression levels of the DEGs and the three metabolites, and a correlation clustering heatmap was drawn based on the Pearson’s correlation coefficients between the DEGs and metabolites (Fig. B). KEGG pathway analysis was performed on the identified differential metabolites, and the results were integrated with the enriched KEGG pathways of DEGs identified through transcriptomic analysis to obtain the shared pathways. CCA analysis was performed between the DEGs and differential metabolites in the shared KEGG pathways, and DEGs were closely linked with citric acid (Fig. C). Four mito-genes were highly correlated with the metabolites (citric acid and ADP) as well, including cholinergic receptor nicotinic alpha 1 subunit ( CHRNA1 ), ornithine transcarbamylase ( OTC ), kit proto-oncogene ( KIT ), and calcium/calmodulin dependent protein kinase II alpha ( CAMK2A ) (Fig. D). Through transcriptomic and metabolomic analyses, we identified three key metabolites (citric acid, 3-phenyllactic-acid, and ADP) and four related mito-genes ( CHRNA1 , OTC , KIT , and CAMK2A ) in PAH pathogenesis. Mitochondrial dysfunction has been implicated in the pathogenesis and progression in a wide range of diseases, such as Alzheimer’s disease, chronic lung diseases, and various malignancies . It also plays an important role in PAH pathophysiology . Mitochondrial dynamic encompass the process of fusion and fission, which plays critical roles in maintaining the functional integrity of mitochondria . Specifically, the expression of dynamin-related protein 1 (DRP1), a central mediator of mitochondrial fission, was markedly up-regulated in pulmonary artery smooth muscle cells (PASMCs) of PAH patients and could induce the proliferation of PASMCs . Increased levels of citric acid cycle intermediates have been found in PAH patients compared with healthy subjects, suggesting possible dysfunction of mitochondrial citric acid cycle in PAH . Nevertheless, the key mitochondrial functional changes in the development of PAH remain to be fully understood. Further elucidation of PAH pathogenesis would be beneficial for therapy development. Herein, we employed integrated transcriptomic and metabolomic analyses to explore the key genes and metabolites involved in the development of PAH. In our analysis of the GEO datasets, KEGG pathway enrichment indicated that the DEGs in calcium signaling and cAMP signaling pathways were significantly enriched in the PAH group. Abnormalities in calcium signaling have been linked with pathological modulation of pulmonary vascular tone and pulmonary artery smooth muscle cell proliferation . Moreover, mitochondria served as sensors and regulators of calcium signaling have been shown to affect the Ca 2+ feedback regulation of channel activity, and an increased uptake of mitochondrial Ca 2+ was observed in pulmonary arterial smooth muscle cells from patients with PAH . The cAMP signaling pathway was also implicated in PAH. A recent study revealed that cAMP signaling pathway played an important role in PAH pathogenesis . In addition, the cAMP-protein kinase A (PKA) signaling can regulate mitochondrial functions, and alterations of mitochondrial cAMP-PKA signaling have implications in the pathogenesis of various diseases . Given the close connections between mitochondria and calcium signaling as well as cAMP signaling pathways, and the growing evidence of the role of mitochondrial dysfunction in PAH, we further examined the alterations of mitochondria-related pathways in the PAH group, and sifted the most critical signals by machine learning. Results revealed that the ETC in mitochondrial oxidative phosphorylation system was significantly down-regulated in PAH, especially the process of electron transport from cytochrome c to oxygen. It was suggested that the main proteins of the ETC involved in mitochondrial dysfunction might contribute to PAH . The mitochondrial ETC (also known as respiratory chain) as a major part of mitochondrial oxidative phosphorylation system contains four enzymatic complexes (complex I to IV), and complex IV (cytochrome c oxidases, COX) receives electrons transferred by cytochrome c to reduce oxygen to water for ATP production . COX4I2 , one of the major gene participating in the process of electron transport from cytochrome c to oxygen, is essential for acute pulmonary oxygen sensing and involved in hypoxia-induced pulmonary hypertension . Our findings suggest that dysfunction of the electron transport from cytochrome c to oxygen in mitochondria might contribute to PAH. To further investigate the findings derived from public datasets, we obtained PAH and non-PAH lung tissue samples from patients for transcriptomic and metabolomic analyses. In the transcriptomic analysis, 14 DEGs were mitochondria-related genes, four of which ( KIT , OTC , CAMK2A , and CHRNA1 ) were highly linked with the differential metabolites identified through subsequent metabolomic profiling. Similar to our findings, quantitative immunohistochemistry in human lung PAH tissues demonstrated an elevation in c-KIT level , and c-KIT-positive cells were reported to participate in vascular remodeling in PAH . OTC, which had higher gene expression levels in PAH lung tissue samples in the present study, also exhibited a significant increase in PAH gut bacteria . As for CAMK2A , it has been reported that alpha1A-adrenoceptor is involved in the proliferation of pulmonary artery smooth muscle cells via CaMKII signaling . In addition, variation in CHRNA1 genetics was highly associated with diastolic blood pressure . These results indicate that mitochondria-related genes are involved in the development of PAH. The regulation of gene expression is intricately linked to cell metabolism. Metabolic pathways provide precursor molecules and ATP that are necessary for gene expression . Mitochondria play a pivotal role in cellular metabolism, and mitochondrial dysfunction has been implicated in the pathogenesis of various metabolic diseases . Hence, we conducted metabolomic profiling to examine the metabolic alterations in PAH, and elevated levels of ADP were observed. It has been proposed that purinergic signaling activated by an alteration of nucleotides contributes to the pathogenesis of PAH . Moreover, activation of purinergic P2Y 1 -receptor (P2Y 1 R) and P2Y 12 R mediated by ADP plays an important role in pulmonary vascular remodeling and inflammation in PAH . Our metabolomic analysis also revealed a significant decrease in the levels of citric acid in the PAH samples. Conversely, increased levels of citric acid or citrate (the conjugate base of citric acid) in PAH patients were observed in other studies . The discrepancy should be further explored. Citrate is a significant intermediate produced in the initial step of the citric acid cycle (also known as the tricarboxylic acid cycle), subsequently undergoing conversion into isocitrate . The citric acid cycle, a vital metabolic pathway that takes play in mitochondria, plays a pivotal role in regulating cellular physiology and maintaining homeostasis, and the metabolites generated during the citric acid cycle exert significant control over various cellular processes, influencing both physiology function and disease development . Dysfunction of the citric acid cycle has been reported to occur in PAH and might potentially contribute to the pathogenesis of PAH . In addition, increased levels of 3-phenyllactic-acid were found in the PAH samples in the current study. 3-phenyllactic acid is a product of phenylalanine catabolism generated by human host and intestinal bacteria . It regulates human immune system and acts as a mediator of bacterial-host interactions . Phenyllactic acid can be accumulated in the blood and tissues of patients with phenylketonuria . It also promotes cell migration and invasion in cervical cancer by up-regulating the expression of the mitochondrial protease MMP9 . In fungi, 3-phenyllactic acid could disrupt cell membrane and interfere with mitochondrial energy metabolism . Our findings suggest that 3-phenyllactic-acid might potentially be a novel biomarker for PAH. Certain limitations existed in the current study. Limited lung tissue samples from Han Chinese patients were available for analyses, which might affect the broader applicability of the results. Access to larger sample sizes and samples from different ethnicities will allow further exploration of the present results. Moreover, future in vitro experiments would provide further insights into the potential pathogenic mechanisms of PAH. The present study revealed a significant down-regulation of mitochondrial ETC in PAH, and the process of electron transport from cytochrome c to oxygen was identified as the most crucial mitochondria-related pathway in PAH. Metabolomic profiling demonstrated decreased levels of citric acid and elevated levels of 3-phenyllactic-acid as well as ADP in PAH lung tissues. The integration of transcriptomic and metabolomic analyses suggested that disruption of mitochondrial ETC and citric acid cycle homeostasis may contribute to the pathogenesis of PAH, and 3-phenyllactic acid is a potential emerging biomarker for the diagnosis of PAH. These findings could provide insights into further understanding of PAH pathogenesis and presenting potential diagnostic biomarkers and therapeutic targets of PAH. Additional file 1: Table S1 The 36 DEGs in the GEO dataset analysis of 164 PAH and 58 control samples, with log 2 (fold change), P value and adjusted P value. Additional file 2: Table S2 The 361 DEGs in the transcriptomic analysis of 8 PAH and 8 control samples, with log 2 (fold change), P value and adjusted P value. Additional file 3: Table S3 The 630 mitochondria-related genes summarized through the mitochondria-related pathways retrieved from the MSigDB. Additional file 4: Table S4 The 63 metabolites detected by metabolomic profiling, with VIP, log 2 (fold change), and P value. |
The association between antigenemia, histology with immunohistochemistry, and mucosal PCR in the diagnosis of ulcerative colitis with concomitant human cytomegalovirus infection | 8813b08b-e35a-40fe-87bb-3b03deebaea8 | 9825535 | Anatomy[mh] | Human cytomegalovirus (HCMV), which is a member of the herpesvirus family, is generally contracted during childhood and can persist as a lifelong latent infection . Latent HCMV is reactivated under inflammatory conditions in immunosuppressed hosts and can cause organ damage . Much attention has been given to HCMV infection in patients with ulcerative colitis (UC) who are refractory to steroids and immunosuppressive drugs as well as to those with Clostridioides difficile infection because HCMV infection causes significant morbidity in UC patients . Therefore, an early diagnosis of HCMV infection in patients with refractory UC and rapid initiation of antiviral agents could help to avoid colectomy. Confirming the presence of HCMV on biopsy is the gold standard for the diagnosis of HCMV enterocolitis . Diagnosis by histological examination has a high specificity (92–100%) but a variable sensitivity (10 to 87%) . The HCMV antigenemia assay is also widely used for the detection of HCMV antigen-positive neutrophils in peripheral blood using C7-HRP and C10/11 monoclonal antibodies against the primary structural antigen (pp65) of HCMV. This method has a sensitivity of 47.0–65.4% and a specificity of 81.7–93.6% for diagnosing HCMV gastrointestinal disease . The usefulness of the mucosal polymerase chain reaction (PCR) method with colon biopsy tissues for diagnosing HCMV infection and determination of the treatment strategy for patients with refractory UC has been demonstrated in previous studies . Consistent with the clinical research, several reports have indicated that the copy number of HCMV-DNA in colonic tissues contributes to the decision in favor of antiviral treatment . However, the mucosal PCR method is not available in all medical institutions, and data on the associations among histopathological findings, antigenemia assays, and mucosal HCMV-PCR are limited. In this study, we aimed to clarify the relationships between the HCMV-DNA copy number in colonic mucosa and other HCMV tests or endoscopic score.
Patients We retrospectively analyzed a total of 82 patients with UC who were suspected of concomitant HCMV colitis and underwent HCMV mucosal PCR at Kyoto University Hospital (78) or Sapporo Medical University Hospital (4) between October 2013 and March 2020. The diagnosis of UC was based on clinical, endoscopic, radiologic, and histologic parameters. Fecal bacterial culture showed no specific pathogens in any of the enrolled patients. We used antiviral therapy when the diagnosis of HCMV infection was compatible based on the results of antigenemia and histopathological tests, endoscopic findings, viral titers by mucosal PCR, and clinical manifestations. Antiviral agents were administered to the patients intravenously or orally for 2–4 weeks and ended after observing clinical improvement. In this study, we posted information about this study on the hospital website, gave participants the opportunity to opt out, and considered that patients who did not opt out provided tacit consent for study participation. Study design The primary outcome was the association between HCMV mucosal PCR and other HCMV tests. The secondary outcomes were the association between HCMV mucosal PCR and the patient characteristics, endoscopic findings, and rate of surgery after HCMV mucosal PCR. Clinical data were obtained retrospectively from electronic medical records. Assessment of endoscopic severity All 82 patients underwent colonoscopy. The endoscopists categorized the disease phenotype based on the Montreal classification (E1/E2/E3) and assessed endoscopic severity according to the Mayo endoscopic subscore (MES) and Ulcerative Colitis Endoscopic Index of Severity (UCEIS). The items composed of the UCEIS (erosion and ulcers, vascular pattern, and bleeding) were also assessed (Table S1) . Although the endoscopic score was not independently assessed by central reviewers, it was evaluated by IBD experts at each facility. Histopathology We took colonic biopsy specimens from the margins of the ulcer or the most severely inflamed mucosa of the left colon or rectum in patients without ulcerations. They were fixed in formalin and embedded in paraffin and then evaluated with hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) using anti-HCMV monoclonal antibodies (Dako Cytomation, Kyoto, Japan). HCMV antigenemia assay The HCMV antigenemia assay directly detects HCMV antigen-positive neutrophils present in peripheral blood using C7-HRP and C10/11 monoclonal antibody (SRL Inc., Tokyo, Japan) against the primary structural antigen of HCMV. A positive result in this assay was defined as one or more HCMV-positive cells per 50,000 leukocytes applied. Quantitative real-time PCR (mucosal PCR) We extracted DNA from colonic tissue with the QIAamp DNA Mini Kit (QIAGEN, Tokyo) according to the manufacturer’s instructions, and detected HCMV-DNA using quantitative real time PCR. This assay was performed using an ABI Prism 7700 Sequence Detector System (Perkin Elmer Applied Biosystems, Foster City, CA, USA). The oligonucleotide primers used for HCMV-DNA amplification were 5_-GACTAGTGTGATGCTGGCCAAG-3_(forward) and 5_-GCTACAATAGCCTCTTCCTCATCTG-3_(reverse), and the 6-carboxyfluorescein-labeled probe was 5_-AGCCTGAGGTTATCAGTGTAATGAAGCGCC-3_. The PCR conditions were 95 °C for 10 min, 50 cycles at 95 °C for 15 s and 62 °C for 1 min as described previously . Ten or more copies/μg of HCMV-DNA was defined as positive in this study . For cases performing multiple mucosal PCR tests, we separately counted the data of each test. Statistical analysis Statistical analysis was performed using IBM SPSS Statistics 25 (IBM SPSS, Chicago, USA). The value of the HCMV-DNA copy number was logarithmically transformed for the statistical analysis (ln HCMV-DNA). Categorical and continuous data were compared using the Mann–Whitney U test and Spearman’s rank correlation coefficient, as appropriate, to calculate the statistical significance of the demographic and clinical variables according to the manufacturer’s instructions. A value of p < 0.05 was considered statistically significant.
We retrospectively analyzed a total of 82 patients with UC who were suspected of concomitant HCMV colitis and underwent HCMV mucosal PCR at Kyoto University Hospital (78) or Sapporo Medical University Hospital (4) between October 2013 and March 2020. The diagnosis of UC was based on clinical, endoscopic, radiologic, and histologic parameters. Fecal bacterial culture showed no specific pathogens in any of the enrolled patients. We used antiviral therapy when the diagnosis of HCMV infection was compatible based on the results of antigenemia and histopathological tests, endoscopic findings, viral titers by mucosal PCR, and clinical manifestations. Antiviral agents were administered to the patients intravenously or orally for 2–4 weeks and ended after observing clinical improvement. In this study, we posted information about this study on the hospital website, gave participants the opportunity to opt out, and considered that patients who did not opt out provided tacit consent for study participation.
The primary outcome was the association between HCMV mucosal PCR and other HCMV tests. The secondary outcomes were the association between HCMV mucosal PCR and the patient characteristics, endoscopic findings, and rate of surgery after HCMV mucosal PCR. Clinical data were obtained retrospectively from electronic medical records.
All 82 patients underwent colonoscopy. The endoscopists categorized the disease phenotype based on the Montreal classification (E1/E2/E3) and assessed endoscopic severity according to the Mayo endoscopic subscore (MES) and Ulcerative Colitis Endoscopic Index of Severity (UCEIS). The items composed of the UCEIS (erosion and ulcers, vascular pattern, and bleeding) were also assessed (Table S1) . Although the endoscopic score was not independently assessed by central reviewers, it was evaluated by IBD experts at each facility.
We took colonic biopsy specimens from the margins of the ulcer or the most severely inflamed mucosa of the left colon or rectum in patients without ulcerations. They were fixed in formalin and embedded in paraffin and then evaluated with hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) using anti-HCMV monoclonal antibodies (Dako Cytomation, Kyoto, Japan).
The HCMV antigenemia assay directly detects HCMV antigen-positive neutrophils present in peripheral blood using C7-HRP and C10/11 monoclonal antibody (SRL Inc., Tokyo, Japan) against the primary structural antigen of HCMV. A positive result in this assay was defined as one or more HCMV-positive cells per 50,000 leukocytes applied.
We extracted DNA from colonic tissue with the QIAamp DNA Mini Kit (QIAGEN, Tokyo) according to the manufacturer’s instructions, and detected HCMV-DNA using quantitative real time PCR. This assay was performed using an ABI Prism 7700 Sequence Detector System (Perkin Elmer Applied Biosystems, Foster City, CA, USA). The oligonucleotide primers used for HCMV-DNA amplification were 5_-GACTAGTGTGATGCTGGCCAAG-3_(forward) and 5_-GCTACAATAGCCTCTTCCTCATCTG-3_(reverse), and the 6-carboxyfluorescein-labeled probe was 5_-AGCCTGAGGTTATCAGTGTAATGAAGCGCC-3_. The PCR conditions were 95 °C for 10 min, 50 cycles at 95 °C for 15 s and 62 °C for 1 min as described previously . Ten or more copies/μg of HCMV-DNA was defined as positive in this study . For cases performing multiple mucosal PCR tests, we separately counted the data of each test.
Statistical analysis was performed using IBM SPSS Statistics 25 (IBM SPSS, Chicago, USA). The value of the HCMV-DNA copy number was logarithmically transformed for the statistical analysis (ln HCMV-DNA). Categorical and continuous data were compared using the Mann–Whitney U test and Spearman’s rank correlation coefficient, as appropriate, to calculate the statistical significance of the demographic and clinical variables according to the manufacturer’s instructions. A value of p < 0.05 was considered statistically significant.
Baseline clinical features of the patients A total of 82 patients with 131 HCMV mucosal PCR tests were enrolled. The median age was 48 years (range 17–90) and the sample consisted of 46 males and 36 females. The median MES and total UCEIS values were 2 and 5, respectively (Table ). Regarding the extent of disease according to the Montreal classification, the number of patients with E1, E2, and E3 was 0, 31, and 100, respectively. Among all enrolled patients, 75.6% were treated with prednisolone (PSL), 48.9% with immunomodulator (IM), 19.8% with tacrolimus (Tac), and 17.6% with biologics (Bio). The positive rates of tests other than HCMV mucosal PCR were 3.5% (4/115) for nuclear inclusion with H&E staining, 22.2% (14/63) for IHC, and 20.9% (9/43) for HCMV antigenemia using C7-HRP and 41.3% (19/46) using C10/11; IHC, C7-HRP, and C10/11 had many missing values (Table ). Relationship between the HCMV-DNA copy number and clinical features We investigated several factors associated with the HCMV-DNA copy number in colonic mucosa. The HCMV-DNA copy number tended to increase with age (Fig. A). Regarding the extent of disease, patients with Montreal E3 had a higher HCMV-DNA copy number than those with E2 (Fig. B). Regarding medication, patients treated with PSL had a higher HCMV-DNA copy number than those without PSL, although there was no significant difference (Fig. S1A, B). Other medical treatments, such as IM, Tac, and Bio, did not affect the HCMV-DNA copy number (Fig. S1C-E). Relationship between the HCMV-DNA copy number and other HCMV tests We examined the relationship between the HCMV-DNA copy number and other HCMV tests. No association between nuclear inclusion bodies on H&E staining and HCMV mucosal PCR was observed (Fig. A), while we found a significant association between the HCMV-DNA copy number and the positivity of IHC ( p < 0.01) (Fig. B). Regarding HCMV antigenemia, there was a positive correlation between HCMV-DNA copy number and the number of positive cells for C7-HRP ( p < 0.001, r s = 0.58) (Fig. A) and C10/11 ( p < 0.001, r s = 0.56) (Fig. B). Next, we generated a receiver operating characteristic (ROC) curve and calculated the area under the curve to determine the best discriminating level of HCMV-DNA copy number for predicting an HCMV antigenemia-positive cell. ROC curve analysis confirmed 1,300 copies/μg as the best diagnostic cut-off value of HCMV-DNA copy number for a positive result of antigenemia (area under the curve = 0.80, 95% CI 0.64–0.90, sensitivity 71.4% and specificity 78.4%) (Fig. A). For IHC, ROC curve analysis confirmed 1,650 copies/μg as the best diagnostic cut-off value of HCMV-DNA copy number for a positive result of IHC (area under the curve = 0.81, 95% CI 0.65–0.98, sensitivity 78.6% and specificity 85.7%) (Fig. B). We compared HCMV-DNA copy numbers between the groups with positive and/or negative results on the antigenemia and histopathological tests (Table S2). Twenty-eight patients with refractory UC who were negative for both the antigenemia and histopathological tests had lower levels of mucosal HCMV-DNA than those who were positive for either one or both (Fig. S2). Among the 28 patients, 13 received antiviral agents, and 15 did not. The clinical outcome of these patients showed that one patient underwent total colectomy in each group (Table S3). Relationship between the HCMV-DNA copy number and endoscopic findings We examined the relationship between the HCMV-DNA copy number and endoscopic findings (MES and UCEIS). Patients with MES 3 tended to have a higher HCMV-DNA copy number than those with MES 1 and 2 (Fig. S3A). For the UCEIS, the total UCEIS score was correlated with the HCMV-DNA copy number ( p = 0.014, r s = 0.22) (Fig. S3B). Among the items included in the UCEIS, the bleeding score was also correlated with the HCMV-DNA copy number ( p = 0.013, r s = 0.22) (Fig. S3C). However, there was no significant correlation between the HCMV-DNA copy number and the other two items, erosion/ulcer and vascular permeability (Fig. S3D, E). Moreover, an analysis using a mixed model that takes into account correlations for each individual patient showed a positive correlation between the total UCEIS score and HCMV-DNA copy number ( p = 0.040) (Fig. A). Furthermore, more cases led to colectomy as the HCMV-DNA copy number increased (Fig. B). On the other hand, there was no similar correlation for MES (no figure).
A total of 82 patients with 131 HCMV mucosal PCR tests were enrolled. The median age was 48 years (range 17–90) and the sample consisted of 46 males and 36 females. The median MES and total UCEIS values were 2 and 5, respectively (Table ). Regarding the extent of disease according to the Montreal classification, the number of patients with E1, E2, and E3 was 0, 31, and 100, respectively. Among all enrolled patients, 75.6% were treated with prednisolone (PSL), 48.9% with immunomodulator (IM), 19.8% with tacrolimus (Tac), and 17.6% with biologics (Bio). The positive rates of tests other than HCMV mucosal PCR were 3.5% (4/115) for nuclear inclusion with H&E staining, 22.2% (14/63) for IHC, and 20.9% (9/43) for HCMV antigenemia using C7-HRP and 41.3% (19/46) using C10/11; IHC, C7-HRP, and C10/11 had many missing values (Table ).
We investigated several factors associated with the HCMV-DNA copy number in colonic mucosa. The HCMV-DNA copy number tended to increase with age (Fig. A). Regarding the extent of disease, patients with Montreal E3 had a higher HCMV-DNA copy number than those with E2 (Fig. B). Regarding medication, patients treated with PSL had a higher HCMV-DNA copy number than those without PSL, although there was no significant difference (Fig. S1A, B). Other medical treatments, such as IM, Tac, and Bio, did not affect the HCMV-DNA copy number (Fig. S1C-E).
We examined the relationship between the HCMV-DNA copy number and other HCMV tests. No association between nuclear inclusion bodies on H&E staining and HCMV mucosal PCR was observed (Fig. A), while we found a significant association between the HCMV-DNA copy number and the positivity of IHC ( p < 0.01) (Fig. B). Regarding HCMV antigenemia, there was a positive correlation between HCMV-DNA copy number and the number of positive cells for C7-HRP ( p < 0.001, r s = 0.58) (Fig. A) and C10/11 ( p < 0.001, r s = 0.56) (Fig. B). Next, we generated a receiver operating characteristic (ROC) curve and calculated the area under the curve to determine the best discriminating level of HCMV-DNA copy number for predicting an HCMV antigenemia-positive cell. ROC curve analysis confirmed 1,300 copies/μg as the best diagnostic cut-off value of HCMV-DNA copy number for a positive result of antigenemia (area under the curve = 0.80, 95% CI 0.64–0.90, sensitivity 71.4% and specificity 78.4%) (Fig. A). For IHC, ROC curve analysis confirmed 1,650 copies/μg as the best diagnostic cut-off value of HCMV-DNA copy number for a positive result of IHC (area under the curve = 0.81, 95% CI 0.65–0.98, sensitivity 78.6% and specificity 85.7%) (Fig. B). We compared HCMV-DNA copy numbers between the groups with positive and/or negative results on the antigenemia and histopathological tests (Table S2). Twenty-eight patients with refractory UC who were negative for both the antigenemia and histopathological tests had lower levels of mucosal HCMV-DNA than those who were positive for either one or both (Fig. S2). Among the 28 patients, 13 received antiviral agents, and 15 did not. The clinical outcome of these patients showed that one patient underwent total colectomy in each group (Table S3).
We examined the relationship between the HCMV-DNA copy number and endoscopic findings (MES and UCEIS). Patients with MES 3 tended to have a higher HCMV-DNA copy number than those with MES 1 and 2 (Fig. S3A). For the UCEIS, the total UCEIS score was correlated with the HCMV-DNA copy number ( p = 0.014, r s = 0.22) (Fig. S3B). Among the items included in the UCEIS, the bleeding score was also correlated with the HCMV-DNA copy number ( p = 0.013, r s = 0.22) (Fig. S3C). However, there was no significant correlation between the HCMV-DNA copy number and the other two items, erosion/ulcer and vascular permeability (Fig. S3D, E). Moreover, an analysis using a mixed model that takes into account correlations for each individual patient showed a positive correlation between the total UCEIS score and HCMV-DNA copy number ( p = 0.040) (Fig. A). Furthermore, more cases led to colectomy as the HCMV-DNA copy number increased (Fig. B). On the other hand, there was no similar correlation for MES (no figure).
This is the first study to evaluate the relationship between HCMV-DNA copy numbers in colonic tissues and other HCMV tests or endoscopic scores. We found a significant correlation between HCMV-DNA copy number in colonic tissues and both the antigenemia assay and histopathological tests by IHC in UC patients with concomitant HCMV colitis. Regarding endoscopic scores, HCMV-DNA copy number by mucosal PCR correlated with both the total UCEIS score and the bleeding score. Moreover, a positive correlation between the change in the total UCEIS score and HCMV-DNA copy number for UC patients taking multiple mucosal PCR tests. Generally, HCMV is contracted during childhood and can persist as a lifelong latent infection, reactivating under inflammatory conditions in an immunosuppressed host . HCMV infection is involved in active UC patients who are refractory to steroids and immunosuppressive drugs . Previous reports showed that steroids are predisposing factors for HCMV reactivation by suppressing anti-HCMV T-cell specific function and immunoglobulin production via B-cells, and by directly activating viral replication . In this study, patients treated with PSL had a higher HCMV-DNA copy number than those treated without PSL. In patients with acute steroid-resistant UC, HCMV infection should be excluded before increasing immunosuppressive drugs . However, the clinical features of HCMV enterocolitis are sometimes indistinguishable from UC relapse . Histological examination, including H&E staining and IHC, on biopsy from colonic tissues is the gold standard for diagnosing HCMV enterocolitis, which yields high specificity but variable sensitivity . IHC has 22.1–30.0% higher sensitivity than H&E staining for diagnosing HCMV infection in IBD patients . In the present study, the positive rate of HCMV antigen by IHC correlated with the HCMV-DNA copy number. Another standard method for detecting HCMV infection is the HCMV antigenemia assay. Previous reports demonstrated that the antigenemia assay has a high specificity for detecting HCMV and a significant association with the subsequent colectomy rate in UC patients with concomitant HCMV enterocolitis . However, the sensitivity for diagnosing HCMV gastrointestinal diseases is only 47.0–67.3%, suggesting that the antigenemia assay alone is considered insufficient for early detection of HCMV . Yoshino et al. reported that a mucosal quantitative real-time PCR assay for detecting HCMV-DNA in the gastrointestinal tract was useful for the early and accurate diagnosis of HCMV infection in active UC patients refractory to immunosuppressive therapies . Roblin et al. reported that UC patients with an HCMV-DNA load higher than 250 copies/mg in colonic tissue required early antiviral treatment . However, mucosal PCR assays cannot be available in all medical institutions, and most physicians should decide to start antiviral treatment based on the results of antigenemia assays or histopathological tests in clinical practice. In the present study, we found a positive correlation between HCMV-DNA copy number and both HCMV antigenemia assay and IHC positivity. ROC analysis showed that 1300 (AUC = 0.80, 95% CI = 0.64–0.90) and 1,650 (AUC = 0.81, 95% CI = 0.65–0.98) copies/µg of HCMV-DNA were the best diagnostic values to detect HCMV antigenemia-positive cells and IHC positivity, respectively. These data suggest that positivity for HCMV antigenemia and IHC signify the increase of HCMV-DNA in the inflamed colon to decide the intervention of antiviral treatment for HCMV. Conversely, among UC patients who were negative for both the antigenemia and histopathological tests, the HCMV-DNA copy numbers were significantly lower than those in the other groups. In this group, the median HCMV-DNA copy number was 300 copies/μg (interquartile range: 39–810 copies/μg); however, there were no significant differences in HCMV-DNA copy numbers and the clinical outcome regardless of the antiviral treatment. These data suggest that a group of patients with a low mucosal HCMV-DNA copy number (less than 800 copies/μg) during treatment is more likely to be controlled with immune regulating therapy alone. However, given that the inflammatory control was poor during treatment, even in patients with a low mucosal HCM-DNA copy number at diagnosis, we must consider that exacerbation of HCMV infection can modify the disease state. Regarding endoscopic findings, Suzuki et al. reported that irregular punched-out or longitudinal ulcers were specific for HCMV infection in UC patients . On the other hand, the endoscopic findings of HCMV colitis are variable and sometimes do not reveal specific features . The present study also showed that the endoscopic scores varied even with the same amount of HCMV-DNA copy numbers. Taken together, previous and current data suggest that the heterogeneity of the immune response to HCMV and mucosal cytokine pattern in each UC patient could contribute to endoscopic features of UC with concomitant HCMV infection . To date, there have been no reports on the relationship between HCMV enterocolitis and endoscopic scores for MES or UCEIS in IBD patients. In this study, the total UCEIS score and the bleeding score were correlated with the HCMV-DNA copy numbers. Moreover, when we focused on patients who underwent multiple HCMV mucosal PCR tests, we found a significant correlation between changes in HCMV-DNA and those in the total UCEIS. At the beginning of HCMV reactivation, HCMV can infect the vascular endothelium and cause ischemic damage to the mucosa, which results in gastrointestinal bleeding . Previous reports have shown that gastrointestinal bleeding is the most frequent symptom of HCMV enterocolitis . Thus, in UC patients refractory to immune suppressive therapy who had a higher total UCEIS score, severe bleeding score, and increasing total UCEIS score during the clinical course, we should consider the involvement of HCMV reactivation in the colon. Furthermore, HCMV-DNA copy number tended to increase in cases who led to colectomy. These results suggest that early initiation of antiviral therapy may avoid the risk of colectomy in cases with elevated UCEIS scores. There are several limitations to this study. First, because of the retrospective study design, not all patients underwent antigenemia assays and histopathological tests with IHC. Additionally, not all HCMV antigenemia assays were performed on the same date as the biopsy for mucosal PCR. Since the difference in dates between HCMV antigenemia and mucosal PCR tests was at most 7 days, of which 71% were within 3 days, the initiation of therapeutic agents (i.e., steroids, biologics, and antiviral agents) might have affected the value of antigenemia in cases where the antigenemia was measured after mucosal PCR was performed. Second, whether to measure HCMV-DNA was made by each attending physician, which could be at risk for selection bias. In conclusion, our data showed that the antigenemia assay and histopathological test with IHC could help estimate the HCMV-DNA copy number in colonic tissues. Furthermore, the changes in UCEIS score in each individual may be helpful for decision-making in UC patients with concomitant HCMV infection. Additional study is required to establish an accurate diagnostic scoring system for the early diagnosis of HCMV infection in UC patients with a combination of HCMV tests and endoscopic score.
Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 925 KB) Supplementary file2 (DOCX 540 KB)
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Metabolic pathways of Alternative Lengthening of Telomeres in pan-carcinoma | 08f5d2c5-c1a2-41d5-b172-2fd2309f7469 | 11845024 | Biochemistry[mh] | Telomeres are nucleoprotein complexes that, in tandem with shelterin complex, protect the ends of eukaryotic chromosomes from degradation, recombination, and end to end fusion . Each round of the cellular cycle results in the shortening of telomeres, a process that, in healthy somatic cells, functions as a natural mechanism of cellular senescence . In cancer, most tumors activate various telomere maintenance mechanisms (TMM) that enable them to elongate their telomeres and achieve cellular immortality . Thus, a variety of therapies aimed at blocking these mechanisms have been implemented in recent years . Despite these efforts, approximately 15% of cancers can activate another TMM known as the Alternative Lengthening of Telomeres (ALT) . ALT is a telomerase-independent mechanism that utilizes homologous recombination to extend telomeres . While ALT is commonly associated with tumors of mesenchymal origin, such as sarcomas , there have been reports of its presence in other tumor types, including carcinomas. ALT+ cells have been identified in carcinomas of breast, liver, lung, bladder, cervix, endometrium, esophagus, and kidney . Additionally, ALT activity has been observed in cell lines derived from patients with breast and colon carcinomas, as well in histological analyses of both pediatric and adult cancers, including neuroblastomas and astrocytomas . In fact, a significant proportion of astrocytomas (34%) exhibit ALT activity . Moreover, in vivo and in vitro experiments have demonstrated the coexistence of ALT with telomerase-dependent mechanisms within the same population of cancer cells . It is believed that some cancer cells may switch from a telomerase-dependent mechanism to ALT under selective pressure from telomerase inhibitors . This phenomenon might add an extra layer of complexity to the diagnosis, prognosis, and treatment of ALT-positive cancer patients . To shed light on the ALT molecular mechanism, Braun et al. (2018) have developed the TelNet database , which compiles information on over 2000 human genes involved in telomere maintenance, including those specifically associated with the ALT pathway. Since the ALT pathway involves ~400 genes, occurs in multiple cancer types, and is linked to clinical outcomes, we previously prioritized ALT-related genes based on their alterations across 31 cancer types. Thus, we were able to identify 20 genes with significant alterations and proposed these as biomarkers for the ALT process. Additionally, we integrated multi-omic data to investigate 71 APBs (ALT-associated PML bodies)-related genes, uncovering 13 key proteins with distinctive mutations, interactions, and functional patterns related to ALT . To further incorporate metabolomics data into our prior findings, here we perform an integrative analysis of ALT-related proteins and metabolites to reveal putative metabolic pathways implicated in the ALT process, with the potential to be used as biomarkers or therapeutic targets for ALT-positive tumors. ALT and CDG gene set ALT related proteins were extracted from TelNet database , ( http://www.cancertelsys.org/telnet/ ): 411 were directly associated with the ALT process, and 71 were linked with the ALT-associated PML bodies . Cancer driver genes (CDGs) were filtered by type ( Pan-cancer ), including pan-cancer_adult and pan-cancer_paediatric subdivisions and by NCG_oncogene . A total of 106 CDGs were filtered. Prioritization of ALT-related proteins A total of 482 ALT-related proteins were prioritized based on their degree of alteration in both pan-cancer and mesenchymal-origin tumors. For pan-cancer analysis, these proteins were evaluated for genomic, transcriptomic, and proteomic alterations across 32 studies from the TCGA Pan Cancer Atlas (n = 10,918 samples), as previously reported . For mesenchymal-origin tumors, genomic alterations (mutations, structural variants, and copy number alterations) were extracted from 17 studies encompassing 12,405 samples (S1 Table in ) using the cBioPortal platform ( https://www.cbioportal.org/ ). Transcriptomic and proteomic data were unavailable in these datasets. Proteins were ranked according to their number of alterations, and the top quartile (Q1) was selected for further analysis . Detection of ALT-related metabolites To determine the metabolites implicated in ALT process, we indirectly identified which metabolites are interacting with our previously prioritized ALT-related proteins. Thus, we interrogated the Human Metabolome Database ( https://hmdb.ca/downloads ) . The interactions of these proteins with their corresponding metabolites were subsequently extracted from the “All Proteins” file using xml2 package (Wickham H, Hester J, Ooms J (2023). xml2: Parse XML. https://xml2.r-lib.org/ , https://github.com/r-lib/xml2 .) in RStudio 2023.06.1+524 "Mountain Hydrangea" Release (547dcf861cac0253a8abb52c135e44e02ba407a1, 2023-07-07) for windows. Mozilla/5.0 (Windows NT 10.0; Win64; x64) AppleWebKit/537.36 (KHTML, like Gecko) RStudio/2023.06.1+524. Chrome/110.0.5481.208 Electron/23.3.0 Safari/537.36. “All Proteins” file, released on 2021-11-09 (Size: 34.7 MB) was downloaded on 11/10/2023. Enrichment analysis of ALT-related metabolites To detect enriched metabolic pathways associated with the natural metabolites previously prioritized (n = 30), an enrichment analysis was performed using MetaboAnalyst 5.0 . The Small Molecule Pathway Database (SMPDB) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY Database were used for this analysis. To detect druggable metabolic pathways against ALT-positive tumors, we also performed an enrichment analysis in MetaboAnalyst 5.0 of the 30 naturally occurring ALT-related metabolites but using 461 metabolite sets based on drug pathways from SMPDB . Joint pathway analysis This integrative analysis was performed in the MetaboAnalyst 5.0 platform using two datasets: 1) 39 ALT-related genes, and 2) 30 naturally occurring metabolites associated with the ALT process. The KEGG PATHWAY Database (Kyoto Encyclopedia of Genes and Genomes) was used for this analysis. Parameters: 1) metabolomics type = targeted (compound list); 2) All pathways (integrated); 3) enrichment analysis = hypergeometric Test; 4) topology measure = degree centrality; 5) integration method = combine queries . Protein-metabolite interaction network Cytoscape v3.9.1 was used to generate an interaction network of the proteins and their associated metabolites. This network was constructed based protein-metabolite and protein-protein interactions (PMIs, PPIs) between ALT-related proteins, pan-cancer oncogenes, and their corresponding metabolites. PPIs were obtained from STRING database . The interactions were sourced from both experiments and databases with an interaction score of 0.9. This is the highest possible confidence of an interaction to be true based on all the available evidence. PMIs were extracted from the “All Proteins” file using xml2 package (Wickham H, Hester J, Ooms J (2023). xml2: Parse XML. https://xml2.r-lib.org/ , https://github.com/r-lib/xml2 .) in RStudio 2023.06.1+524 "Mountain Hydrangea" Release (547dcf861cac0253a8abb52c135e44e02ba407a1, 2023-07-07) for windows. Mozilla/5.0 (Windows NT 10.0; Win64; x64) AppleWebKit/537.36 (KHTML, like Gecko) RStudio/2023.06.1+524. Chrome/110.0.5481.208 Electron/23.3.0 Safari/537.36. ALT related proteins were extracted from TelNet database , ( http://www.cancertelsys.org/telnet/ ): 411 were directly associated with the ALT process, and 71 were linked with the ALT-associated PML bodies . Cancer driver genes (CDGs) were filtered by type ( Pan-cancer ), including pan-cancer_adult and pan-cancer_paediatric subdivisions and by NCG_oncogene . A total of 106 CDGs were filtered. A total of 482 ALT-related proteins were prioritized based on their degree of alteration in both pan-cancer and mesenchymal-origin tumors. For pan-cancer analysis, these proteins were evaluated for genomic, transcriptomic, and proteomic alterations across 32 studies from the TCGA Pan Cancer Atlas (n = 10,918 samples), as previously reported . For mesenchymal-origin tumors, genomic alterations (mutations, structural variants, and copy number alterations) were extracted from 17 studies encompassing 12,405 samples (S1 Table in ) using the cBioPortal platform ( https://www.cbioportal.org/ ). Transcriptomic and proteomic data were unavailable in these datasets. Proteins were ranked according to their number of alterations, and the top quartile (Q1) was selected for further analysis . To determine the metabolites implicated in ALT process, we indirectly identified which metabolites are interacting with our previously prioritized ALT-related proteins. Thus, we interrogated the Human Metabolome Database ( https://hmdb.ca/downloads ) . The interactions of these proteins with their corresponding metabolites were subsequently extracted from the “All Proteins” file using xml2 package (Wickham H, Hester J, Ooms J (2023). xml2: Parse XML. https://xml2.r-lib.org/ , https://github.com/r-lib/xml2 .) in RStudio 2023.06.1+524 "Mountain Hydrangea" Release (547dcf861cac0253a8abb52c135e44e02ba407a1, 2023-07-07) for windows. Mozilla/5.0 (Windows NT 10.0; Win64; x64) AppleWebKit/537.36 (KHTML, like Gecko) RStudio/2023.06.1+524. Chrome/110.0.5481.208 Electron/23.3.0 Safari/537.36. “All Proteins” file, released on 2021-11-09 (Size: 34.7 MB) was downloaded on 11/10/2023. To detect enriched metabolic pathways associated with the natural metabolites previously prioritized (n = 30), an enrichment analysis was performed using MetaboAnalyst 5.0 . The Small Molecule Pathway Database (SMPDB) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY Database were used for this analysis. To detect druggable metabolic pathways against ALT-positive tumors, we also performed an enrichment analysis in MetaboAnalyst 5.0 of the 30 naturally occurring ALT-related metabolites but using 461 metabolite sets based on drug pathways from SMPDB . This integrative analysis was performed in the MetaboAnalyst 5.0 platform using two datasets: 1) 39 ALT-related genes, and 2) 30 naturally occurring metabolites associated with the ALT process. The KEGG PATHWAY Database (Kyoto Encyclopedia of Genes and Genomes) was used for this analysis. Parameters: 1) metabolomics type = targeted (compound list); 2) All pathways (integrated); 3) enrichment analysis = hypergeometric Test; 4) topology measure = degree centrality; 5) integration method = combine queries . Cytoscape v3.9.1 was used to generate an interaction network of the proteins and their associated metabolites. This network was constructed based protein-metabolite and protein-protein interactions (PMIs, PPIs) between ALT-related proteins, pan-cancer oncogenes, and their corresponding metabolites. PPIs were obtained from STRING database . The interactions were sourced from both experiments and databases with an interaction score of 0.9. This is the highest possible confidence of an interaction to be true based on all the available evidence. PMIs were extracted from the “All Proteins” file using xml2 package (Wickham H, Hester J, Ooms J (2023). xml2: Parse XML. https://xml2.r-lib.org/ , https://github.com/r-lib/xml2 .) in RStudio 2023.06.1+524 "Mountain Hydrangea" Release (547dcf861cac0253a8abb52c135e44e02ba407a1, 2023-07-07) for windows. Mozilla/5.0 (Windows NT 10.0; Win64; x64) AppleWebKit/537.36 (KHTML, like Gecko) RStudio/2023.06.1+524. Chrome/110.0.5481.208 Electron/23.3.0 Safari/537.36. Identifying ALT-related metabolic pathways ALT-related metabolites To elucidate the metabolites implicated in the ALT process and further identify key metabolic pathways in pan-cancer, an indirect analysis was conducted. This analysis aimed to determine which specific metabolites engage in interactions with the ALT-related proteins that had been prioritized in previous stages of our research . These proteins were prioritized by multi-omic in silico analyses from our previous studies across 32 cancer types . To identify the metabolites linked with these proteins, the Human Metabolome Database was downloaded and interrogated . Thus, we identified a total of 39 ALT-related proteins interacting with 42 distinct metabolites (30 natural and 12 unnatural metabolites) (S2 and S3 Tables in ). Deregulation of some of these metabolites has been correlated to cancer, specifically to colorectal cancer, bladder cancer, and oral squamous cell carcinoma (S2 Table in ). ALT metabolic pathways To detect specific metabolic pathways related with these natural metabolites (n = 30), we next performed an enrichment analysis using MetaboAnalyst 5.0 with two databases, KEGG and SMPDB . The platform did not find pathway records for three metabolites: HMDB0251846, HMDB0059656 and HMDB0256910. Regarding the KEGG database, we have identified 6 statistically significant metabolic pathways: 1) nicotinate and nicotinamide metabolism (p-val ≤ 0.001), 2) purine metabolism (p-val ≤ 0.001), 3) citrate cycle (TCA cycle) (p-val ≤ 0.05), 4) glutathione metabolism (p-val ≤ 0.05), 5) glyoxylate and dicarboxylate metabolism (p-val ≤ 0.05), and 6) cysteine and methionine metabolism (p-val ≤ 0.05) ( ; S4 Table in ). Concerning the analysis of the SMPDB, we have identified 92 statistically significant metabolic pathways, 5 with p-values ≤ 0.05, 8 with p-values ≤ 0.01, and 79 with p-values ≤ 0.001 ( ; S4 Table in ). We finally merged both statistically significant pathway lists from SMPDB and KEGG databases and obtained a final list of 5 metabolic pathways ( ; S4 Table in ). shows the enriched metabolites associated with these pathways. Given that the ALT process has been observed in tumors of mesenchymal origin, we also prioritized the 482 ALT genes based on their genomic alterations, specifically within a cohort of 12,405 mesenchymal tissue tumor samples (S1 Table in ). We then selected the top quartile (Q1) based on their genomic alterations (S5 Table in ) to indirectly identify metabolic pathways associated with ALT in this tumor type (S6 and S7 Tables in ). Although transcriptomic and proteomic data were not available, we observed similar results . Thus, by integrating statistically significant pathways from the SMPDB and KEGG databases, we identified five key metabolic pathways: purine metabolism, citrate cycle (TCA cycle), pyruvate metabolism, glycolysis/gluconeogenesis, and nicotinate and nicotinamide metabolism. . Joint-pathway analysis of ALT-related proteins and metabolites To detect enriched metabolic and cellular pathways based on the presence of both genes and metabolites, we performed a joint pathway analysis using MetaboAnalyst 5.0 . We interrogated the database with the 39 ALT-related proteins along with their 30 naturally occurring metabolites. We detected 37 highly significant metabolic and cellular pathways (S8 Table in ; ). Three metabolic pathways have been primarily related with the ALT mechanism: 1) purine metabolism, 2) cysteine and methionine metabolism, and 3) nicotinate and nicotinamide metabolism (S8 Table in ; ). Networking analysis of ALT-related proteins, pan-cancer oncogenes, and metabolites Networking analysis has proved useful in identifying key elements among complex interactions. On this basis, to identify ALT-related metabolic pathways enhanced by pan cancer oncogenes, we next predict protein-metabolite (PMI) and protein-protein interactions (PPIs) between ALT-related proteins, pan-cancer oncogenes, and their corresponding metabolites ( and S9 Table in ). The interactions shown in are supported by experimental evidence from various cell lines , but remain predictive and require further validation in ALT-specific experimental models. In our detailed network analysis ( and S9 Table in ), we detected intricate interactions between ALT-related proteins, pan-cancer oncogenes, and associated metabolites. Three metabolic pathways emerged as principal components of the network: nicotinate and nicotinamide metabolism, cysteine/methionine metabolism, and purine metabolism. n the Nicotinate and nicotinamide metabolism pathway, key metabolites like Niacinamide, NAD, and NADH revealed pronounced interactions with ALT-related proteins, including pivotal roles for proteins such as SIRT2, PARP1, and PARP2. The Cysteine/methionine metabolism pathway, prominently featured by S-Adenosylmethionine and S-Adenosylhomocysteine metabolites, could be influenced by pan-cancer oncogenes EZH2 and SETDB1. The Purine metabolism pathway, vital for DNA replication and repair , showcased a dense interaction network. Metabolites related with this pathway interacts extensively with ALT-related proteins and pan-cancer oncogenes. Targeting ALT-related metabolic pathways To finally detect druggable metabolic pathways aiming to develop treatment strategies for ALT-positive tumors, we next performed an enrichment analysis in MetaboAnalyst 5.0 of the 30 naturally occurring ALT-related metabolites using 461 metabolite sets based on drug pathways from SMPDB . The platform did not find pathway records for three metabolites: HMDB0251846, HMDB0059656 and HMDB0256910. We have found 191 enriched pathways with p-values ≤ 0.0001 (S10 Table in ). illustrates the ten most promising druggable metabolic pathways, with purine metabolism prominently positioned as a prime target for possible therapeutic intervention. This central role highlights the potential for utilizing specific agents, such as mercaptopurine, thioguanine, and azathioprine. Azathioprine, thioguanine, and mercaptopurine are purine antimetabolite prodrugs exerting cytotoxic effects by incorporating their metabolites into DNA and RNA, inhibiting purine nucleotide synthesis, and targeting Ras-related C3 botulinum toxin substrate 1, leading to T cell apoptosis. All undergo metabolic conversions in cells, impacting purine pathways and cellular processes. The shared mechanism involves the inhibition of cellular functions and DNA/RNA incorporation, affecting cell survival and function . ALT-related metabolites To elucidate the metabolites implicated in the ALT process and further identify key metabolic pathways in pan-cancer, an indirect analysis was conducted. This analysis aimed to determine which specific metabolites engage in interactions with the ALT-related proteins that had been prioritized in previous stages of our research . These proteins were prioritized by multi-omic in silico analyses from our previous studies across 32 cancer types . To identify the metabolites linked with these proteins, the Human Metabolome Database was downloaded and interrogated . Thus, we identified a total of 39 ALT-related proteins interacting with 42 distinct metabolites (30 natural and 12 unnatural metabolites) (S2 and S3 Tables in ). Deregulation of some of these metabolites has been correlated to cancer, specifically to colorectal cancer, bladder cancer, and oral squamous cell carcinoma (S2 Table in ). ALT metabolic pathways To detect specific metabolic pathways related with these natural metabolites (n = 30), we next performed an enrichment analysis using MetaboAnalyst 5.0 with two databases, KEGG and SMPDB . The platform did not find pathway records for three metabolites: HMDB0251846, HMDB0059656 and HMDB0256910. Regarding the KEGG database, we have identified 6 statistically significant metabolic pathways: 1) nicotinate and nicotinamide metabolism (p-val ≤ 0.001), 2) purine metabolism (p-val ≤ 0.001), 3) citrate cycle (TCA cycle) (p-val ≤ 0.05), 4) glutathione metabolism (p-val ≤ 0.05), 5) glyoxylate and dicarboxylate metabolism (p-val ≤ 0.05), and 6) cysteine and methionine metabolism (p-val ≤ 0.05) ( ; S4 Table in ). Concerning the analysis of the SMPDB, we have identified 92 statistically significant metabolic pathways, 5 with p-values ≤ 0.05, 8 with p-values ≤ 0.01, and 79 with p-values ≤ 0.001 ( ; S4 Table in ). We finally merged both statistically significant pathway lists from SMPDB and KEGG databases and obtained a final list of 5 metabolic pathways ( ; S4 Table in ). shows the enriched metabolites associated with these pathways. Given that the ALT process has been observed in tumors of mesenchymal origin, we also prioritized the 482 ALT genes based on their genomic alterations, specifically within a cohort of 12,405 mesenchymal tissue tumor samples (S1 Table in ). We then selected the top quartile (Q1) based on their genomic alterations (S5 Table in ) to indirectly identify metabolic pathways associated with ALT in this tumor type (S6 and S7 Tables in ). Although transcriptomic and proteomic data were not available, we observed similar results . Thus, by integrating statistically significant pathways from the SMPDB and KEGG databases, we identified five key metabolic pathways: purine metabolism, citrate cycle (TCA cycle), pyruvate metabolism, glycolysis/gluconeogenesis, and nicotinate and nicotinamide metabolism. . Joint-pathway analysis of ALT-related proteins and metabolites To detect enriched metabolic and cellular pathways based on the presence of both genes and metabolites, we performed a joint pathway analysis using MetaboAnalyst 5.0 . We interrogated the database with the 39 ALT-related proteins along with their 30 naturally occurring metabolites. We detected 37 highly significant metabolic and cellular pathways (S8 Table in ; ). Three metabolic pathways have been primarily related with the ALT mechanism: 1) purine metabolism, 2) cysteine and methionine metabolism, and 3) nicotinate and nicotinamide metabolism (S8 Table in ; ). To elucidate the metabolites implicated in the ALT process and further identify key metabolic pathways in pan-cancer, an indirect analysis was conducted. This analysis aimed to determine which specific metabolites engage in interactions with the ALT-related proteins that had been prioritized in previous stages of our research . These proteins were prioritized by multi-omic in silico analyses from our previous studies across 32 cancer types . To identify the metabolites linked with these proteins, the Human Metabolome Database was downloaded and interrogated . Thus, we identified a total of 39 ALT-related proteins interacting with 42 distinct metabolites (30 natural and 12 unnatural metabolites) (S2 and S3 Tables in ). Deregulation of some of these metabolites has been correlated to cancer, specifically to colorectal cancer, bladder cancer, and oral squamous cell carcinoma (S2 Table in ). To detect specific metabolic pathways related with these natural metabolites (n = 30), we next performed an enrichment analysis using MetaboAnalyst 5.0 with two databases, KEGG and SMPDB . The platform did not find pathway records for three metabolites: HMDB0251846, HMDB0059656 and HMDB0256910. Regarding the KEGG database, we have identified 6 statistically significant metabolic pathways: 1) nicotinate and nicotinamide metabolism (p-val ≤ 0.001), 2) purine metabolism (p-val ≤ 0.001), 3) citrate cycle (TCA cycle) (p-val ≤ 0.05), 4) glutathione metabolism (p-val ≤ 0.05), 5) glyoxylate and dicarboxylate metabolism (p-val ≤ 0.05), and 6) cysteine and methionine metabolism (p-val ≤ 0.05) ( ; S4 Table in ). Concerning the analysis of the SMPDB, we have identified 92 statistically significant metabolic pathways, 5 with p-values ≤ 0.05, 8 with p-values ≤ 0.01, and 79 with p-values ≤ 0.001 ( ; S4 Table in ). We finally merged both statistically significant pathway lists from SMPDB and KEGG databases and obtained a final list of 5 metabolic pathways ( ; S4 Table in ). shows the enriched metabolites associated with these pathways. Given that the ALT process has been observed in tumors of mesenchymal origin, we also prioritized the 482 ALT genes based on their genomic alterations, specifically within a cohort of 12,405 mesenchymal tissue tumor samples (S1 Table in ). We then selected the top quartile (Q1) based on their genomic alterations (S5 Table in ) to indirectly identify metabolic pathways associated with ALT in this tumor type (S6 and S7 Tables in ). Although transcriptomic and proteomic data were not available, we observed similar results . Thus, by integrating statistically significant pathways from the SMPDB and KEGG databases, we identified five key metabolic pathways: purine metabolism, citrate cycle (TCA cycle), pyruvate metabolism, glycolysis/gluconeogenesis, and nicotinate and nicotinamide metabolism. . To detect enriched metabolic and cellular pathways based on the presence of both genes and metabolites, we performed a joint pathway analysis using MetaboAnalyst 5.0 . We interrogated the database with the 39 ALT-related proteins along with their 30 naturally occurring metabolites. We detected 37 highly significant metabolic and cellular pathways (S8 Table in ; ). Three metabolic pathways have been primarily related with the ALT mechanism: 1) purine metabolism, 2) cysteine and methionine metabolism, and 3) nicotinate and nicotinamide metabolism (S8 Table in ; ). Networking analysis has proved useful in identifying key elements among complex interactions. On this basis, to identify ALT-related metabolic pathways enhanced by pan cancer oncogenes, we next predict protein-metabolite (PMI) and protein-protein interactions (PPIs) between ALT-related proteins, pan-cancer oncogenes, and their corresponding metabolites ( and S9 Table in ). The interactions shown in are supported by experimental evidence from various cell lines , but remain predictive and require further validation in ALT-specific experimental models. In our detailed network analysis ( and S9 Table in ), we detected intricate interactions between ALT-related proteins, pan-cancer oncogenes, and associated metabolites. Three metabolic pathways emerged as principal components of the network: nicotinate and nicotinamide metabolism, cysteine/methionine metabolism, and purine metabolism. n the Nicotinate and nicotinamide metabolism pathway, key metabolites like Niacinamide, NAD, and NADH revealed pronounced interactions with ALT-related proteins, including pivotal roles for proteins such as SIRT2, PARP1, and PARP2. The Cysteine/methionine metabolism pathway, prominently featured by S-Adenosylmethionine and S-Adenosylhomocysteine metabolites, could be influenced by pan-cancer oncogenes EZH2 and SETDB1. The Purine metabolism pathway, vital for DNA replication and repair , showcased a dense interaction network. Metabolites related with this pathway interacts extensively with ALT-related proteins and pan-cancer oncogenes. To finally detect druggable metabolic pathways aiming to develop treatment strategies for ALT-positive tumors, we next performed an enrichment analysis in MetaboAnalyst 5.0 of the 30 naturally occurring ALT-related metabolites using 461 metabolite sets based on drug pathways from SMPDB . The platform did not find pathway records for three metabolites: HMDB0251846, HMDB0059656 and HMDB0256910. We have found 191 enriched pathways with p-values ≤ 0.0001 (S10 Table in ). illustrates the ten most promising druggable metabolic pathways, with purine metabolism prominently positioned as a prime target for possible therapeutic intervention. This central role highlights the potential for utilizing specific agents, such as mercaptopurine, thioguanine, and azathioprine. Azathioprine, thioguanine, and mercaptopurine are purine antimetabolite prodrugs exerting cytotoxic effects by incorporating their metabolites into DNA and RNA, inhibiting purine nucleotide synthesis, and targeting Ras-related C3 botulinum toxin substrate 1, leading to T cell apoptosis. All undergo metabolic conversions in cells, impacting purine pathways and cellular processes. The shared mechanism involves the inhibition of cellular functions and DNA/RNA incorporation, affecting cell survival and function . In prior studies, we have identified key genes associated with the Alternative Lengthening of Telomeres (ALT) using multi-omic data integration strategies. These genes may be useful as targets for diagnosing and treating ALT-positive cancers . The objective of this study was to identify targetable metabolic pathways for ALT-positive tumors by integrating metabolomics data with our previous findings. Thus, we first identified 39 ALT-related proteins interacting with 42 distinct metabolites. These metabolites were significantly enriched in five metabolic pathways: 1) nicotinate and nicotinamide metabolism, 2) purine metabolism, 3) citrate cycle (TCA cycle), 4) cysteine and methionine metabolism, and 5) glutathione metabolism . To narrow down the identification of ALT-metabolic pathways, we performed a joint pathway analysis including both genes and metabolites and prioritized three metabolic pathways: 1) nicotinate and nicotinamide metabolism, 2) purine metabolism, and 3) cysteine and methionine metabolism . Noteworthy, we also found pan-cancer oncoproteins that could influence these ALT metabolic pathways . Finally, we identified purine metabolism as a prime target against ALT-positive tumors by using specific agents such as mercaptopurine, thioguanine, and azathioprine . The comparison of metabolic pathways in mesenchymal-origin tumors vs. those across pan-cancer highlights both shared and unique features of the ALT mechanism. While key pathways such as purine metabolism, nicotinate and nicotinamide metabolism, and citrate cycle metabolism emerged prominently in both mesenchymal and broader tumor contexts, certain metabolic dependencies may be more pronounced in mesenchymal-origin tumors due to their specific cellular background. This alignment suggests that while some metabolic routes are universally associated with ALT-positive tumors, the mesenchymal context may amplify or modulate these pathways, potentially impacting tumor behavior and response to targeted therapies. PPIs and PMIs presented in were detected experimentally and validated using multiple sources (e.g. PubMed and KEGG). These interactions are curated by experts who review experimental findings and ensure the reliability of the data. Most of these interactions are well-documented and have been identified in cancer cell lines . Specifically, interactions between PARP1, PARP2, and niacinamide are well-known in the synthesis and regulation of NAD+, a crucial coenzyme involved in DNA repair and cellular metabolism. PARP1 and PARP2 use NAD+ as a substrate to carry out poly-ADP-ribosylation, a process critical for DNA damage repair and genomic stability. Niacinamide plays a key role in the NAD+ salvage pathway, where it helps regenerate NAD+ from nicotinamide, thus fueling the activities of PARP enzymes during cellular stress and DNA repair responses . Regarding purine metabolism, it is well-stablished that deoxyadenosine triphosphate (dATP), thymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP) are substrates for DNA polymerases POLH, POLE, and POLD2. These components are essential for synthesizing new DNA strands during replication and repair processes in cancer cells . However, it is important to note that these interactions are predictive in nature and still require experimental validation to confirm their occurrence in ALT-specific models. Mass spectrometry-based protein-protein interaction networks could help address this need. Thus, techniques such as affinity purification mass spectrometry (MS), proximity labeling, and cross-linking MS could be used to study PPIs globally . On the other hand, PMIs could also be detected through chemical and structural proteomics approaches or by thermal proteome profiling (TPP) . Purine metabolism is regulated by purinosomes, which are multi-enzyme complexes—composed of nine enzymes—that promotes cancer progression through uncontrolled cell proliferation and increased tumor viability . In glioblastoma, alterations in purine metabolism have been associated not only to genomic instability and cell death but also to uncontrolled cell growth . Within purine metabolism, the metabolites dCTP and dGTP are associated with cancer progression. For instance, the dCTPase has been observed to be highly expressed in multiple carcinomas , while several studies have demonstrated that dGTP and its derivatives are involved in various aspects of cancer, such as cancer cell survival under oxidative stress, and as targets in chemotherapy and apoptosis induction . Interestingly, we found that dCTP and dGTP interact with ALT-related proteins POLE, POLH, and POLD2, which in turn interact with various ALT proteins. . In addition, several studies have shown that mutations in these proteins promote tumor progression in several cancer types . In glioma, inhibition of purine synthesis has been shown to impaired the self-renewal of brain tumor-initiating cells . This highlights the importance of purine metabolism in cancer treatment and suggest that manipulating this pathway or purinosomes may offer new ways to impair ALT mechanism . In this context, mercaptopurine, thioguanine, and azathioprine could be used to inhibit purine metabolism and induce cancer cell death . Regarding the nicotinate and nicotinamide metabolism pathway, the nicotinamide adenine dinucleotide (NAD+) coenzyme regulates various cellular processes such as cellular energy production, DNA repair, telomerase activity, cell growth, and cell death, thereby linking it to tumor growth and survival ; its deregulation, a common phenomenon in diverse types of cancer, can trigger several events that favor tumor development and progression . This is exemplified in glioblastoma, where the overexpression of nicotinamide N-methyltransferase (NNMT) depletes the methyl donor S-adenosylmethionine (SAM), thereby promoting tumor growth . It has also been shown that the expression of nicotinamide phosphoribosyltransferase (NAMPT), an essential enzyme for NAD+ synthesis, is increased in the most aggressive and invasive cancers and in tumor metastases . Additionally, NAD+ participates in the base excision repair (BER) pathway, which operates through the PARP-NAD-SIRT axis . This aligns with our results presented in , where NAD+ interacts with ALT-related proteins: PARP1, PARP2, and SIRT2. Thus, targeting NAD+ metabolism could induce cytotoxicity in cancer cells . Concerning cysteine and methionine metabolism, it has been observed that alterations in this pathway contributes to tumorigenesis by facilitating epigenetic regulation of gene expression . Furthermore, this has also been observed in different cancer cell lines, suggesting a link with oncogenesis . Moreover, some cancer types are auxotrophic for methionine due to alterations in methionine metabolism, which has been used for therapeutic approaches . In our analysis related with this pathway , ALT-related proteins DNMT1, DNMT3A, DNMT3B, CARM1, and PRMT2 interact with S-Adenosylmethionine and S-Adenosylhomocysteine metabolites. Noteworthy, several studies have shown that alterations in these proteins contribute to tumorigenesis . For instance, the CARM1 protein plays a multifaceted role in cancer metabolism, affecting metabolic pathways, regulating gene expression, and promoting metastasis . Conversely, the PRMT2 protein is implicated in cancer metabolism through the modulation of hormonal receptor signaling, gene expression, and cell cycle regulation . Moreover, this pathway could be influenced by two CDGs, EZH2 and SETDB1. Indeed, SETDB1, a histone methyltransferase, promotes heterochromatin formation at telomeres. This, in turn, stimulates transcriptional elongation at telomeres and the recruitment of essential ALT factors . Moreover, targeting EZH2 for cancer therapy is currently a highly active area of research, and various types of EZH2 inhibitors have been developed . This study indirectly prioritized three metabolic pathways related to the ALT process and identified potential therapeutic targets. These findings point to the need for more research into these metabolic pathways and how they affect the growth of ALT-positive tumors. Thus, this research could lead to the development of new therapeutic approaches to combat this tumoral process. While this work provides a wider understanding of the metabolomic aspects of ALT-positive cancers, further experimental research is needed to elucidate the functional roles of these metabolites in this process. S1 File A single file containing S1 to S10 Tables. Each table is numbered and labeled within the document. (XLSX) S1 Fig Identification of ALT proteins engaged in protein-metabolite interactions. A workflow detailing the identification, filtration, and prioritization of ALT-related genes and their interactions with metabolites in pan-cancer and tumors from mesenchymal origin. ALT = Alternative Lengthening of Telomeres; APB = ALT-associated PML Bodies; PMIs = Protein-Metabolite interactions. (PDF) S2 Fig ALT-related metabolic pathways in soft tissue tumors. Enrichment analysis of ALT-related metabolites using KEGG (A) and SMPDB (B) databases. Metabolite pathways are listed on the y-axis with their respective significance levels shown on the x-axis, represented as the negative logarithm (base 10) of the p-value. The size of the circles is proportional to the enrichment ratio, with larger circles indicating higher enrichment. (C) Venn diagram displaying shared statistically significant pathways between SMPDB and KEGG. (PDF) |
MIlitary Combat Mental Health Framework | 25d81113-0339-49d7-a265-3aa0b05c83ae | 8223620 | Preventive Medicine[mh] | Armed forces and veteran populations may have exposures to combat that cause a severe impact on their physical, mental or social health. The primary UK long-term cohort study of a sample of UK military personnel and veterans from the wars in Iraq and Afghanistan has shown a prevalence of symptoms of 21.9% for common mental disorders, 10.0% for alcohol misuse and 6.2% for probable post-traumatic stress disorder (PTSD). PTSD was the most common clinical keyword in the global military medical literature between 1988 and 2017. While there is a substantial body of research on military service and mental health, there are very few models that integrate this evidence to inform policy and practice. The purpose of this paper is to offer an explanatory framework to support further debate on risk, treatment and social interventions for combat-related mental ill-health in armed forces personnel and veterans. However, it should be noted exposure to combat is not the primary cause of mental ill-health in this population and that the overall incidence of mental ill-health is less than an age-matched group of the overall UK population. In October 2019, the North Atlantic Treaty Organisation published Standardisation Agreements for military medical services on deployment mental health. While not all mental illness resulting from combat exposure is PTSD, a meta-analysis of risk factors for combat-related PTSD and a comprehensive review of military-related PTSD are valuable summaries of the research evidence. These papers are the basis for the Military Combat Mental Health Framework shown in . Exposure to one or more ‘potential trauma events’ (PTE) is essential for causation. A PTE is an event occurring as a result of combat that involved actual or threatened death or serious injury, or threat to the physical integrity of self or others. The framework extends from before military service through to postmilitary life as the mental health consequences of exposure to combat-related stress may be delayed and first present in veterans. An individual’s mental well-being may fluctuate on a continuum between ‘Fit’, ‘Reacting’, ‘Injured’ and ‘Ill’ defined by function within their communities. This ‘community’ context is important because it is family, friends and coworkers who may notice the consequences of mental ill-health before the individual recognises or accepts their condition. The framework uses the following definitions: Fit (or healthy) Individuals in this zone have a state of balanced ‘well-being’ across all domains of health and are functional for their role within work, personal life and social environment (communities). This extrapolates the concepts behind physical fitness (trained optimal performance) into mental and social fitness. Reacting Individuals within this zone are experiencing mild, transient stress reactions as a result of their combat-related experiences. These are a common human reaction to the realities of combat and are recognisable if explained to third party. Injured This state includes more severe, persistent symptoms that result in abnormalities in an individual’s behaviour and are not getting better. A third party may notice this change because of changes in the individual’s functioning within their communities, but their condition would not meet the criteria for a formal clinical diagnosis. Ill Illness occurs when an individual’s combat experience has a significant impact on their function within their communities, indicating a severe disorder that can be clinically diagnosed, such as major depression, anxiety or PTSD. The framework shows a hypothetical trajectory of an individual’s mental health course through their life after joining the armed forces. The initial trajectory assumes improved mental fitness compared with entry to the armed forces as military training is designed to increase their physical fitness and provide psychological preparation for their military role. This experience and their social community help to develop personal resilience factors . Until the first PTE, military personnel have no combat-related mental ill-health and have the potential to be mentally Fit. The first PTE may result in a stress reaction (reacting), but they may be able to return to fitness because of their resilience factors. They could then experience a second PTE, which may compound the effects of the first PTE. After a variable duration, this exposure may cause a progressive deterioration of their mental health becoming injured or ill as a result of precipitating factors . Successful therapeutic interventions to improve from illness will depend on treatment factors , though long-term mental health will depend on recovery factors . Unfortunately, a permanent ‘cure’ may not be possible, and individuals may deteriorate again as a result of the re-emergence of precipitating factors. This is shown as happening in the individual’s life course after military service as a veteran. Individuals in this zone have a state of balanced ‘well-being’ across all domains of health and are functional for their role within work, personal life and social environment (communities). This extrapolates the concepts behind physical fitness (trained optimal performance) into mental and social fitness. Individuals within this zone are experiencing mild, transient stress reactions as a result of their combat-related experiences. These are a common human reaction to the realities of combat and are recognisable if explained to third party. This state includes more severe, persistent symptoms that result in abnormalities in an individual’s behaviour and are not getting better. A third party may notice this change because of changes in the individual’s functioning within their communities, but their condition would not meet the criteria for a formal clinical diagnosis. Illness occurs when an individual’s combat experience has a significant impact on their function within their communities, indicating a severe disorder that can be clinically diagnosed, such as major depression, anxiety or PTSD. The framework shows a hypothetical trajectory of an individual’s mental health course through their life after joining the armed forces. The initial trajectory assumes improved mental fitness compared with entry to the armed forces as military training is designed to increase their physical fitness and provide psychological preparation for their military role. This experience and their social community help to develop personal resilience factors . Until the first PTE, military personnel have no combat-related mental ill-health and have the potential to be mentally Fit. The first PTE may result in a stress reaction (reacting), but they may be able to return to fitness because of their resilience factors. They could then experience a second PTE, which may compound the effects of the first PTE. After a variable duration, this exposure may cause a progressive deterioration of their mental health becoming injured or ill as a result of precipitating factors . Successful therapeutic interventions to improve from illness will depend on treatment factors , though long-term mental health will depend on recovery factors . Unfortunately, a permanent ‘cure’ may not be possible, and individuals may deteriorate again as a result of the re-emergence of precipitating factors. This is shown as happening in the individual’s life course after military service as a veteran. There is strong epidemiological evidence that an individual’s risk of military combat-related mental illness can be associated with a range of factors that accrue during their life course. These are categorised into the following groups with examples in each. Prejoining vulnerability factors Prejoining vulnerability factors are adverse life experiences that occur prior to military service. The existence of these is fixed but their impact will vary according to subsequent life events. They are common in the population that seek to join the armed forces and include: childhood adversity and childhood antisocial behaviour; low educational attainment (which has an influence in choice of military employment, the likelihood of being employed in a combat role and socioeconomic status as a veteran); and pre-service mental ill-health (though most militaries exclude applicants with a previous mental health diagnosis). While these risk factors are not suitable as screening criteria for exclusion from military service, there may be value in identifying them in those that seek help after a PTE in order to inform clinical interventions. Resilience factors Resilience factors are those, post-entry training, organisational and personal interventions that reduce (or the absence of which increase) the risk that an individual may experience mental illness as the result of exposure to a PTE. These may include: not being deployed as a reservist; absence of alcohol or substance abuse; being a non-smoker; absence of previous mental ill-health; not in a combat role (though this may be solely due to reduction in risk of exposure to a PTE); being in a personal relationship; unit cohesion and leadership; rest and recuperation during operational deployment; post-tour third location decompression; higher rank; recipient of Trauma Risk Management support (TRiM); deployment length less than 6 months; and lower total number of deployments. Many of these factors are amenable to organisational interventions at the group level. Precipitating factors A severe PTE may result in an acute stress disorder (immediate onset of debilitating mental illness). However, there may also be a time lag between exposure, the onset of mental ill-health and seeking help. Precipitating factors are those that trigger a deterioration to the extent that the individual becomes mentally injured or ill. These may include: concurrent physical illness or injury, severity of PTE, poor sleep, physically aggressive behaviour and concurrent alcohol misuse. There may be differences between the factors that precipitate combat-related mental ill-health while in military service and those factors that apply to veterans who seek help. Thus, a separate ‘Precipitating Factors’ box is shown after transition to veteran. Treatment factors Although there are clearly established clinical modalities for the treatment of combat mental illness, there is variation in the clinical outcomes of these therapies for individual patients. Some of this variation can be explained by adverse clinical and non-clinical treatment factors such as: presence of non-PTE concurrent mental illness; poor outcomes from concurrent physical illness; chronic pain; older age; duration of functional impairment prior to seeking treatment; severity of functional impairment; perception of stigma to seeking help; and lack of an internal locus of control. Recovery factors The final group of factors is labelled recovery factors. These are the psychosocial factors that facilitate recovery from the injured through to the fit zones and mitigate against the precipitating factors causing a recurrence of a deterioration in mental well-being for the individual. All military personnel will transition to veteran at some point in their life and so these factors apply both during and after military service. Positive recovery factors include: not smoking; no substance or alcohol abuse; no risk-taking behaviour; no homelessness; no criminal activity; post-traumatic growth; remaining in military service; being in employment; having a personal relationship; and no decline in cognition in later age. Social networks play an important role in personal resilience with an increase in common mental disorders and PTSD symptoms in service leavers after they have left their social network in the military. This can extend to non-healthcare social interventions such as adventurous activities, surfing and ‘animal therapies’. Prejoining vulnerability factors are adverse life experiences that occur prior to military service. The existence of these is fixed but their impact will vary according to subsequent life events. They are common in the population that seek to join the armed forces and include: childhood adversity and childhood antisocial behaviour; low educational attainment (which has an influence in choice of military employment, the likelihood of being employed in a combat role and socioeconomic status as a veteran); and pre-service mental ill-health (though most militaries exclude applicants with a previous mental health diagnosis). While these risk factors are not suitable as screening criteria for exclusion from military service, there may be value in identifying them in those that seek help after a PTE in order to inform clinical interventions. Resilience factors are those, post-entry training, organisational and personal interventions that reduce (or the absence of which increase) the risk that an individual may experience mental illness as the result of exposure to a PTE. These may include: not being deployed as a reservist; absence of alcohol or substance abuse; being a non-smoker; absence of previous mental ill-health; not in a combat role (though this may be solely due to reduction in risk of exposure to a PTE); being in a personal relationship; unit cohesion and leadership; rest and recuperation during operational deployment; post-tour third location decompression; higher rank; recipient of Trauma Risk Management support (TRiM); deployment length less than 6 months; and lower total number of deployments. Many of these factors are amenable to organisational interventions at the group level. A severe PTE may result in an acute stress disorder (immediate onset of debilitating mental illness). However, there may also be a time lag between exposure, the onset of mental ill-health and seeking help. Precipitating factors are those that trigger a deterioration to the extent that the individual becomes mentally injured or ill. These may include: concurrent physical illness or injury, severity of PTE, poor sleep, physically aggressive behaviour and concurrent alcohol misuse. There may be differences between the factors that precipitate combat-related mental ill-health while in military service and those factors that apply to veterans who seek help. Thus, a separate ‘Precipitating Factors’ box is shown after transition to veteran. Although there are clearly established clinical modalities for the treatment of combat mental illness, there is variation in the clinical outcomes of these therapies for individual patients. Some of this variation can be explained by adverse clinical and non-clinical treatment factors such as: presence of non-PTE concurrent mental illness; poor outcomes from concurrent physical illness; chronic pain; older age; duration of functional impairment prior to seeking treatment; severity of functional impairment; perception of stigma to seeking help; and lack of an internal locus of control. The final group of factors is labelled recovery factors. These are the psychosocial factors that facilitate recovery from the injured through to the fit zones and mitigate against the precipitating factors causing a recurrence of a deterioration in mental well-being for the individual. All military personnel will transition to veteran at some point in their life and so these factors apply both during and after military service. Positive recovery factors include: not smoking; no substance or alcohol abuse; no risk-taking behaviour; no homelessness; no criminal activity; post-traumatic growth; remaining in military service; being in employment; having a personal relationship; and no decline in cognition in later age. Social networks play an important role in personal resilience with an increase in common mental disorders and PTSD symptoms in service leavers after they have left their social network in the military. This can extend to non-healthcare social interventions such as adventurous activities, surfing and ‘animal therapies’. This framework adds to those that already exist by illustrating mental health across the life course from before military service to becoming a veteran. It categorises risk factors to inform individual, organisational and therapeutic interventions to mitigate and manage the risks from exposure to a PTE on an individual’s mental health. The actual experience of mental well-being or ill-health for an individual may not exactly follow the trajectory shown. The framework de-emphasises the notion of a permanent ‘cure’ for mental ill-health and recognises the inter-relationships between physical, mental and social domains of health. This reinforces the importance of the social domain of health and the need to consider ‘social illness’ and ‘social interventions’ alongside mental illness and mental interventions. It also highlights the potential for community and social interventions during the ‘Reacting’ and ‘Injured’ stage when interventions by clinical professionals may not be required or appropriate. The framework has created categories for the various factors that influence the risk of combat-related mental ill-health. These categories may need further refinement to develop the best descriptors for each factor and to identify those factors with positive attributes alongside negative risk factors. The prejoining vulnerability factors are not sufficiently specific or sensitive for use as screening criteria to exclude individuals from joining the armed forces. There is also evidence that postdeployment screening for mental ill-health in military personnel was not effective reducing the prevalence of mental disorders in the UK population. However, this does not remove the need for general awareness of the symptoms and signs of mental ill-health across the military population as demonstrated by the Trauma Risk Management programme and briefings during Third Location Decompression. Knowledge of these factors can also inform the inter-relationship of the responsibilities between the individual, their social circumstances, the armed forces as an employer, the government for statutory services for veterans, and the contribution of charities and the wider the voluntary sector for mitigating mental ill-health in this population. The time course emphasises the importance of maintaining the observational research cohorts from the deployment to Iraq and Afghanistan in order to observe risk factors of combat-related mental ill-health over the long term. It is also necessary to ensure the replenishment of the cohorts with new entrants to the armed forces so that cohort studies can be rapidly initiated at the beginning of the next combat operation. Finally, the model could apply to non-military populations with similar exposures such as front-line emergency responders, journalists and humanitarian aid workers. The model does not illustrate the balance of non-PTE exposures (eg, non-combat deployments, separation from personal relationships and work-related stress) as risk factors nor does it accommodate the emerging concept of ‘moral injury’ as a source of mental ill-health in military populations. This paper described the Military Combat Mental Health Framework to aid understanding of combat-related mental illness in armed forces personnel and veterans. It is based on a life course approach that maps an individual’s mental well-being against four ‘states’: fit, reacting, injured and ill. The paper described five categories of factors that influence the risk of mental illness from exposure to PTEs: prejoining vulnerability, resilience, precipitating, treatment and recovery. This framework provides a structure to debate current knowledge, inform policy and therapeutic interventions and to guide future research into this subject. |
Effect of porcine collagen matrix versus rotated pedicle palatal connective tissue flap on the soft-tissue closure after the immediate posterior implantation: study protocol for a randomised controlled trial | 99f1b867-355d-4580-b2ce-44724af2be31 | 11749683 | Dentistry[mh] | A high success rate can be expected when dental implants are placed according to standard procedure. To further improve outcomes, clinical workflows should be optimised to achieve minimally invasive procedures, higher success rates and higher patient satisfaction. Owing to advancements in innovative surface modification technologies and implant designs, recent protocols have arisen, shortening the time intervals by means of immediate implantation and immediate restoration. Implant placement in fresh extraction sockets in conjunction with appropriate guided bone regeneration is well documented. A systematic review reported that immediate implantation in molar extraction socket might be considered a predictable technique, as demonstrated by high survival and success rates, with minimal marginal bone loss. The immediate implantation still faces obstacles, primarily due to the lack of hard tissue. Initial implant stability is the key to osseointegration, which mostly comes from the contact between basal bone and implant. It is challenging to achieve adequate bone and implant contact in molar immediate implantation due to the lack of apical and lateral bone compared with other tooth extraction sites. Smith and Tarnow proposed a classification system dividing molar extraction sockets into three categories, in which type B socket has sufficient septal bone to achieve initial stability but not enough to fully contain the implant. Early researchers demonstrated that the implant site should be grafted when the gap between an implant and the adjacent bone is over 2 mm. The ultra-wide-diameter (6–9 mm) implants serve to increase the implant and bone contact and decrease the gap. However, Ketabi found that implant failures were higher in ultra-wide-diameter (6–9 mm) implants than wide-diameter implants (4–6 mm). Soft-tissue defect is commonly seen in immediate implantation because of tooth extraction wound and tension from bone graft. Bone graft materials exposure has a significant detrimental influence on bone augmentation. For peri-implant bone defects, the sites without membrane exposure had 27% more defect reduction than those with exposure. Hence, primary wound closure is necessary to create a stable environment to avoid plaque attachment, infection, bone absorption and implant failure. Various soft-tissue management techniques in immediate implantation procedure have been discussed, among which pedicled flap is considered safe and predictable. Rotated pedicle palatal connective tissue flap (RPPCTF) helps achieve primary closure, combines with soft-tissue augmentation and maintains the vascularity of the flap. A drawback of the soft-tissue graft is the discomfort from the secondary surgical area. Therefore, xenogenic collagen matrix is proposed to be used as a substitute. There are many studies comparing the soft-tissue augmentation of xenogenic collagen matrix with various soft-tissue grafts, most of which showed that the effect of xenogenic collagen matrix was similar to or slightly lower than soft-tissue graft, with less postoperative reaction and reduced operative times. However, these studies did not compare the effect of RPPCTF and porcine collagen matrix on maxillary immediate implant sites. Further evidence should be explored to guide wound closure after immediate posterior implantation. Moreover, previous studies did not use the intraoral scanner and 3D method to accurately evaluate the soft-tissue and hard-tissue augmentation and the volume changes at different follow-up time points. Additionally, Bevilacqua suggested new randomised clinical trials would be desirable for a histological evaluation. The aim of this randomised controlled trial is to assess the effect of using xenogenic collagen matrix compared with RPPCTF on achieving primary wound closure in sites with insufficient soft-tissue volume after maxillary immediate posterior implantation by using 3D integration technology. Study design and setting This is a single-centre, prospective, evaluator-blinded, randomised, two-arm parallel-group controlled trial. It commenced in October 2023 and will conclude in October 2025 at the Department of Implantology and Department of Prosthodontics in Shanghai Stomatological Hospital. According to the sample size calculation, we plan to recruit 54 patients in need of immediate implant placement in the first upper molar extraction sockets. The patients will be randomly assigned to receive the porcine collagen matrix (Mucograft, Geistlich Pharma AG) or RPPCTF on the soft-tissue closure after the immediate posterior implantation. The treatment process is presented in . The project has been registered on the ClinicalTrials.gov website ( https://clinicaltrials.gov/ ; identifier no. ChiCTR2300067770) and National Medical Research Registration and Filing Information System ( https://www.medicalresearch.org.cn/ ). We will approach participants who meet the inclusion criteria after tooth extraction to inquire about their interest regarding this study. If interested, potential participants will be referred to our study team members who will provide a detailed description of the study procedures and invite them to participate. Written informed consent will be obtained prior to the collection of any study data. Sample size The required sample size for detecting a clinically significant effect size in the primary endpoint, which is the changes in soft-tissue volume between two groups, was determined using a two-sample t-test. The calculation was based on a two-tailed test with a significance level of 0.05 and a power of 0.8. A clinically significant difference was defined as a mean keratinised soft-tissue width exceeding 1.5 mm. Assuming an SD of approximately 1.8 mm and accounting for a 10% dropout rate, it was estimated a total of 54 patients (27 in each group) would be required to detect an effect of that size. Inclusion criteria Adult patients aged over 18 years old requiring extraction of first upper molar for caries, fracture or prosthetic reason. The presence of type B extraction socket with the intact buccal bone over 1 mm evaluated on the preoperative cone beam CT (CBCT). Sufficient residual bone to maintain one implant with at least 4.3 mm in diameter, 10 mm in length. Systemically healthy patients not smoking more than 10 cigarettes per day. Patients with adequate oral hygiene with full-mouth plaque score less than 25%. Exclusion criteria Uncontrolled diabetes with HbA1c over 7%, osteoporosis or any other systemic or local disease or condition that would compromise postoperative healing. Patients with a history of malignancy, radiotherapy or chemotherapy for the treatment of malignancy. Pregnant patient or intending to become pregnant or nursing at the time of study inclusion. Patients taking medications or having treatments with an effect on healing in general, including steroids use, large doses of anti‐inflammatory drugs and bisphosphonates. Periapical inflammation. Allocation, randomisation and blinding This study is a single-centre, prospective, evaluator-blinded, randomised, two-arm parallel-group controlled trial. All eligible patients were randomly assigned to two groups after CBCT assessment and obtaining informed consent. The allocation sequence was generated using computer software (SAS) and sealed in opaque envelopes by WZ who would not participate in subsequent studies. These envelopes were opened immediately before intervention by WL. WL did not attend the enrolment of the participants, assignment of the interventions or assessment of the outcome. KH and SF enrolled the participants. The clinical operators will not have access to the statistical analysis process. The outcome indicators were assessed using a single-blind method. The data collectors and outcome evaluators did not know the group information during the whole research process. Interventions and observation The upper first molar will be extracted using a minimally invasive approach to preserve the integrity of the buccal bone and surrounding hard tissue. After CBCT assessment of the residual bone and socket type, patients will be randomly assigned to two groups: Control group: n=27, immediate implant placement with deproteinised bovine bone mineral (Bio-oss, Geistlich Pharma AG) grafted into the gap, up to the buccal bone crest, covered by type I collagen membrane (Bio-Gide, Geistlich Pharma AG). Wound will be closed by RPPCTF. Experimental group: n=27, immediate implant placement with deproteinised bovine bone mineral grafted into the gap, up to the buccal bone crest, covered by type I collagen membrane. Wound will be closed by porcine collagen matrix. Subsequently, the soft-tissue borders will be de-epithelialised. Single interrupted resorbable suture and horizontal mattress suture will be used to stabilise the grafted materials. The patients will be recalled timely when unexpected circumstances occur, such as bleeding, surgical area infection and implant loss. When the wound is infected or the soft tissue is necrotic, the doctor will perform a timely debridement. During this period, the cost associated with debridement, bone grafting and secondary surgery will all be free. In addition, considering that the secondary surgery would increase revisiting frequency, a transportation subsidy will also be provided as compensation. The 3Shape intraoral scanner (Trios 3, 3Shape) will be used to obtain the 3D soft-tissue morphology after surgery, at 2 weeks, 1 month, 3 months, 6 months and 12 months to evaluate the soft-tissue healing and the volume collapse. All the 3D soft-tissue data will be superimposed in the Geomagic Studio software (Raindrop Geomagic, USA) to compare volume and thickness changes. First, standard tessellation language (STL) files will be aligned and superimposed by selecting eight reproducible anatomical points in the Geomagic Studio software. A standardised region of interest will be delimited with a digital pen at the peri-implant aspect. Then the change in thickness, peri-implant soft-tissue contour area and its corresponding contraction rates between the corresponding time points will be assessed by applying the computer aid design (CAD) comparison tool. Each analysis will be performed in triplicate by one calibrated examiner. Additional follow-up propaganda and education will be conducted when patients fail to follow-up on time. The CBCT will be performed before tooth extraction to evaluate the socket type and the residual bone volume. The vertical height and horizontal width of the bone crest at buccal midpoint, buccal mesial point and buccal distal point will then be measured at the postoperation baseline, 3 months, 6 months and 12 months. The operation time of two different groups will be recorded and compared, which starts from the cover screw delivery and ends in the wound closure. The patient’s postoperative painkiller taking time and dosage will also be recorded. Visual analogue scale (VAS) will be used to evaluate comfort and satisfaction at 2 weeks after surgery. 6 months after the surgical procedure, a 5 mm diameter micro-punch biopsy of the implantation area will be performed. The tissue will be histologically evaluated, graded into four categories: immature, mature, fragmented and oedematous collagen tissue. The elastic fibres will also be examined and graded in three categories: with a normal structure, fragmented rare and fragmented multiplied. The photographs will be taken with a digital single-lens reflex after the crown delivery and evaluated under similar conditions. The Pink Esthetics Scores (PES) will be recorded by three dentists (KH, ZD and CZ) with experience and prior training in using PES index in research settings. Examiner ZD is an oral implantologist with 20 years of clinical experience. Examiners KH and CZ are oral implantologists with 6 years of clinical experience. Before commencing PES assessment, all the examiners would calibrate the assessment procedure to ensure high intraexaminer and interexaminer reliability. Outcomes Primary endpoint The primary outcome is the soft-tissue volume change because this parameter is imperative in evaluating the long-term stability of the two methods for wound closure. The soft-tissue volume change evaluation will be conducted during postoperative 6 months. Soft-tissue healing conditions at baseline, 2 weeks, 1 month, 3 months and 6 months will be recorded and evaluated using a 3D intraoral scanner. Then the soft-tissue data at different time points will be integrated into 3D software for the purpose of comparing tissue collapse. Secondary endpoints Marginal bone change in postoperative 12 months, operation time, patient comfort and satisfaction, and the final aesthetics will be assessed and recorded. The marginal bone changes at baseline, 3 months, 6 months and 12 months will be compared in both groups via CBCT and 3D data software. The differences in operation time, postoperative patient comfort and satisfaction, and the final aesthetics between the two groups will also be recorded. Data collection, data management and monitoring All participants were identified by their random number ID. All these data have been collected by data collectors according to the record form and have been documented on paper, which would be entered using EpiData software. The user name and password were saved and operated by KH. Data management was conducted by two experimenters independently every month. The monitoring team consisting of two experienced clinicians, two biostatisticians and one ethicist met every 3 months to ensure the trial followed the protocol and the provisions of good clinical practice. The publication of the trial data will be managed by a data manager appointed by the principal investigators. Statistical analysis Statistical analysis will be conducted using Statistical Package for Social Sciences software (IBM). Continuous variables, if normally distributed, will be presented as the mean±SD, and if skewed, as the median and IQR. Categorical variables will be presented as counts and percentages. All analyses will be based on the intent-to-treat principle. Mixed-effect model will be adopted to evaluate the differences of soft-tissue volume change and marginal bone-level change between the experimental group and control group. The time point will be set as repeated measures with treatment as a fixed effect. A two-sample t-test or Mann-Whitney U test will be used to compare the differences of width of keratinised gingiva, operation time and VAS scores of patient comfort and satisfaction, and the final aesthetics. Multiple imputations will be used as a sensitivity analysis for missing values of primary outcome. All tests will be performed at the both-sided 0.05 significance level unless otherwise noted. Patient and public involvement Patients will not be involved in the design, recruitment, conduct or reporting of the study. Ethical consideration and dissemination of results The study protocol has been approved by the Ethics Committee of Shanghai Stomatological Hospital (Shanghai, China; approval no. SHSHIRB-2022016; approval date October 8, 2022). If there are any changes in eligibility criteria, outcomes or analyses, new plans will be reported to the ethics committee. The study was conducted in accordance with the Declaration of Helsinki, the Clinical Trials Act and other current legal regulations in China. Written informed consent was obtained from all the participants after they received a comprehensive explanation of this study . Data from the present research were registered with the International Clinical Trials Registry Platform. Additionally, the results will be disseminated at medical conferences and through journal publications. This is a single-centre, prospective, evaluator-blinded, randomised, two-arm parallel-group controlled trial. It commenced in October 2023 and will conclude in October 2025 at the Department of Implantology and Department of Prosthodontics in Shanghai Stomatological Hospital. According to the sample size calculation, we plan to recruit 54 patients in need of immediate implant placement in the first upper molar extraction sockets. The patients will be randomly assigned to receive the porcine collagen matrix (Mucograft, Geistlich Pharma AG) or RPPCTF on the soft-tissue closure after the immediate posterior implantation. The treatment process is presented in . The project has been registered on the ClinicalTrials.gov website ( https://clinicaltrials.gov/ ; identifier no. ChiCTR2300067770) and National Medical Research Registration and Filing Information System ( https://www.medicalresearch.org.cn/ ). We will approach participants who meet the inclusion criteria after tooth extraction to inquire about their interest regarding this study. If interested, potential participants will be referred to our study team members who will provide a detailed description of the study procedures and invite them to participate. Written informed consent will be obtained prior to the collection of any study data. The required sample size for detecting a clinically significant effect size in the primary endpoint, which is the changes in soft-tissue volume between two groups, was determined using a two-sample t-test. The calculation was based on a two-tailed test with a significance level of 0.05 and a power of 0.8. A clinically significant difference was defined as a mean keratinised soft-tissue width exceeding 1.5 mm. Assuming an SD of approximately 1.8 mm and accounting for a 10% dropout rate, it was estimated a total of 54 patients (27 in each group) would be required to detect an effect of that size. Adult patients aged over 18 years old requiring extraction of first upper molar for caries, fracture or prosthetic reason. The presence of type B extraction socket with the intact buccal bone over 1 mm evaluated on the preoperative cone beam CT (CBCT). Sufficient residual bone to maintain one implant with at least 4.3 mm in diameter, 10 mm in length. Systemically healthy patients not smoking more than 10 cigarettes per day. Patients with adequate oral hygiene with full-mouth plaque score less than 25%. Uncontrolled diabetes with HbA1c over 7%, osteoporosis or any other systemic or local disease or condition that would compromise postoperative healing. Patients with a history of malignancy, radiotherapy or chemotherapy for the treatment of malignancy. Pregnant patient or intending to become pregnant or nursing at the time of study inclusion. Patients taking medications or having treatments with an effect on healing in general, including steroids use, large doses of anti‐inflammatory drugs and bisphosphonates. Periapical inflammation. This study is a single-centre, prospective, evaluator-blinded, randomised, two-arm parallel-group controlled trial. All eligible patients were randomly assigned to two groups after CBCT assessment and obtaining informed consent. The allocation sequence was generated using computer software (SAS) and sealed in opaque envelopes by WZ who would not participate in subsequent studies. These envelopes were opened immediately before intervention by WL. WL did not attend the enrolment of the participants, assignment of the interventions or assessment of the outcome. KH and SF enrolled the participants. The clinical operators will not have access to the statistical analysis process. The outcome indicators were assessed using a single-blind method. The data collectors and outcome evaluators did not know the group information during the whole research process. The upper first molar will be extracted using a minimally invasive approach to preserve the integrity of the buccal bone and surrounding hard tissue. After CBCT assessment of the residual bone and socket type, patients will be randomly assigned to two groups: Control group: n=27, immediate implant placement with deproteinised bovine bone mineral (Bio-oss, Geistlich Pharma AG) grafted into the gap, up to the buccal bone crest, covered by type I collagen membrane (Bio-Gide, Geistlich Pharma AG). Wound will be closed by RPPCTF. Experimental group: n=27, immediate implant placement with deproteinised bovine bone mineral grafted into the gap, up to the buccal bone crest, covered by type I collagen membrane. Wound will be closed by porcine collagen matrix. Subsequently, the soft-tissue borders will be de-epithelialised. Single interrupted resorbable suture and horizontal mattress suture will be used to stabilise the grafted materials. The patients will be recalled timely when unexpected circumstances occur, such as bleeding, surgical area infection and implant loss. When the wound is infected or the soft tissue is necrotic, the doctor will perform a timely debridement. During this period, the cost associated with debridement, bone grafting and secondary surgery will all be free. In addition, considering that the secondary surgery would increase revisiting frequency, a transportation subsidy will also be provided as compensation. The 3Shape intraoral scanner (Trios 3, 3Shape) will be used to obtain the 3D soft-tissue morphology after surgery, at 2 weeks, 1 month, 3 months, 6 months and 12 months to evaluate the soft-tissue healing and the volume collapse. All the 3D soft-tissue data will be superimposed in the Geomagic Studio software (Raindrop Geomagic, USA) to compare volume and thickness changes. First, standard tessellation language (STL) files will be aligned and superimposed by selecting eight reproducible anatomical points in the Geomagic Studio software. A standardised region of interest will be delimited with a digital pen at the peri-implant aspect. Then the change in thickness, peri-implant soft-tissue contour area and its corresponding contraction rates between the corresponding time points will be assessed by applying the computer aid design (CAD) comparison tool. Each analysis will be performed in triplicate by one calibrated examiner. Additional follow-up propaganda and education will be conducted when patients fail to follow-up on time. The CBCT will be performed before tooth extraction to evaluate the socket type and the residual bone volume. The vertical height and horizontal width of the bone crest at buccal midpoint, buccal mesial point and buccal distal point will then be measured at the postoperation baseline, 3 months, 6 months and 12 months. The operation time of two different groups will be recorded and compared, which starts from the cover screw delivery and ends in the wound closure. The patient’s postoperative painkiller taking time and dosage will also be recorded. Visual analogue scale (VAS) will be used to evaluate comfort and satisfaction at 2 weeks after surgery. 6 months after the surgical procedure, a 5 mm diameter micro-punch biopsy of the implantation area will be performed. The tissue will be histologically evaluated, graded into four categories: immature, mature, fragmented and oedematous collagen tissue. The elastic fibres will also be examined and graded in three categories: with a normal structure, fragmented rare and fragmented multiplied. The photographs will be taken with a digital single-lens reflex after the crown delivery and evaluated under similar conditions. The Pink Esthetics Scores (PES) will be recorded by three dentists (KH, ZD and CZ) with experience and prior training in using PES index in research settings. Examiner ZD is an oral implantologist with 20 years of clinical experience. Examiners KH and CZ are oral implantologists with 6 years of clinical experience. Before commencing PES assessment, all the examiners would calibrate the assessment procedure to ensure high intraexaminer and interexaminer reliability. Primary endpoint The primary outcome is the soft-tissue volume change because this parameter is imperative in evaluating the long-term stability of the two methods for wound closure. The soft-tissue volume change evaluation will be conducted during postoperative 6 months. Soft-tissue healing conditions at baseline, 2 weeks, 1 month, 3 months and 6 months will be recorded and evaluated using a 3D intraoral scanner. Then the soft-tissue data at different time points will be integrated into 3D software for the purpose of comparing tissue collapse. Secondary endpoints Marginal bone change in postoperative 12 months, operation time, patient comfort and satisfaction, and the final aesthetics will be assessed and recorded. The marginal bone changes at baseline, 3 months, 6 months and 12 months will be compared in both groups via CBCT and 3D data software. The differences in operation time, postoperative patient comfort and satisfaction, and the final aesthetics between the two groups will also be recorded. The primary outcome is the soft-tissue volume change because this parameter is imperative in evaluating the long-term stability of the two methods for wound closure. The soft-tissue volume change evaluation will be conducted during postoperative 6 months. Soft-tissue healing conditions at baseline, 2 weeks, 1 month, 3 months and 6 months will be recorded and evaluated using a 3D intraoral scanner. Then the soft-tissue data at different time points will be integrated into 3D software for the purpose of comparing tissue collapse. Marginal bone change in postoperative 12 months, operation time, patient comfort and satisfaction, and the final aesthetics will be assessed and recorded. The marginal bone changes at baseline, 3 months, 6 months and 12 months will be compared in both groups via CBCT and 3D data software. The differences in operation time, postoperative patient comfort and satisfaction, and the final aesthetics between the two groups will also be recorded. All participants were identified by their random number ID. All these data have been collected by data collectors according to the record form and have been documented on paper, which would be entered using EpiData software. The user name and password were saved and operated by KH. Data management was conducted by two experimenters independently every month. The monitoring team consisting of two experienced clinicians, two biostatisticians and one ethicist met every 3 months to ensure the trial followed the protocol and the provisions of good clinical practice. The publication of the trial data will be managed by a data manager appointed by the principal investigators. Statistical analysis will be conducted using Statistical Package for Social Sciences software (IBM). Continuous variables, if normally distributed, will be presented as the mean±SD, and if skewed, as the median and IQR. Categorical variables will be presented as counts and percentages. All analyses will be based on the intent-to-treat principle. Mixed-effect model will be adopted to evaluate the differences of soft-tissue volume change and marginal bone-level change between the experimental group and control group. The time point will be set as repeated measures with treatment as a fixed effect. A two-sample t-test or Mann-Whitney U test will be used to compare the differences of width of keratinised gingiva, operation time and VAS scores of patient comfort and satisfaction, and the final aesthetics. Multiple imputations will be used as a sensitivity analysis for missing values of primary outcome. All tests will be performed at the both-sided 0.05 significance level unless otherwise noted. Patients will not be involved in the design, recruitment, conduct or reporting of the study. The study protocol has been approved by the Ethics Committee of Shanghai Stomatological Hospital (Shanghai, China; approval no. SHSHIRB-2022016; approval date October 8, 2022). If there are any changes in eligibility criteria, outcomes or analyses, new plans will be reported to the ethics committee. The study was conducted in accordance with the Declaration of Helsinki, the Clinical Trials Act and other current legal regulations in China. Written informed consent was obtained from all the participants after they received a comprehensive explanation of this study . Data from the present research were registered with the International Clinical Trials Registry Platform. Additionally, the results will be disseminated at medical conferences and through journal publications. A well-represented band of keratinised gingiva (at least 2 mm) has a positive influence on peri-implant health. Having an adequate thickness of peri-implant mucosa is important to guarantee marginal bone stability over time and provide a coordinated appearance. Machtei demonstrated that sites with no membrane exposure yielded almost six times more new bone regeneration than sites that became exposed. The porcine collagen matrix is effective in increasing keratinised gingival. However, to the best of our knowledge, the effectiveness of porcine collagen matrix on the maxillary immediate implant site has not been previously studied. Additionally, there is limited evidence comparing the difference between collagen matrix and RPPCTF after the posterior immediate implantation. Hence, this study aims to reveal the clinical difference between these two methods for soft-tissue closure and provide results to guide the selection of clinical procedures. Traditional methods for measuring the volume of soft-tissue change are limited and segmentary. This study proposed using a 3D technique to thoroughly evaluate the effect of collagen matrix and RPPCTF. By superimposing digital scanning data in software, even slight variations, including both linear and volume changes, will be presented clearly and accurately. The colourful and 3D images will also give us a better understanding of the contraction of soft tissue over time. Additionally, histological analysis will be adopted to assess the microscopic differences. This study has some limitations in evaluating the effect of porcine collagen matrix. First, the outline of basal bone beneath the soft tissue or collagen matrix, which may affect the soft-tissue contour, is difficult to control. The adjacent bone form also has an evident impact on the outline. Additionally, dietary and oral hygiene habits may vary greatly among patients. While we will conduct oral health education after surgery, the results may still be disturbed. Trial status Recruitment began in October 2023, and this study is expected to be completed before October 2025. 14 patients have been recruited so far. We expect to submit the research manuscript to a peer-reviewed journal before 31 December 2025. At the time of the manuscript preparation, the recruitment is ongoing. We aim to complete recruitment by 30 September 2024. The anticipated final data collection date for the primary outcome measure is 30 September 2025. Recruitment began in October 2023, and this study is expected to be completed before October 2025. 14 patients have been recruited so far. We expect to submit the research manuscript to a peer-reviewed journal before 31 December 2025. At the time of the manuscript preparation, the recruitment is ongoing. We aim to complete recruitment by 30 September 2024. The anticipated final data collection date for the primary outcome measure is 30 September 2025. 10.1136/bmjopen-2024-090302 online supplemental file 1 10.1136/bmjopen-2024-090302 online supplemental file 2 |
Investigation on the mechanisms of guiqi huoxue capsule for treating cervical spondylosis based on network pharmacology and molecular docking | 0d4163d3-6269-4c2f-b94c-de70009f8913 | 8447999 | Pharmacology[mh] | Introduction Cervical spondylosis (CS) is also called cervical vertebrae syndrome in medicine, which is a general term for cervical osteoarthritis, proliferative cervical spondylitis, cervical nerve root syndrome, and cervical disc herniation. It is a degenerative joint disease, and the causes of the disease include degeneration, trauma, strain, inflammation, developmental spinal stenosis, and congenital deformity. Generally, CS can be divided into different types such as neck type cervical spondylopathy, cervical spondylotic radiculopathy (CSR), cervical spondylotic myelopathy, vertebral artery type of cervical spondylosis, sympathetic type of cervical spondylosis, esophagus-type cervical spondylosis, and mixed cervical spondylosis. CSR is one of the most common types of cervical spondylosis. Traditional Chinese medicine (TCM) theory believes that the CS belongs to the category of “bi syndrome”. “BI syndrome” in traditional Chinese medicine refers to a disease characterized by pain, swelling, stiffness, deformation, and other symptoms and signs of limited movement in the body's joints and skeletal muscles. Also, Vertigo, nuchal arthralgia, headache, and shoulder-neck pain tend to describe this disease. The cause of CS is a clear orifice. The inducements are qi deficiency in clear orifice and meridians obstruction due to blood, which nature is asthenia in origin and asthenia in superficiality. Treatment for CS include operative and non-operative methods. Non-operative methods include drug treatment, physical therapy, exercise therapy, small needle knife therapy, acupuncture therapy, and massage therapy. Oral TCM therapy is an effective means of conservative treatment. Guiqi huoxue capsule (GQHXC) is a Chinese patent medicine composed of 13 Chinese herbal medicines: Astragali Radix (HQ), Angelicae Sinensis Radix (DG), Paeoniae Radix Alba (BS), Polygoni Multiflori Radix Praeparata (ZHSW), Lycii Fructus (GQZ), Viscum Coloratum (HJS), Cervi Cornu Pantotrichum (LR), Drynariae Rhizoma (GSB), Clematidis Radix et Rhizoma (WLX), Tuberculate Speranskia Herb (ZZTGC), Artificial Musk (RGSX), Puerariae Lobatae Radix (GG), Chuanxiong Rhizoma (CX). Its usage and dosage are as follows: orally, 3 capsules at a time, 3 times a day, and the course of treatment is 4 weeks. The main effects of GQHXC are promoting blood circulation for removing obstruction in the channel, tonifying qi and tonifying the kidney. According to TCM, qi is the most essential substance that constitutes the human body and the most essential substance that maintains the life activity of the human body. GQHXC is used for treating a deficiency of both liver and kidney and blood stasis syndrome due to qi deficiency, which is caused by CS (CSR, mixed cervical spondylosis mainly composed of nerve root type). This study focused on the network pharmacology of GQHXC for treating CSR. TCM obtains its superior therapeutic efficacy in the biological network of the human body system, which has the characteristics of multicomponent, multi-target, and multi-pathway synergism. However, its medicinal basis and mechanism of action are unclear, making it difficult to carry out comprehensive system research from the general to the cellular and molecular level. Based on the “disease-gene-target-drug” interaction network, network pharmacology systematically and comprehensively observes the intervention and influence of drugs on the disease network, thus revealing the mystery of the synergetic effect of drugs in the human body. The application of network pharmacology in the research of TCM conforms to the treatment, which is based on syndrome differentiation of the overall view of traditional Chinese medicine, and the characteristics of TCM include the multicomponent, multi-target, and multi-pathways. Chinese herbal medicines such as HQ, DG, CX, and RGSX have therapeutic effects on CSR. Some literature suggests that GQHXC has therapeutic effects on CSR, but its molecular mechanism has not fully been elucidated. Therefore, this study adopted network pharmacology to explore and predict the molecular mechanism of GQHXC against CSR. The detailed workflow of the network pharmacology research is illustrated in Figure .
Materials and methods 2.1 Collection of guiqi huoxue capsule active ingredients To retrieve all the chemical components in GQHXC, we relied on TCM systems pharmacology Database and Analysis Platform (TCMSP, http://tcmspw.com/tcmsp.php ), Shanghai Institute of Organic Chemistry of CAS (Chemistry Database [DB/OL], http://www.organchem.csdb.cn . [1978–2020]), China National Knowledge Infrastructure Database (CNKI, http://www.cnki.net/ ), and PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ). The relevant compounds were collected by entering the name of the Chinese herbal medicine into the TCMSP and Chemistry Database. Among them, LR, the compounds of ZHSW and ZZTGC were not found in the above database. Thus, research articles on components related to those Chinese herbal medicines were searched in CNKI to collect relevant compound information. A total of 980 compounds were collected, of which 87 were HQ, 125 were DG, 85 were BS, 24 were ZHSW, 188 were GQZ, 40 were HJS, 72 were LR, 71 were GSB, 57 were WLX, 11 were ZZTGC, 13 were RGSX, 18 were GG, and 189 were CX. Finally, the collected compounds were integrated to screen the candidate compounds. 2.2 Candidate active compounds and related targets After deleting duplicate data and compounds without structural information, 112 compounds were summarized (see Table S1, Supplemental Content which illustrates the information of 112 active compounds) with oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18, which were regarded as candidate compounds. The numbers of active compounds in HQ, DG, BS, ZHSW, GQZ, HJS, LR, GSB, WLX, ZZTGC, RGSX, GG, and CX were 20, 2, 13, 4, 45, 7, 6, 18, 7, 3, 2, 4, and 7, respectively. Table S1 lists the basic information of these compounds. After removing the compounds with no corresponding targets, 84 compounds were retained. There are 260 targets associated with them. In addition, the targets of these compounds were obtained through TCMSP. The Uniprot database ( https://www.uniprot.org/ ) was utilized to find corresponding genes. 2.3 Cervical spondylotic radiculopathy targets The target search terms used were “Cervical spondylotic radiculopathy,” “cervical spondylosis of nerve root type,” “nerve root cervical spondylosis” and “nerve root cervical spondylopathy” to find the targets in the disease gene search engine ( http://210.107.182.61/geneSearch/ ), DisGeNET ( http://www.disgenet.org/search ), and Therapeutic Target Database (, https://db.idrblab.org/ttd/ ). Finally, we obtained 57 protein targets of CSR. 2.4 Construction of protein-protein interaction network PPI data were collected from the STRING database (Search Tool for the Retrieval of Interacting Genes/Proteins, https://string-db.org/ ), which is known for predicted interactions, including direct and indirect interactions of proteins. The confidence ranges of PPIs are defined by the database for interaction score (low confidence: score <0.4; medium confidence: 0.4–0.7; high confidence: > 0.7). When related targets were entered into the search tool for the retrieval of interacting genes database, we selected the high confidence, and the species was limited to “Homo sapiens”. Furthermore, “the 1st shell” and “the 2nd shell” were set to “no more than 20 interactors” in potential therapeutic targets PPI network. 2.5 Network establishment All the above networks were constructed based on Cytoscape 3.7.2 software ( http://www.cytoscape.org/ ), an open-source software platform, which is made available for interactive network analysis, integration, and visualization of networks and network data. In the interaction network, each node has a meaningful parameter “Degree” to evaluate its topological characteristics. There is a positive correlation between the degree value and the importance of the node in the networks. The following 5 networks were established in this study. 1. Compound-putative target network of GQHXC: This network was built by contacting candidate active compounds of GQHXC and corresponding targets. 2. PPI network related to CSR: The PPI network was established by linking CSR-related targets and other human proteins that connected or interacted with CSR targets. 3. Compound-CSR target network: The above 2 networks were intersected to build this network by the merge function in Cytoscape 3.7.2. 4. Potential therapeutic targets PPI network: This PPI network was constructed by linking the proteins obtained from the network of (3) and other related humans proteins. 5. Herb-compound-target-pathway network: The comprehensive network was based on connecting herbs, compounds, corresponding targets, and pathways. There is a positive proportional relationship between the node size and degree of networks. 2.6 Gene ontology functional enrichment analysis and kyoto encyclopedia of genes and genomes pathway enrichment analysis To evaluate the role of the key potential targets in gene function and signaling pathway, gene ontology (GO) knowledgebase ( http://geneontology.org/ ), kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis ( https://www.genome.jp/kegg/ ) were performed using the R 3.6.1 software ( https://cran.r-project.org/doc/FAQ/R-FAQ.html#Citing-R ) with the Bioconductor package. Both the P value and q-value parameters are set to less than .01. 2.7 Molecular docking verification AutoDockTools 1.5.6 was employed to perform the docking analysis. The compounds were retrieved from TCMSP and Pubchem database and the target proteins retrieved from the protein data bank database ( https://www.rcsb.org/ ), For the ligand and proteins, the parameters such as rotatable bonds, merging charge, dehydration, and hydrogenation were set by default. The screening conditions were set as follows: 1. the protein structure is obtained by X-crystal diffraction; 2. the crystal resolution of the protein is less than 3 Å; 3. preferential selection of protein structures reported in the literature related to molecular docking; 4. the organism comes from Homo sapiens. Based on the above conditions, 7 core target protein protein data bank IDs were gathered, and the water molecules and co-crystallized pro-ligand molecules were removed by the Notepad++ ( https://notepad-plus-plus.org/ ) and AutoDockTools. Subsequently, the format of a compound and target protein was converted into ∗pdbqt format in Auto Dock software. Finally, molecular docking calculations were performed using Autodock Vina 1.1.2. The PyMol 2.3.2 ( https://pymol.org/2/ ) software was wielded to visualize the docking results. The negative binding energy (<0) indicates that the ligand and the receptor can spontaneously bind without consuming energy. At present, there is no uniform standard for the target screening of active molecules. According to the literature report, the active ingredients has been selected with a binding energy of −5.0 kcal/mol or less as the basis for screening GQHXC therapeutic targets. To better analyze the docking results, we took the binding energy and the active pocket of the target predicted by POCASA 1.1 ( http://altair.sci.hokudai.ac.jp/g6/service/pocasa/ ) into consideration.
Collection of guiqi huoxue capsule active ingredients To retrieve all the chemical components in GQHXC, we relied on TCM systems pharmacology Database and Analysis Platform (TCMSP, http://tcmspw.com/tcmsp.php ), Shanghai Institute of Organic Chemistry of CAS (Chemistry Database [DB/OL], http://www.organchem.csdb.cn . [1978–2020]), China National Knowledge Infrastructure Database (CNKI, http://www.cnki.net/ ), and PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ). The relevant compounds were collected by entering the name of the Chinese herbal medicine into the TCMSP and Chemistry Database. Among them, LR, the compounds of ZHSW and ZZTGC were not found in the above database. Thus, research articles on components related to those Chinese herbal medicines were searched in CNKI to collect relevant compound information. A total of 980 compounds were collected, of which 87 were HQ, 125 were DG, 85 were BS, 24 were ZHSW, 188 were GQZ, 40 were HJS, 72 were LR, 71 were GSB, 57 were WLX, 11 were ZZTGC, 13 were RGSX, 18 were GG, and 189 were CX. Finally, the collected compounds were integrated to screen the candidate compounds.
Candidate active compounds and related targets After deleting duplicate data and compounds without structural information, 112 compounds were summarized (see Table S1, Supplemental Content which illustrates the information of 112 active compounds) with oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18, which were regarded as candidate compounds. The numbers of active compounds in HQ, DG, BS, ZHSW, GQZ, HJS, LR, GSB, WLX, ZZTGC, RGSX, GG, and CX were 20, 2, 13, 4, 45, 7, 6, 18, 7, 3, 2, 4, and 7, respectively. Table S1 lists the basic information of these compounds. After removing the compounds with no corresponding targets, 84 compounds were retained. There are 260 targets associated with them. In addition, the targets of these compounds were obtained through TCMSP. The Uniprot database ( https://www.uniprot.org/ ) was utilized to find corresponding genes.
Cervical spondylotic radiculopathy targets The target search terms used were “Cervical spondylotic radiculopathy,” “cervical spondylosis of nerve root type,” “nerve root cervical spondylosis” and “nerve root cervical spondylopathy” to find the targets in the disease gene search engine ( http://210.107.182.61/geneSearch/ ), DisGeNET ( http://www.disgenet.org/search ), and Therapeutic Target Database (, https://db.idrblab.org/ttd/ ). Finally, we obtained 57 protein targets of CSR.
Construction of protein-protein interaction network PPI data were collected from the STRING database (Search Tool for the Retrieval of Interacting Genes/Proteins, https://string-db.org/ ), which is known for predicted interactions, including direct and indirect interactions of proteins. The confidence ranges of PPIs are defined by the database for interaction score (low confidence: score <0.4; medium confidence: 0.4–0.7; high confidence: > 0.7). When related targets were entered into the search tool for the retrieval of interacting genes database, we selected the high confidence, and the species was limited to “Homo sapiens”. Furthermore, “the 1st shell” and “the 2nd shell” were set to “no more than 20 interactors” in potential therapeutic targets PPI network.
Network establishment All the above networks were constructed based on Cytoscape 3.7.2 software ( http://www.cytoscape.org/ ), an open-source software platform, which is made available for interactive network analysis, integration, and visualization of networks and network data. In the interaction network, each node has a meaningful parameter “Degree” to evaluate its topological characteristics. There is a positive correlation between the degree value and the importance of the node in the networks. The following 5 networks were established in this study. 1. Compound-putative target network of GQHXC: This network was built by contacting candidate active compounds of GQHXC and corresponding targets. 2. PPI network related to CSR: The PPI network was established by linking CSR-related targets and other human proteins that connected or interacted with CSR targets. 3. Compound-CSR target network: The above 2 networks were intersected to build this network by the merge function in Cytoscape 3.7.2. 4. Potential therapeutic targets PPI network: This PPI network was constructed by linking the proteins obtained from the network of (3) and other related humans proteins. 5. Herb-compound-target-pathway network: The comprehensive network was based on connecting herbs, compounds, corresponding targets, and pathways. There is a positive proportional relationship between the node size and degree of networks.
Gene ontology functional enrichment analysis and kyoto encyclopedia of genes and genomes pathway enrichment analysis To evaluate the role of the key potential targets in gene function and signaling pathway, gene ontology (GO) knowledgebase ( http://geneontology.org/ ), kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis ( https://www.genome.jp/kegg/ ) were performed using the R 3.6.1 software ( https://cran.r-project.org/doc/FAQ/R-FAQ.html#Citing-R ) with the Bioconductor package. Both the P value and q-value parameters are set to less than .01.
Molecular docking verification AutoDockTools 1.5.6 was employed to perform the docking analysis. The compounds were retrieved from TCMSP and Pubchem database and the target proteins retrieved from the protein data bank database ( https://www.rcsb.org/ ), For the ligand and proteins, the parameters such as rotatable bonds, merging charge, dehydration, and hydrogenation were set by default. The screening conditions were set as follows: 1. the protein structure is obtained by X-crystal diffraction; 2. the crystal resolution of the protein is less than 3 Å; 3. preferential selection of protein structures reported in the literature related to molecular docking; 4. the organism comes from Homo sapiens. Based on the above conditions, 7 core target protein protein data bank IDs were gathered, and the water molecules and co-crystallized pro-ligand molecules were removed by the Notepad++ ( https://notepad-plus-plus.org/ ) and AutoDockTools. Subsequently, the format of a compound and target protein was converted into ∗pdbqt format in Auto Dock software. Finally, molecular docking calculations were performed using Autodock Vina 1.1.2. The PyMol 2.3.2 ( https://pymol.org/2/ ) software was wielded to visualize the docking results. The negative binding energy (<0) indicates that the ligand and the receptor can spontaneously bind without consuming energy. At present, there is no uniform standard for the target screening of active molecules. According to the literature report, the active ingredients has been selected with a binding energy of −5.0 kcal/mol or less as the basis for screening GQHXC therapeutic targets. To better analyze the docking results, we took the binding energy and the active pocket of the target predicted by POCASA 1.1 ( http://altair.sci.hokudai.ac.jp/g6/service/pocasa/ ) into consideration.
Results 3.1 Compound-putative target network of guiqi huoxue capsule As shown in Figure , the compound-putative target network of GQHXC includes 344 nodes (84 compounds and 260 targets) and 928 edges (see Table S2, Supplemental Content which illustrates the information of nodes in the compound-putative target network of GQHXC). In addition, each edge represents an interaction between compound molecules and targets. The degree of a node is equal to the number of edges connected to the node in the network, and the size of the node is positively related to the degree value. The average degree value of the nodes in this network was 5.4, the average number of targets per compound was 12, and each target interacted with an average of 4 compounds. Thus, in GQHXC, we not only found that 1 compound can interact with multiple targets but also different compounds can work together on the same target. These findings reflected the mechanism of interaction between multiple components and multiple targets of GQHXC. From a compound perspective, 28.57% of the compounds interact with 10 or more targets, and 18 compounds can interact with 20 or more targets. Among them, quercetin in HQ and GQZ has the highest degree of connectivity and can interact with 145 target proteins. It is followed by kaempferol, luteolin, and 7-O-methylisomucronulatol, which can interact with 58 targets, 57 targets, and 41 targets, respectively. Besides, isorhamnetin, formononetin, beta-sitosterol, and naringenin can interact with more than 30 target proteins. For the targets, 5 targets can interact with 20 or more compounds. The highest degree value was PTGS2 corresponding to 43 compounds, followed by NCOA2, which corresponded to 36 compounds; PGR corresponded to 34 compounds, PTGS1 corresponded to 32, and HSP90 corresponded to 31. These corresponding interactions also explain the pharmacological mechanism of the multiple components and multiple targets of GQHXC. 3.2 Protein-protein interaction network related to cervical spondylotic radiculopathy The 57 CSR-related target proteins were entered into the String 11.0 database for search, and protein interaction relationship data with a high-confidence interval score >0.7 was selected to ensure the reliability of the data. Then, the retrieved data was imported into Cytoscape 3.7.2 software to build a PPI network (Fig. ). PPI network related to CSR included 40 CSR-related proteins and 125 interactions among CSR-related proteins (see Table S3, Supplemental Content which illustrates the topology characteristics of nodes from the PPI network related to CSR). There are 15 nodes with a degree value greater than the average degree value of 6.25 in the network. 3.3 Compound- cervical spondylotic radiculopathy target network After linking the compound-putative target network of GQHXC with the PPI network related to CSR using the merge function in Cytoscape 3.7.2, 11 potential targets (vascular endothelial growth factor A [VEGFA], mitogen-activated protein kinase 1, AP-1 transcription factor subunit [JUN], tumor necrosis factor [TNF], IL6, Tissue-type plasminogen activator, prostaglandin-endoperoxide synthase 2 [PTGS2], interferon gamma, IL1B, CXCL8, nitric oxide synthase 3 [NOS3]) for GQHXC treatment of CSR were intuitively obtained. The connection principle among nodes is that when the predicted targets of the active ingredients in GQHXC were the same as the targets of the CSR, the predicted targets were associated with the targets of the CSR, and these linked targets were considered as potential targets for the treatment of CSR by GQHXC. The compound-CSR target network was shown in Figure (see Table S4, Supplemental Content which illustrates the topology characteristics of nodes from the Compound-CSR target network). There are 6 proteins (PTGS2, NOS3, JUN, TNF, IL6, mitogen-activated protein kinase 1) with a degree value greater than or equal to an average degree value of 2.69, and the highest degree value of PTGS2 is 43, which means that they are likely to be considered as the key therapeutic targets for GQHXC for treating CSR. The degree value of quercetin, luteolin, kaempferol, aloe-emodin, and formononetin was 11, 7, 4, 3, and 3, respectively, which are all higher than the average degree. They are expected to be the main active ingredients for treating CSR. 3.4 Module analysis of potential therapeutic targets protein-protein interaction network The 11 potential targets obtained in the Compound-CSR target network (3.3) were entered into the String 11.0 database with high confidence of parameter, and the “1st shell” and “the 2nd shell” were set to “no more than 20 interactors”. The species was limited to “Homo sapiens” and high confidence was selected. The collected data were then imported into Cytoscape 3.7.2, and the molecular complex detection function was utilized to perform a module analysis on potential targets. Potential therapeutic targets PPI network is shown in Figure A (see Table S5, Supplemental Content which illustrates the information of nodes in the Potential therapeutic targets PPI network). Four modules were obtained through clustering the network. Module 1 (Fig. B) has 16 nodes and 115 edges, and module 2 has 14 nodes and 43 edges. The degree value of 68.75% of target proteins in module 1 was greater than the average degree value of 14.375. The degree value of 35.71% of target proteins in module 2 (Fig. C) was higher than the average degree value of 6.14. 7 targets (TNF, VEGFA, JUN, NOS3, IL6, CXCL8, and PTGS2) were identified from module 1 and module 2 as the predicted most critical targets. Module 3 (Fig. D) has 3 nodes and 3 edges, and module 4 (Fig. E) has 6 nodes and 7 edges. We selected module 1 and module 2 with higher scores for the subsequent analysis. 3.5 Gene ontology function enrichment and kyoto encyclopedia of genes and genomes pathway enrichment analysis We used R 3.6.1 ( https://cran.r-project.org/doc/FAQ/R-FAQ.html#Citing-R ) software to perform GO and KEGG enrichment analysis on the protein targets of the 3 networks in (a) (b) and (c) in Figure (see Table S6, Supplemental Content which illustrates the list of GO enrichment results to genes in all enrichment analysis; see Table S7, Supplemental Content which illustrates the list of pathway enrichment results to genes in all enrichment analysis). In the bubble chart, the X-axis represents the number of target genes (Gene Ratio), and the Y-axis represents the KEGG pathway or GO term where the target gene is significantly enriched. The size of the dots intuitively reflects the size of the Gene Ratio, and the color depth of the dots reflects different p-value ranges. 1. Analysis of potential therapeutic targets PPI network: In the GO enrichment analysis (Fig. F), a total of 954 terms were found, of which 913 terms were related to biological processes (BP), 15 terms were related to cell composition (CC), and 26 terms were related to molecular function (MF). KEGG pathway enrichment analysis (Fig. G) found 180 pathways, of which 113 pathways had a P value and q-value less than .01. 2. Analysis of module 1: In the GO enrichment analysis (Fig. H), a total of 303 terms were found, of which 277 terms were related to BP, 12 terms were related to CC, and 14 terms were related to MF. KEGG pathway enrichment analysis (Fig. I) found 97 pathways, of which 56 pathways had a P value and q-value less than .01. 3. Analysis of module 2: In the GO enrichment analysis (Fig. J), a total of 125 terms were found, of which 192 terms were related to BP, 9 terms were related to CC, and 14 terms were related to MF. KEGG pathway enrichment analysis (Fig. K) found 130 pathways, of which 41 pathways had a P value and q-value less than .01. 3.6 Herb-compound-target-pathway network Herb-compound-target-pathway network (Fig. A) consists of 94 nodes and 277 edges (7 nodes of key potential targets, 30 nodes of the pathway, 44 nodes of the compound, 12 nodes of herbs, and 1 of “GQHXC”). For the pathways, the degree value of the AGE-RAGE signaling pathway in diabetic complications was 6, and the degree value of Human cytomegalovirus infection, Kaposi sarcoma-associated herpesvirus infection, and IL-17 signaling pathway was 5. From the analysis of the target protein, PTGS2 degree value was 50, TNF degree value was 31, JUN degree value was 28, and IL6 degree value was 20. The highest degree value of quercetin in the compound was 8, the degree value of luteolin was 6, the degree value of kaempferol was 5, and finally the degree value of formononetin was 4. The pathological processes of CSR are closely associated with inflammatory responses and blood vessels. Seven putative targets and transcription factor p65 [NF-κB] are the most active factors that participate in these key pathways, which imply an important role in the occurrence and promotion of CSR. KEGG Database ( https://www.kegg.jp/kegg/kegg1.html ) and software of Pathway Builder Tool 2.0 were used to generate the figure. As showed in Figure B, putative major signaling pathways of GQHXC were constructed. 3.7 Molecular docking verification We initially selected 44 compounds and the 7 target proteins in Figure A as ligands and receptors for molecular docking verification. We then selected the top 10 of the 44 compounds ranked according to the degree value in table S2 as the final ligands. Because Atropine belongs to table S2 but did not belong to these 44 compounds, the final docking ligands were 9 (quercetin, luteolin, kaempferol, beta-sitosterol, formononetin, isorhamnetin, 7-O-methylisomucronulatol, Stigmasterol, and naringenin). The active pockets of target proteins were predicted on the POCASA 1.1 website. VEGFA and JUN target proteins did not receive any active prediction sites on the website, so their docking results were selected for the successful docking and highest score. For the target proteins in the activity prediction pocket, the docking results with the highest score and success in the active site were selected. The lower the affinity between compound and target, the better the binding activity between them. As shown in Table , 27 pairs of target-compound combinations were delivered to Vina for docking, and their docking score was less than or equal to -5.3 kcal/mol, which indicates that they have good binding activity. We can conclude that PTGS2 and NOS3 have a better affinity with the corresponding compound. Using Pymol software, these compounds were observed to enter the active pocket of the protein. Detailed verification target-compound interactions were given in Figure .
Compound-putative target network of guiqi huoxue capsule As shown in Figure , the compound-putative target network of GQHXC includes 344 nodes (84 compounds and 260 targets) and 928 edges (see Table S2, Supplemental Content which illustrates the information of nodes in the compound-putative target network of GQHXC). In addition, each edge represents an interaction between compound molecules and targets. The degree of a node is equal to the number of edges connected to the node in the network, and the size of the node is positively related to the degree value. The average degree value of the nodes in this network was 5.4, the average number of targets per compound was 12, and each target interacted with an average of 4 compounds. Thus, in GQHXC, we not only found that 1 compound can interact with multiple targets but also different compounds can work together on the same target. These findings reflected the mechanism of interaction between multiple components and multiple targets of GQHXC. From a compound perspective, 28.57% of the compounds interact with 10 or more targets, and 18 compounds can interact with 20 or more targets. Among them, quercetin in HQ and GQZ has the highest degree of connectivity and can interact with 145 target proteins. It is followed by kaempferol, luteolin, and 7-O-methylisomucronulatol, which can interact with 58 targets, 57 targets, and 41 targets, respectively. Besides, isorhamnetin, formononetin, beta-sitosterol, and naringenin can interact with more than 30 target proteins. For the targets, 5 targets can interact with 20 or more compounds. The highest degree value was PTGS2 corresponding to 43 compounds, followed by NCOA2, which corresponded to 36 compounds; PGR corresponded to 34 compounds, PTGS1 corresponded to 32, and HSP90 corresponded to 31. These corresponding interactions also explain the pharmacological mechanism of the multiple components and multiple targets of GQHXC.
Protein-protein interaction network related to cervical spondylotic radiculopathy The 57 CSR-related target proteins were entered into the String 11.0 database for search, and protein interaction relationship data with a high-confidence interval score >0.7 was selected to ensure the reliability of the data. Then, the retrieved data was imported into Cytoscape 3.7.2 software to build a PPI network (Fig. ). PPI network related to CSR included 40 CSR-related proteins and 125 interactions among CSR-related proteins (see Table S3, Supplemental Content which illustrates the topology characteristics of nodes from the PPI network related to CSR). There are 15 nodes with a degree value greater than the average degree value of 6.25 in the network.
Compound- cervical spondylotic radiculopathy target network After linking the compound-putative target network of GQHXC with the PPI network related to CSR using the merge function in Cytoscape 3.7.2, 11 potential targets (vascular endothelial growth factor A [VEGFA], mitogen-activated protein kinase 1, AP-1 transcription factor subunit [JUN], tumor necrosis factor [TNF], IL6, Tissue-type plasminogen activator, prostaglandin-endoperoxide synthase 2 [PTGS2], interferon gamma, IL1B, CXCL8, nitric oxide synthase 3 [NOS3]) for GQHXC treatment of CSR were intuitively obtained. The connection principle among nodes is that when the predicted targets of the active ingredients in GQHXC were the same as the targets of the CSR, the predicted targets were associated with the targets of the CSR, and these linked targets were considered as potential targets for the treatment of CSR by GQHXC. The compound-CSR target network was shown in Figure (see Table S4, Supplemental Content which illustrates the topology characteristics of nodes from the Compound-CSR target network). There are 6 proteins (PTGS2, NOS3, JUN, TNF, IL6, mitogen-activated protein kinase 1) with a degree value greater than or equal to an average degree value of 2.69, and the highest degree value of PTGS2 is 43, which means that they are likely to be considered as the key therapeutic targets for GQHXC for treating CSR. The degree value of quercetin, luteolin, kaempferol, aloe-emodin, and formononetin was 11, 7, 4, 3, and 3, respectively, which are all higher than the average degree. They are expected to be the main active ingredients for treating CSR.
Module analysis of potential therapeutic targets protein-protein interaction network The 11 potential targets obtained in the Compound-CSR target network (3.3) were entered into the String 11.0 database with high confidence of parameter, and the “1st shell” and “the 2nd shell” were set to “no more than 20 interactors”. The species was limited to “Homo sapiens” and high confidence was selected. The collected data were then imported into Cytoscape 3.7.2, and the molecular complex detection function was utilized to perform a module analysis on potential targets. Potential therapeutic targets PPI network is shown in Figure A (see Table S5, Supplemental Content which illustrates the information of nodes in the Potential therapeutic targets PPI network). Four modules were obtained through clustering the network. Module 1 (Fig. B) has 16 nodes and 115 edges, and module 2 has 14 nodes and 43 edges. The degree value of 68.75% of target proteins in module 1 was greater than the average degree value of 14.375. The degree value of 35.71% of target proteins in module 2 (Fig. C) was higher than the average degree value of 6.14. 7 targets (TNF, VEGFA, JUN, NOS3, IL6, CXCL8, and PTGS2) were identified from module 1 and module 2 as the predicted most critical targets. Module 3 (Fig. D) has 3 nodes and 3 edges, and module 4 (Fig. E) has 6 nodes and 7 edges. We selected module 1 and module 2 with higher scores for the subsequent analysis.
Gene ontology function enrichment and kyoto encyclopedia of genes and genomes pathway enrichment analysis We used R 3.6.1 ( https://cran.r-project.org/doc/FAQ/R-FAQ.html#Citing-R ) software to perform GO and KEGG enrichment analysis on the protein targets of the 3 networks in (a) (b) and (c) in Figure (see Table S6, Supplemental Content which illustrates the list of GO enrichment results to genes in all enrichment analysis; see Table S7, Supplemental Content which illustrates the list of pathway enrichment results to genes in all enrichment analysis). In the bubble chart, the X-axis represents the number of target genes (Gene Ratio), and the Y-axis represents the KEGG pathway or GO term where the target gene is significantly enriched. The size of the dots intuitively reflects the size of the Gene Ratio, and the color depth of the dots reflects different p-value ranges. 1. Analysis of potential therapeutic targets PPI network: In the GO enrichment analysis (Fig. F), a total of 954 terms were found, of which 913 terms were related to biological processes (BP), 15 terms were related to cell composition (CC), and 26 terms were related to molecular function (MF). KEGG pathway enrichment analysis (Fig. G) found 180 pathways, of which 113 pathways had a P value and q-value less than .01. 2. Analysis of module 1: In the GO enrichment analysis (Fig. H), a total of 303 terms were found, of which 277 terms were related to BP, 12 terms were related to CC, and 14 terms were related to MF. KEGG pathway enrichment analysis (Fig. I) found 97 pathways, of which 56 pathways had a P value and q-value less than .01. 3. Analysis of module 2: In the GO enrichment analysis (Fig. J), a total of 125 terms were found, of which 192 terms were related to BP, 9 terms were related to CC, and 14 terms were related to MF. KEGG pathway enrichment analysis (Fig. K) found 130 pathways, of which 41 pathways had a P value and q-value less than .01.
Herb-compound-target-pathway network Herb-compound-target-pathway network (Fig. A) consists of 94 nodes and 277 edges (7 nodes of key potential targets, 30 nodes of the pathway, 44 nodes of the compound, 12 nodes of herbs, and 1 of “GQHXC”). For the pathways, the degree value of the AGE-RAGE signaling pathway in diabetic complications was 6, and the degree value of Human cytomegalovirus infection, Kaposi sarcoma-associated herpesvirus infection, and IL-17 signaling pathway was 5. From the analysis of the target protein, PTGS2 degree value was 50, TNF degree value was 31, JUN degree value was 28, and IL6 degree value was 20. The highest degree value of quercetin in the compound was 8, the degree value of luteolin was 6, the degree value of kaempferol was 5, and finally the degree value of formononetin was 4. The pathological processes of CSR are closely associated with inflammatory responses and blood vessels. Seven putative targets and transcription factor p65 [NF-κB] are the most active factors that participate in these key pathways, which imply an important role in the occurrence and promotion of CSR. KEGG Database ( https://www.kegg.jp/kegg/kegg1.html ) and software of Pathway Builder Tool 2.0 were used to generate the figure. As showed in Figure B, putative major signaling pathways of GQHXC were constructed.
Molecular docking verification We initially selected 44 compounds and the 7 target proteins in Figure A as ligands and receptors for molecular docking verification. We then selected the top 10 of the 44 compounds ranked according to the degree value in table S2 as the final ligands. Because Atropine belongs to table S2 but did not belong to these 44 compounds, the final docking ligands were 9 (quercetin, luteolin, kaempferol, beta-sitosterol, formononetin, isorhamnetin, 7-O-methylisomucronulatol, Stigmasterol, and naringenin). The active pockets of target proteins were predicted on the POCASA 1.1 website. VEGFA and JUN target proteins did not receive any active prediction sites on the website, so their docking results were selected for the successful docking and highest score. For the target proteins in the activity prediction pocket, the docking results with the highest score and success in the active site were selected. The lower the affinity between compound and target, the better the binding activity between them. As shown in Table , 27 pairs of target-compound combinations were delivered to Vina for docking, and their docking score was less than or equal to -5.3 kcal/mol, which indicates that they have good binding activity. We can conclude that PTGS2 and NOS3 have a better affinity with the corresponding compound. Using Pymol software, these compounds were observed to enter the active pocket of the protein. Detailed verification target-compound interactions were given in Figure .
Discussion Owing to the complexity of GQHXC components, its mechanism for treating CSR has not yet been elucidated. In this study, a network pharmacological approach was used to determine the pharmacological mechanism of potential compounds and targets in GQHXC on CSR. Therefore, the compound-putative target network of GQHXC, PPI network related to CSR, compound-CSR target network, potential therapeutic targets PPI network, and the herb-compound-target-pathway network, were established to systematically analyze the mechanism of GQHXC action on CSR. It is well known that after CSR occurs, a large number of inflammatory factors are released at the damaged tissue, which mediate nerve root pain. Some studies have shown that the pathogenesis of CSR is closely related to the expression of pro-inflammatory factors such as IL-1, IL-6, IL-18, interleukin 1 beta [IL-1β], and TNF-α, . It is also related to the content of nitric oxide (NO), ET (endothelin), IgA, IgM, IgG, C3, C4, PGE2(prostaglandin E2), and so on in plasma. CSR is increased by the release of PGE2, a pro-inflammatory mediator. The disease progression of CSR will be exacerbated when the activity of NO decreases and excessive secretion of ET. C3 and C4 in CSR patients are lower than in normal people, while IgA, IgM, and IgG are higher than in normal people. The network pharmacology study predicted the following 7 potential targets: TNF, VEGFA, JUN, NOS3, IL6, CXCL8, and PTGS2, which may be the key targets for GQHXC for treating CSR. PTGS2 is responsible for the prostanoid biosynthesis involved in inflammation and mitogenesis. PTGS2 may additionally be associated with activating the NF kappa B signaling pathway. PTGS2 is an essential target for nonsteroidal anti-inflammatory drugs, and down-regulating PTGS2 expression can help reduce the inflammatory response. PTGS2 is a key enzyme for PGE2 formation, which affects the release of PGE2. The protein encoded by CXCL8 is a significant mediator of the inflammatory response. The encoded protein is secreted primarily by neutrophils, where it serves as a chemotactic factor by guiding the neutrophils to the site of infection. This chemokine is also a potent angiogenic factor. VEGFA is a member of the PDGF/VEGF growth factor family. It encodes a heparin-binding protein, which exists as a disulfide-linked homodimer. This growth factor induces proliferation and migration of vascular endothelial cells and has an essential role in physiological and pathological angiogenesis. Furthermore, VEGFA expression may be regulated by PI3K/AKT pathway. The TNF gene encodes a multifunctional proinflammatory cytokine that belongs to the TNF superfamily. This cytokine participates in regulating a broad spectrum of biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. TNF gene transcription produces inflammatory factors such as TNF-α. Its production is generally the combination of some stimulating factors such as LPS and specific receptors of monocytes or macrophages. These stimulating factors activate transcription factors through a series of signal transduction and initiate downstream TNF gene transcription. IL6 gene encodes a cytokine that functions in inflammation and the maturation of B cells. The protein is primarily produced at sites of acute and chronic inflammation, where it is secreted into the serum and induces a transcriptional inflammatory response through the interleukin 6 receptor, alpha. The functioning of this gene is implicated in a wide variety of inflammation-associated disease states IL-6. JUN is linked to human malignant tumors. Variations in the NOS3 are made in association with susceptibility to coronary spasm. NO mediates vasodilation, and the release of NO is regulated by endothelial NOS3. CSR pathogenesis is associated with inflammation and blood vessels, and these possible key targets can regulate inflammatory factors and angiogenesis. From the above analysis, it can be recognized that the 7 targets screened in this study have relevant research support. By analyzing the compound-putative target network of GQHXC and the herb-compound-target-pathway network, 9 target compounds (quercetin, luteolin, kaempferol, beta-sitosterol, formononetin, 7-O-methylisomucronulatol, stigmasterol, isorhamnetin, naringenin) are considered as the key components of GQHXC for treating CSR. For example, quercetin can block the activation of NF-κB, a transcription factor associated with the inflammatory response, and inhibit the production of inflammatory factors. At the same time, it also inhibits the generation of blood vessels. S. H. Kim observed that kaempferol not only can suppress the release of NO and PGE2 but also reduce the expression levels of tumor necrosis factor superfamily, member 2 (TNF-α) and NF-κB. Luteolin and quercetin have obvious inhibitory effects on IL-6 and TNF-α and have beneficial anti-inflammatory effects. F. Yao verified that beta-sitosterol could reduce the content of TNF-α and IL-6 in BALF of ALI mice and down-regulate the activation of NF-κB p65 signal transduction pathway. Furthermore, formononetin can significantly reduce the expression of inducible nitric oxide synthase, TNF-α and IL-1β, and other inflammatory factors. Another study suggested that kaempferol, quercetin, and isorhamnetin inhibit iNOS protein and mRNA expression and NO production in a dose-dependent manner, thereby inhibiting the activation of NF-κB. This is a significant transcription factor of iNOS, which plays a role in anti-inflammatory and protecting the integrity of vascular endothelium. Stigmasterol can inhibit the degradation of various inflammatory mediators and matrices and exert its inhibitory effect by blocking the IL-1β-induced NF-κB pathway. MAPK signaling pathway in MCF-7 cells can be inhibited by naringenin, while PI3K/Akt signaling protein can be activated. Even though there are no large literature reports on 7-O-methylisomucronulatol, it has good oral bioavailability (OB=74.69%) and drug-like properties (DL=0.30). It can be perceived that the 9 screened key compounds mainly affect inflammatory response. Theoretically, they can reduce the inflammatory factors released at the tissues damaged by CSR, ameliorate inflammation and pain, and regulate angiogenesis and vascular tone. In this study, we performed GO enrichment analysis to elucidate various mechanisms of GQHXC treatment of CSR at a systemic level. The bubble chart showed that the BP enrichment results of modules 1 and 2 are better than CC and MF. From thatwe can know that GQHXC mainly treats CSR through biological processes. BP entries in module 1 include regulations of I-kappaB kinase / NF-kappaB signaling, DNA–binding transcription factor activity, and tumor necrosis factor–mediated signaling pathway. In addition, the positive regulations of NF–kappaB transcription factor activity, DNA–binding transcription factor activity, and I–kappaB kinase/NF–kappaB signaling are also included in GO entries. Furthermore they also included stress–activated MAPK cascade, pattern recognition receptor, and toll–like receptor signaling pathways. The I–kappaB kinase/NF–kappaB signaling not only ranks first in the GO entries but also some of the above-mentioned related regulations are related to it. These BP entries are mainly involved in the regulation of the inflammatory response. It is believed that GQHXC can treat the damaged tissues of CSR patients. BP entries in module 2 include positive regulations of angiogenesis, positive chemotaxis, endothelial cell proliferation, and vasculature development. Additionally here were the regulations of vasculature development, positive chemotaxis, angiogenesis regulation of positive chemotaxis, and endothelial cell proliferation. Vascular endothelial growth factor signaling pathways and endothelial cell proliferation were also among the GO entries. BP entries in module 2 are mainly involved in the regulations of angiogenesis and vascular endothelium. We speculated that this is related to the regulation of NO, ET, IgA, IgM, IgG, PGE2, etc. in the plasma of CSR patients. From KEGG enrichment analysis of module 1, we found that most of the pathways involved inflammation and immunity, such as the NF-kappa B signaling pathway, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, and so on. In addition, it is speculated that Shigellosis, Pathogenic Escherichia coli, Herpes simplex virus 1, and other infections are related to inflammation. There are vascular-related pathways in module 2, such as the VEGF signaling pathway, Oxytocin signaling pathway, and Relaxin signaling pathway. The Ras signaling pathway, Rap1 signaling pathway, focal adhesion, and EGFR tyrosine kinase inhibitor resistance are linked with cell regulation. Pathways in both modules such as MAPK signaling pathway, Toll-like receptor signaling pathway, IL-17 signaling pathway, T cell receptor signaling pathway, and TNF signaling pathway, are all connected with inflammation and immunity. The infections in both modules are also detected in association with inflammation. Thus we can see that the results of the KEGG enrichment analysis of inflammation and immunity in these 2 modules are very useful. There are pathways related to blood vessels and cell regulation in module 2. For instance, the AGE-RAGE signaling pathway can activate NF-κB and stimulate the production of vascular endothelial growth factor VEGF. Focal plaques are constituted by the interconnection of extracellular membrane adhesion molecules (ECM), integrin on the cell membrane, and intracellular cytoskeleton proteins. Adhesive plaques have functions of mechanical structure and signal transmission, which can maintain the normal structure of cells and exert their normal functions. Quercetin can exert anti-inflammatory and immune regulation effects by inhibiting MAPK and NF-κB signaling pathways. Formononetin can regulate the PI3K-Akt pathway, VEGF pathway, and MAPK signaling pathway to intervene in the inflammatory response. We speculate that the components of GQHXC may play an important role in treating CSR through key factors of these signaling pathways. Although our research discussed the molecular mechanism of GQHXC, there are still some limitations. First, research data comes from existing databases, so the authenticity and completeness of the results depend on the quality of the data. Second, the results do not reflect all the natural cellular network characteristics in the organism, so further experiments will be needed to confirm the presented data.
Conclusion In this study, 9 active compounds and 7 key genes were selected using network pharmacology. The enrichment analysis of GO and KEGG was conducted to find the potential mechanism of GQHXC in the treatment of CSR. In conclusion, we predicted that GQHXC affects the inflammatory response and vascular regulation process and may achieve the goal of curing CSR. Further experiments are, however, needed to confirm the results of this prediction in GQHXC.
YYL and JRW conceived and designed the study; GLC, XKL, and WZ provided significant suggestions on the methodology; JYZ, CGF, and SYG collected the compounds of GQHXC and targets of CSR; SSJ, and, BBL performed the network pharmacology analysis, HJW, JLL and SL performed molecular docking verification. JYZ review the manuscript, Stalin A polished the manuscript, YYL was a major contributor in writing the manuscript. All authors read and approved the final of the manuscript. Thanks to all authors for their contribution to this manuscript.
Conceptualization: Jiarui Wu. Data curation: Changgeng Fu, Siyu Guo. Formal analysis: Shanshan Jia, Bingbing Li, Haojia Wang, Jialin Li, Shan Lu. Methodology: Xinkui Liu, Wei Zhou, Guoliang Cheng. Supervision: Jiarui Wu, Guoliang Cheng. Writing – original draft: Yingying Liu. Writing – review & editing: Yingying Liu, Jingyuan Zhang, Antony Stalin.
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99b11f75-55e3-476f-ab79-32e49c0d7365 | 9564454 | Anatomy[mh] | INTRODUCTION Programed cell death ligand 1 (PD‐L1) is an immune regulatory molecule which can act as a coregulatory signal through binding to the inhibitory programed cell death 1 (PD‐1) receptor. Binding leads to inhibition of cytokine production and cytolytic activity of PD‐1‐expressing tumor infiltrating CD4 + and CD8 + T cells. During pregnancy, this mechanism has a crucial role in suppressing immune responses and allows development of the partly allogenic fetus and placenta. , Previous studies showed that PD‐L1 is strongly expressed by trophoblast cells in different types of gestational trophoblastic disease (GTD), , , evading immune responses, and thereby allowing proliferation of these (pre‐)malignant cells. GTD comprises a group of pregnancy‐related disorders, originating from placental tissue. It potentially progresses into gestational trophoblastic neoplasia (GTN), a malignant form of GTD. Although GTN is highly curable with current chemotherapies, the mortality rate is still 5% due to chemotherapy resistance, necessitating novel treatment approaches. Blockade of the PD‐1/PD‐L1 pathway with immune checkpoint inhibitors (ICI) has emerged as a novel therapy for various types of cancer. , In cancer patients with, for example, head and neck malignancies or melanoma, durable responses after treatment with PD‐1/PD‐L1 ICIs of up to 2 years in 20%–40% of patients have been reported. , In patients with unresectable chemotherapy‐resistant GTN, pembrolizumab, an antibody against PD‐1, can induce complete responses as described in several case reports. , , , , Selection of patients eligible for treatment with anti PD‐L1 ICIs, however, is challenging. Currently, there are multiple methods to assess expression of PD‐L1 on tumor cells using different antibodies, platforms, scoring systems and cutoff values, often linked to a specific ICI. , , In most studies, patients eligible for anti‐PD‐L1 treatment are selected based on the immunohistochemical (IHC) expression of PD‐L1 on tumor cells, inflammatory cells, or both. However, substantial response rates in patients with tumors lacking PD‐L1, and minimal response rates in patients with tumors highly expressing PD‐L1 have been reported. , Studies analyzing PD‐L1 expression in patients with GTD showed considerable variation in reported PD‐L1 expression patterns. , This variation may be explained not only by tumor heterogeneity but also by the use of different commercially available PD‐L1 antibodies for IHC analysis. , , , Therefore, it is currently insufficiently clear which PD‐L1 antibody is most suitable for detection of PD‐L1‐expressing trophoblast cells, to assess whether PD‐L1 would be a good marker for response to ICI in GTD patients. Therefore, the aim of this study was to identify the antibody most specific for detection of PD‐L1‐expressing cells, by evaluating the most frequently used commercially available PD‐L1 antibodies for IHC.
MATERIAL AND METHODS 2.1 Patient material Formalin‐fixed paraffin embedded (FFPE) samples of four patients with a complete hydatidiform mole (CHM) which progressed to post‐molar GTN after suction curettage, and four patients with a choriocarcinoma were selected from the Radboudumc pathology archives. Selection was based on the availability of FFPE specimens of the first uterine suction curettage at the Radboudumc, and concerned trophoblast tissue removed from patients before start of chemotherapy. Post‐molar GTN was defined according to the FIGO 2000 guideline (ie mola hydatidosa with serum hCG plateauing for three consecutive weeks or rising over a period of two consecutive weeks). 2.2 Flow cytometry Chinese hamster ovarian (CHO) cells (85 050 302, Sigma Aldrich) were transfected using Lipofectamine 3000 (L3000‐015; Invitrogen) with the cDNA constructs cloned into the mammalian expression vector pcDNA3.1+/C‐(K)‐DYK encoding for PD‐L1 and PD‐L2 (OHu22144D and OHu04434D, respectively, both from Genscript) as described before. CHO cells were used since these cells have been the most commonly used mammalian host for (large)‐scale commercial production of therapeutic proteins in the past decades. Flow cytometry was performed on (transfected) CHO cells with conjugated antibodies suitable for flow cytometric assays: anti–PD‐L1‐BV421 (clone MIH1, 563 738, BD Biosciences) and anti‐PD‐L2‐PE (clone MIH18, 558 066, BD Biosciences), with the FACS Verse (BD Biosciences). Flow cytometry data were analyzed using FlowJo software (v10; Tree Star). Transfection efficiency was ~80% for both PD‐L1 and PD‐L2 (Figure ). The remainder of cells were embedded in paraffin with AgarCyto cell block preparation and used for staining. 2.3 Immunohistochemistry Sections of 4‐μm thickness were cut and mounted on glass slides (900 226, VWR). Seven commercially available PD‐L1 antibodies were validated on wildtype Chinese ovarian hamster cells (CHO), and CHO cells transfected with PD‐L1 and PD‐L2. Tonsil tissue was used as a positive control, since PD‐L1 and PD‐L2 are expressed in lymphoid tissue. Primary antibodies included; anti‐PD‐L1 (clone E1L3N, 13 684, Cell Signaling), PD‐L1 Antibody Panel (clones 73–10, CAL10, SP142, 28–8, ab239749, Abcam), anti‐PD‐L1 (clone 22C3, M365329, DAKO) and anti‐PD‐L1 (clone SP263, Roche). IHC with anti‐PD‐L1 clone SP263 was performed on a VENTANA BenchMark ULTRA automated slide stainer as described by Hurkmans et al. Other IHC protocols were carried out manually with antibody dilutions and epitope retrievals according to the manufacturers’ instructions (Appendix ). The IHC‐stained CHO cell and tonsil slides were assessed individually by two pathologists specialized in gynecological oncology. Positivity of the PD‐L1 protein was defined by membranous staining. The percentage of positive cells was determined. Additionally, cytoplasmatic staining and staining intensity were documented. We scored staining intensity as weak (+/−), mild , strong or very strong . Scoring results of the slides were revealed after both pathologists scored all slides individually. Antibodies considered specific for PD‐L1 protein staining based on the results of the tonsil and transfected CHO samples, were used to stain four FFPE tumor tissue samples of patients with a CHM, and four FFPEs of patients with a choriocarcinoma. Samples were again analyzed by the two pathologists, and scoring results were revealed after they both scored all slides individually. To evaluate the agreement between the two pathologists in PD‐L1‐positive scored cells of the CHM and choriocarcinoma samples, we determined the intraclass correlation coefficient (ICC) for all tested antibodies individually. 2.4 Statistical analyses To calculate ICC, the two‐way random‐effects model with consistency was used. The ICCs were determined with both average and single measures. Average measures are used when measures of ≥2 raters are averaged to derive the result of a test. Single measures are used when a single rater performs the test, and is likely to be used in daily clinical practice. An ICC value of <0.50 is considered poor, between 0.50 and 0.75 moderate, between 0.75 and 0.90 good, and >0.90 red excellent. Analyses were performed with IBM SPSS statistics, version 25. 2.5 Ethics statement This study was approved by the local ethical committee of the Radboud University Medical Center (reference number 2018–4132) on June 7, 2018.
Patient material Formalin‐fixed paraffin embedded (FFPE) samples of four patients with a complete hydatidiform mole (CHM) which progressed to post‐molar GTN after suction curettage, and four patients with a choriocarcinoma were selected from the Radboudumc pathology archives. Selection was based on the availability of FFPE specimens of the first uterine suction curettage at the Radboudumc, and concerned trophoblast tissue removed from patients before start of chemotherapy. Post‐molar GTN was defined according to the FIGO 2000 guideline (ie mola hydatidosa with serum hCG plateauing for three consecutive weeks or rising over a period of two consecutive weeks).
Flow cytometry Chinese hamster ovarian (CHO) cells (85 050 302, Sigma Aldrich) were transfected using Lipofectamine 3000 (L3000‐015; Invitrogen) with the cDNA constructs cloned into the mammalian expression vector pcDNA3.1+/C‐(K)‐DYK encoding for PD‐L1 and PD‐L2 (OHu22144D and OHu04434D, respectively, both from Genscript) as described before. CHO cells were used since these cells have been the most commonly used mammalian host for (large)‐scale commercial production of therapeutic proteins in the past decades. Flow cytometry was performed on (transfected) CHO cells with conjugated antibodies suitable for flow cytometric assays: anti–PD‐L1‐BV421 (clone MIH1, 563 738, BD Biosciences) and anti‐PD‐L2‐PE (clone MIH18, 558 066, BD Biosciences), with the FACS Verse (BD Biosciences). Flow cytometry data were analyzed using FlowJo software (v10; Tree Star). Transfection efficiency was ~80% for both PD‐L1 and PD‐L2 (Figure ). The remainder of cells were embedded in paraffin with AgarCyto cell block preparation and used for staining.
Immunohistochemistry Sections of 4‐μm thickness were cut and mounted on glass slides (900 226, VWR). Seven commercially available PD‐L1 antibodies were validated on wildtype Chinese ovarian hamster cells (CHO), and CHO cells transfected with PD‐L1 and PD‐L2. Tonsil tissue was used as a positive control, since PD‐L1 and PD‐L2 are expressed in lymphoid tissue. Primary antibodies included; anti‐PD‐L1 (clone E1L3N, 13 684, Cell Signaling), PD‐L1 Antibody Panel (clones 73–10, CAL10, SP142, 28–8, ab239749, Abcam), anti‐PD‐L1 (clone 22C3, M365329, DAKO) and anti‐PD‐L1 (clone SP263, Roche). IHC with anti‐PD‐L1 clone SP263 was performed on a VENTANA BenchMark ULTRA automated slide stainer as described by Hurkmans et al. Other IHC protocols were carried out manually with antibody dilutions and epitope retrievals according to the manufacturers’ instructions (Appendix ). The IHC‐stained CHO cell and tonsil slides were assessed individually by two pathologists specialized in gynecological oncology. Positivity of the PD‐L1 protein was defined by membranous staining. The percentage of positive cells was determined. Additionally, cytoplasmatic staining and staining intensity were documented. We scored staining intensity as weak (+/−), mild , strong or very strong . Scoring results of the slides were revealed after both pathologists scored all slides individually. Antibodies considered specific for PD‐L1 protein staining based on the results of the tonsil and transfected CHO samples, were used to stain four FFPE tumor tissue samples of patients with a CHM, and four FFPEs of patients with a choriocarcinoma. Samples were again analyzed by the two pathologists, and scoring results were revealed after they both scored all slides individually. To evaluate the agreement between the two pathologists in PD‐L1‐positive scored cells of the CHM and choriocarcinoma samples, we determined the intraclass correlation coefficient (ICC) for all tested antibodies individually.
Statistical analyses To calculate ICC, the two‐way random‐effects model with consistency was used. The ICCs were determined with both average and single measures. Average measures are used when measures of ≥2 raters are averaged to derive the result of a test. Single measures are used when a single rater performs the test, and is likely to be used in daily clinical practice. An ICC value of <0.50 is considered poor, between 0.50 and 0.75 moderate, between 0.75 and 0.90 good, and >0.90 red excellent. Analyses were performed with IBM SPSS statistics, version 25.
Ethics statement This study was approved by the local ethical committee of the Radboud University Medical Center (reference number 2018–4132) on June 7, 2018.
RESULTS 3.1 IHC on CHO and tonsil tissue All antibodies showed positive membranous staining of the PD‐L1‐transfected CHO cells varying from 1% to 90%. The highest percentages of cells that scored positive for PD‐L1 in the PD‐L1‐transfected CHO cells were observed for the EL13N antibody (70%–90%) and 22C3 antibody (60%–70%),whereas the 73–10, CAL10, SP142, 28–8 and SP263 antibodies scored low percentages (Figure ; Table ). None of the PD‐L1 antibodies showed positive membranous staining on the CHO cells transfected with PD‐L2, or the wildtype CHO cells; however, the E1L3N antibody showed slight cytoplasmatic staining in the CHO PD‐L2 cells and, according to one pathologist, also in the wildtype CHO cells. The E1L3N, 73–10, 22C3, SP142 and SP263 antibodies showed strong membranous staining of the tonsil tissue (Figure ; Table ). The CAL10 and 28–8 antibodies showed weak to mild membranous staining of the tonsil tissue (Figure ; Table ). 3.2 PD‐L1 staining of complete hydatidiform moles and choriocarcinomas We observed qualitative differences in PD‐L1 staining patterns between the antibodies in the same CHM samples (Figure ; Table ). Expression of PD‐L1 was predominantly seen on the membrane of syncytiotrophoblast cells. Staining intensity was strongest with the SP263 antibody; however, besides membranous staining, strong cytoplasmatic staining was observed for all CHM samples with this antibody (Figure ). The 73–10, 22C3 and SP142 antibodies showed mild staining intensity, and staining intensity was weakest with the CAL10 and 28–8 antibodies (Figure ). Comparable to the results in CHM tissue, expression of PD‐L1 on choriocarcinoma tissue was predominantly seen on the membrane of syncytiotrophoblast cells. Percentages of tumor cells positive for PD‐L1 appeared to be higher in the choriocarcinoma samples than the CHM samples (Table ). Staining intensity was comparable to the results in the CHM samples: intensity was strongest with the SP263 antibody, mild with the 73–10, 22C3 and SP142 antibodies, and weakest with the CAL10 and 28–8 antibodies (Figure ). Again, cytoplasmatic staining was observed for choriocarcinoma samples stained with the SP263 antibody. 3.3 Pathologists’ agreement The ICCs determined with average measures were excellent for the 73–10, 22C3, 28–8 and SP263 antibodies, and good for the CAL10 and SP142 antibodies (Table ). ICCs determined with single measures were considered excellent for the 22C3 and SP263 antibodies, good for the 73–10 and 28–8 antibodies, and moderate for the CAL10 and SP142 antibodies (Table ).
IHC on CHO and tonsil tissue All antibodies showed positive membranous staining of the PD‐L1‐transfected CHO cells varying from 1% to 90%. The highest percentages of cells that scored positive for PD‐L1 in the PD‐L1‐transfected CHO cells were observed for the EL13N antibody (70%–90%) and 22C3 antibody (60%–70%),whereas the 73–10, CAL10, SP142, 28–8 and SP263 antibodies scored low percentages (Figure ; Table ). None of the PD‐L1 antibodies showed positive membranous staining on the CHO cells transfected with PD‐L2, or the wildtype CHO cells; however, the E1L3N antibody showed slight cytoplasmatic staining in the CHO PD‐L2 cells and, according to one pathologist, also in the wildtype CHO cells. The E1L3N, 73–10, 22C3, SP142 and SP263 antibodies showed strong membranous staining of the tonsil tissue (Figure ; Table ). The CAL10 and 28–8 antibodies showed weak to mild membranous staining of the tonsil tissue (Figure ; Table ).
PD‐L1 staining of complete hydatidiform moles and choriocarcinomas We observed qualitative differences in PD‐L1 staining patterns between the antibodies in the same CHM samples (Figure ; Table ). Expression of PD‐L1 was predominantly seen on the membrane of syncytiotrophoblast cells. Staining intensity was strongest with the SP263 antibody; however, besides membranous staining, strong cytoplasmatic staining was observed for all CHM samples with this antibody (Figure ). The 73–10, 22C3 and SP142 antibodies showed mild staining intensity, and staining intensity was weakest with the CAL10 and 28–8 antibodies (Figure ). Comparable to the results in CHM tissue, expression of PD‐L1 on choriocarcinoma tissue was predominantly seen on the membrane of syncytiotrophoblast cells. Percentages of tumor cells positive for PD‐L1 appeared to be higher in the choriocarcinoma samples than the CHM samples (Table ). Staining intensity was comparable to the results in the CHM samples: intensity was strongest with the SP263 antibody, mild with the 73–10, 22C3 and SP142 antibodies, and weakest with the CAL10 and 28–8 antibodies (Figure ). Again, cytoplasmatic staining was observed for choriocarcinoma samples stained with the SP263 antibody.
Pathologists’ agreement The ICCs determined with average measures were excellent for the 73–10, 22C3, 28–8 and SP263 antibodies, and good for the CAL10 and SP142 antibodies (Table ). ICCs determined with single measures were considered excellent for the 22C3 and SP263 antibodies, good for the 73–10 and 28–8 antibodies, and moderate for the CAL10 and SP142 antibodies (Table ).
DISCUSSION We observed substantial heterogeneity in the percentage of cells stained positive for PD‐L1 within the same tonsil, CHO, CHM and choriocarcinoma samples between the different PD‐L1 antibodies. Based on our results, the 22C3 antibody is the most suitable for adequate detection of PD‐L1‐positive cells in tissues of CHM, and in choriocarcinoma patients. With the use of the 22C3 antibody, we observed a high to very high membranous staining intensity positive for PD‐L1 in 60%–70% within the, for 80% PD‐L1‐transfected CHO cells. No positive staining on the CHO cells transfected with PD‐L2 or wildtype CHO cells was observed. The latter two observations underscore the specificity of the antibody. Additionally, with the 22C3 antibody, ICCs were excellent for the CHO, CHM and choriocarcinoma samples. Based on the results with the transfected CHO cells, we excluded the E1L3N antibody, since unexpected positive cytoplasmatic staining was observed in the CHO cells transfected with PD‐L2. Although according to the manufacturer cells with pure cytoplasmic immunoreaction (without membranous staining) should be ignored, we cannot fully exclude the possibility that E1L3N recognizes either a PD‐L1 variant, or another structurally related protein like for instance PD‐L2, and may therefore incorrectly indicate the presence of PD‐L1‐positive cells. Concerning this cytoplasmatic staining with the E1L3N antibody of the CHO cells that were transfected with PD‐L2, the pathologists scored 30%–40% of CHO cells that were transfected with PD‐L2 as falsely positive for PD‐L1, underlining the risk of overestimation and incorrect observation of PD‐L1‐positive cells. Low percentages of PD‐L1‐positive cells with PD‐L1‐transfected CHO cells were observed upon staining with the antibodies 73–10, CAL10, SP142 and SP263. This suggests that these antibodies have a lower affinity for the PD‐L1 protein than do 22C3 and 28–8 antibodies. Many ongoing studies in patients with GTD, analyzing PD‐L1 expression by trophoblast cells in order to predict clinical outcome or to select patients for treatment with PD‐L1 ICIs, are based on percentage of PD‐L1 expression in tumor samples of these patients, stained with, among others, the E1L3N, 28–8, SP263 and 22C3 PD‐L1 antibodies. , , , , , , Since different antibodies are used to stain the same type of tumor samples, we compared the percentage of PD‐L1‐positive cells within the same CHM and choriocarcinoma samples using the 73–10, 22C3, CAL10, SP142, 28–8 and SP263 antibodies, to assess whether comparable results are obtained. As expected, PD‐L1 expression was different in the CHM and choriocarcinoma samples. Fewer PD‐L1‐positive cells were present in CHM samples compared with the choriocarcinoma samples. Strikingly, we also observed a substantial variety in detection of PD‐L1‐expressing cells within the same CHM and choriocarcinoma samples when we compared staining results with the different antibodies. These results suggest that the reported varieties in staining patterns and trophoblast subtypes expressing PD‐L1 can be explained by the use of different PD‐L1 antibodies. , , As treatment eligibility of patients is frequently based on the percentage of PD‐L1 expression within tumor samples, , , , , and since different PD‐L1 antibodies showed various levels of PD‐L1‐expressing cells within the same samples, patient eligibility for treatment with anti‐PD‐L1 ICIs is actually determined by the choice of PD‐L1 antibody, rather than by the actual expression percentages of PD‐L1. This is the first study to compare seven frequently used commercially available antibodies in order to select the antibody most suitable for detection of PD‐L1‐expressing cells in CHO cells and tissue of CHM and choriocarcinoma patients. , , , , To assess the specificity of the PD‐L1 antibodies, we needed a “model” whose PD‐L1 and PD‐L2 expression percentages were known beforehand, in order afterwards to assess the percentage of PD‐L1‐expressing cells the antibody stained positive for expression of PD‐L1. By transfecting CHO cells with PD‐L1 and PD‐L2, we were able to compare the IHC results with the flow cytometry results, here used as the gold standard. This gold standard is needed, since choriocarcinoma cell lines already express PD‐L1 and PD‐L2, and we cannot visualize the difference between cells transfected with PD‐L1 and PD‐L2, and the non‐transfected (ie negative control) cells. Additionally, since the true percentage of PD‐L1‐expressing cells within the trophoblast tissues is unknown, it is not feasible to determine antibody specificity based on staining results in trophoblast samples. Selection of the 22C3 antibody was primarily based on the staining results of the CHO samples, and was underlined by the staining results of the CHM and choriocarcinoma samples. Although we used a low number of CHM and choriocarcinoma cases, we observed substantial variety in staining patterns using different PD‐L1 antibodies within the same CHM and choriocarcinoma samples, emphasizing the need for uniformity in usage of a single PD‐L1 antibody to detect PD‐L1‐expressing cells in patients with GTN. One could argue that the variety in staining patterns may partially be explained by PD‐L1 heterogeneity within the tissues. This was also observed in other studies in which PD‐L1 expression was determined on different tumor or trophoblast cells. , , , We anticipated this possible inter‐sample heterogeneity using subsequent samples of the same tissue‐blocks for staining with the different antibodies. A possible limitation of our study is that except for SP263, samples were stained manually and therefore were more prone to staining inaccuracies. However, manufacturer protocols were strictly followed, and all samples were stained simultaneously. Secondly, in clinical settings, antibody staining protocols are frequently optimized to enhance staining intensity for easier detection of positively stained cells. For example, the CAL10 antibody hardly stained the tonsil or the PD‐L1‐transfected CHO cells and may have improved optimization. A limitation of “in‐house” optimization of immunohistochemical staining methods is that these procedures should be described thoroughly to facilitate comparison between published studies, and to facilitate reproducibility in future studies. Therefore, we adhered to the manufacturer’s recommended protocols to determine the most optimal and reliable PD‐L1 antibody, resulting in 22C3 being our recommended choice for future studies.
CONCLUSION Using different antibodies to detect PD‐L1‐expressing cells in CHM and choriocarcinoma samples leads to substantial variation in observed tumor cells expressing PD‐L1. Based on our results, the 22C3 antibody seems most suitable for detecting PD‐L1‐positive cells in CHO cells transfected with PD‐L1, and should therefore be used in future studies analyzing expression of PD‐L1 in samples of patients with GTD. Clinical correlation with treatment response should be the next step.
All authors have been involved in the study design. YH collected the data. YH, MS, JB, MG, NO, JdeV and FS interpreted the data. YH wrote the manuscript and all authors were equally involved in reviewing and editing the manuscript.
None.
Appendix S1 Click here for additional data file.
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The Mechanical Properties of Orthodontic Aligners of Clear Aligner After Intraoral Use in Different Time Periods | 3ba18aa4-a1f0-4030-a5e7-6ad9c309b88e | 11897417 | Dentistry[mh] | Introduction The use of orthodontic aligners to achieve the desired tooth movement has become increasingly popular over the years. Numerous manufactures and techniques have emerged, offering a variety of options, such as Invisalign, Clear Aligner, K‐Line, Duran or in‐house three‐dimensional (3D) printed aligners. In comparison with conventional fixed appliances, orthodontic aligners have the advantage of decreased chair time, better oral hygiene condition, gingival health, less pain and more concealed treatment . However, many researchers argue that aligners may result to less predictable and accurate orthodontic outcomes, as specific orthodontic movements, such as extrusion and rotation, are difficult to be accomplished and more than half of the cases may require refinement . Polyester, polyurethane and polypropylene are the predominant thermoplastic materials in polymer blends used to manufacture orthodontic aligners. Polyurethane is one of the most versatile thermoplastic engineering compounds, with excellent physical properties, chemical and abrasion resistance, and ease of processing. However, it is not an inert material, and its properties may be affected by heat, moisture or contact with salivary enzymes . Invisalign aligners are made of a polyurethane‐based material , whereas Clear Aligner by polyethylene glycol terephthalate (PETG) . Fang et al. evaluated the Invisalign thermoformed aligner material condition before and after clinical application, including its mechanical properties, surface morphology, internal structure, and chemical composition and concluded that the mechanical properties of aligners were not significantly affected by the oral environment. On the other hand, Bradley et al. performed similar research and identified significant changes in the mechanical properties of the aligners after extended oral use. Can et al. studied the alterations of the mechanical properties of in‐house three‐dimensional (3D) printed orthodontic aligners and concluded that the mechanical properties of the aligners were not affected after 1 week of intraoral use. In 2019, Jindal et al. found that the 3D printed aligners could deform elastically and reversibly for lower displacements, whereas larger displacements result to plastic, hence irreversible, deformation. According to the authors, the ideal aligner should exert continuous light force, its material should be stiff enough to withstand the applied force, and the stress relaxation curve should be quite flat. Despite the increasing interest in orthodontic aligner treatment, the mechanical properties of clinically worn thermoformed aligners have not been thoroughly investigated and more studies are needed to evaluate the effect of ageing on their mechanical properties . The aim of this study was to investigate the tensile properties and hardness of Clear Aligner orthodontic aligners (Scheu‐Dental, Iserlohn, Germany), after their intraoral use for 7, 10 and 14 days and to compare them with unused aligners (as‐received [0 days]), and their unprocessed material (PETG). The null hypothesis tested is that intraoral ageing of the aligners does not adversely affect the tensile properties and hardness of the aligners.
Materials and Methods 2.1 Patient Population After obtaining approval from the Ethic approval committee of the National and Kapodistrian University of Athens, we prospectively enrolled in the study patients presented at the Department of Orthodontics of the National and Kapodistrian University of Athens (Athens, Greece). We considered eligible all patients who were seeking initial full orthodontic treatment with Clear Aligner on both arches, had full dental arches with absence of transverse and vertical occlusal discrepancies, had no periodontal disease, parafunctional habits or other systemic diseases or syndromes, and were willing to provide informed consent and follow the provided instructions. We excluded patients who had extracted or missing teeth, other than the third molars, had to use transmaxillary elastics, and whose aligners presented signs of abuse, such as cracks or breaks. We did not apply any exclusion criteria on the basis of age, race or gender. The exclusion criteria were applied on the basis of the results of previous studies which have shown that the aforementioned factors could affect the mastication ability and create uneven distribution of forces between the patients. Particularly, temporomandibular disorder (TMD), as also sleep bruxism, can alter the masticatory muscles' functions and eventually cause longer acting and greater occlusal forces to be exerted on the aligners . Periodontitis and extractions also affect the mastication force causing a negative impact on masticatory cycle efficiency and molar bite force, according to the residual number of occlusal support areas . Patients using transmaxillary elastics were excluded from our sample to eliminate any possible influence elastics have on jaw movement . Last, patients with systematic diseases that affect mastication, such as diabetes, stroke, chronic obstructive pulmonary disease and Sjögren's syndrome , were excluded. Patients who agreed to participate were instructed to use the aligners for at least 22 h a day, and to remove them only during mastication, liquid consumption and oral hygiene care. They were also requested to use a diary to record the daily use of the aligners to confirm the exact duration of their use. Participants were asked to use the first set of aligners for 7 days, the second one for 10 days and the third for 14 days. They were then asked to return them to their orthodontist. An unused set (as‐received) from each patient was taken for analysis. 2.2 Sample Preparation In our study, aligners of similar thicknesses (0.75 mm) were used, as Kwon et al. concluded that the forces delivered by aligners are influenced by the type and the thickness of the material. The importance of the aligners' thickness is correlated with the amount of load exerted on the periodontal ligament and with the orthodontic force delivered to achieve the planned sequential dental movements . Appliances fabricated from thicker material (0.75 or 0.8 mm), according to Kohda et al. produce significantly greater force than those fabricated from thinner material (0.4 or 0.5 mm). Following retrieval, the aligners underwent ultrasound cleaning, and each aligner was divided into three different sections, two posterior and one anterior. Specifically, the posterior segments spanned from the second molar to the first premolar, and the anterior included the canine‐to‐canine section. The right and left segments of the aligners were studied together and are referred as posterior. From each segment a specimen was made that included the occlusal surface for the posterior segments, and the buccal surfaces for the anterior. The segments were prepared using a sharp tool with straight edges to lower the possibility of shear stress microcracks forming. Each segment was measured in the three dimensions of length, width and thickness. In collaboration with the Chemical Engineering School of the National Technical University of Athens (Athens, Greece), the different samples of the orthodontic aligners were subjected to a study of their tensile properties and hardness. Separate specimens from the unprocessed material of Clear Aligner were also submitted to tensile testing. Ten specimens were prepared using a water jet machine and six with the same sharp tool as the one used for the aligners, both according to ISO 6892‐1:2019 specifications (Figure ) . 2.3 Assessment of Mechanical Properties and Analysis Tensile tests were conducted to assess the mechanical properties of the samples, specifically elastic modulus ( E ), ultimate tensile stress (UTS) and yield stress (YS). Elastic modulus provides the correlation of applied force to resulting deformation and is defined as the ratio of stress to strain. Materials with low elastic modulus tend to be less brittle than materials with high elastic modulus, and to exert lower elastic forces . Yield stress is the point at which a predetermined amount of permanent deformation occurs, and a high value indicates better resistance to deactivation caused by plastic deformation, which favours applications relying on elastic forces. Ultimate tensile stress is the maximum stress that a material can withstand, without breaking, while being stretched or pulled and signifies the probability of a material fracturing under load . All mechanical properties were measured according to international standard specifications (ISO 14577‐2016) . Elastic modulus and tensile stress were tested with a SAUTER TVO 500N500S tensile machine by Sauter (GmbH, Wutöschingen, Germany, error = ±2%) with a load cell of 500 N and 15 mm/min displacement speed. As sliding and minor strain during the placing of the segments at the machine grips contribute to measurement error, we aimed towards accurate placement with minimum application of force. For the hardness measurement a Durometer Type D by Instron (Boston, Massachusetts, USA, error = ±2%) was used, according to the ASTM D2240 standard . Specific anterior and posterior points were measured for hardness, prior to tensile properties measurements, and in particular the buccal surface of the central incisors and the mesial buccal cusp of the first molars. Samples with identified flaws were excluded from the tensile analysis. From each measurement, a stress–strain diagram was obtained and studied. The elastic modulus was defined as the slope of the obtained linear fit regression graph of each diagram. From each diagram a regression, graph was manually created and through parallel shifting along the x ‐axis until intersection with the stress–strain curve at 0.1% relative strain, the 0.1% offset yield strength was defined. Ultimate tensile stress was the highest stress value of each diagram (Figure ). To estimate the required sample size, we conducted a power analysis. Based on the previous literature, the minimum sample size to detect an effective size of 0.5 for all the properties tested, setting alpha to 0.05 and power to 0.80 was at least 15 segments of aligners for each period. The results from the different samples were compared using the two‐tailed Mann–Whitney test. For all the statistical tests, the level of significance was set at 95% ( p = 0.05).
Patient Population After obtaining approval from the Ethic approval committee of the National and Kapodistrian University of Athens, we prospectively enrolled in the study patients presented at the Department of Orthodontics of the National and Kapodistrian University of Athens (Athens, Greece). We considered eligible all patients who were seeking initial full orthodontic treatment with Clear Aligner on both arches, had full dental arches with absence of transverse and vertical occlusal discrepancies, had no periodontal disease, parafunctional habits or other systemic diseases or syndromes, and were willing to provide informed consent and follow the provided instructions. We excluded patients who had extracted or missing teeth, other than the third molars, had to use transmaxillary elastics, and whose aligners presented signs of abuse, such as cracks or breaks. We did not apply any exclusion criteria on the basis of age, race or gender. The exclusion criteria were applied on the basis of the results of previous studies which have shown that the aforementioned factors could affect the mastication ability and create uneven distribution of forces between the patients. Particularly, temporomandibular disorder (TMD), as also sleep bruxism, can alter the masticatory muscles' functions and eventually cause longer acting and greater occlusal forces to be exerted on the aligners . Periodontitis and extractions also affect the mastication force causing a negative impact on masticatory cycle efficiency and molar bite force, according to the residual number of occlusal support areas . Patients using transmaxillary elastics were excluded from our sample to eliminate any possible influence elastics have on jaw movement . Last, patients with systematic diseases that affect mastication, such as diabetes, stroke, chronic obstructive pulmonary disease and Sjögren's syndrome , were excluded. Patients who agreed to participate were instructed to use the aligners for at least 22 h a day, and to remove them only during mastication, liquid consumption and oral hygiene care. They were also requested to use a diary to record the daily use of the aligners to confirm the exact duration of their use. Participants were asked to use the first set of aligners for 7 days, the second one for 10 days and the third for 14 days. They were then asked to return them to their orthodontist. An unused set (as‐received) from each patient was taken for analysis.
Sample Preparation In our study, aligners of similar thicknesses (0.75 mm) were used, as Kwon et al. concluded that the forces delivered by aligners are influenced by the type and the thickness of the material. The importance of the aligners' thickness is correlated with the amount of load exerted on the periodontal ligament and with the orthodontic force delivered to achieve the planned sequential dental movements . Appliances fabricated from thicker material (0.75 or 0.8 mm), according to Kohda et al. produce significantly greater force than those fabricated from thinner material (0.4 or 0.5 mm). Following retrieval, the aligners underwent ultrasound cleaning, and each aligner was divided into three different sections, two posterior and one anterior. Specifically, the posterior segments spanned from the second molar to the first premolar, and the anterior included the canine‐to‐canine section. The right and left segments of the aligners were studied together and are referred as posterior. From each segment a specimen was made that included the occlusal surface for the posterior segments, and the buccal surfaces for the anterior. The segments were prepared using a sharp tool with straight edges to lower the possibility of shear stress microcracks forming. Each segment was measured in the three dimensions of length, width and thickness. In collaboration with the Chemical Engineering School of the National Technical University of Athens (Athens, Greece), the different samples of the orthodontic aligners were subjected to a study of their tensile properties and hardness. Separate specimens from the unprocessed material of Clear Aligner were also submitted to tensile testing. Ten specimens were prepared using a water jet machine and six with the same sharp tool as the one used for the aligners, both according to ISO 6892‐1:2019 specifications (Figure ) .
Assessment of Mechanical Properties and Analysis Tensile tests were conducted to assess the mechanical properties of the samples, specifically elastic modulus ( E ), ultimate tensile stress (UTS) and yield stress (YS). Elastic modulus provides the correlation of applied force to resulting deformation and is defined as the ratio of stress to strain. Materials with low elastic modulus tend to be less brittle than materials with high elastic modulus, and to exert lower elastic forces . Yield stress is the point at which a predetermined amount of permanent deformation occurs, and a high value indicates better resistance to deactivation caused by plastic deformation, which favours applications relying on elastic forces. Ultimate tensile stress is the maximum stress that a material can withstand, without breaking, while being stretched or pulled and signifies the probability of a material fracturing under load . All mechanical properties were measured according to international standard specifications (ISO 14577‐2016) . Elastic modulus and tensile stress were tested with a SAUTER TVO 500N500S tensile machine by Sauter (GmbH, Wutöschingen, Germany, error = ±2%) with a load cell of 500 N and 15 mm/min displacement speed. As sliding and minor strain during the placing of the segments at the machine grips contribute to measurement error, we aimed towards accurate placement with minimum application of force. For the hardness measurement a Durometer Type D by Instron (Boston, Massachusetts, USA, error = ±2%) was used, according to the ASTM D2240 standard . Specific anterior and posterior points were measured for hardness, prior to tensile properties measurements, and in particular the buccal surface of the central incisors and the mesial buccal cusp of the first molars. Samples with identified flaws were excluded from the tensile analysis. From each measurement, a stress–strain diagram was obtained and studied. The elastic modulus was defined as the slope of the obtained linear fit regression graph of each diagram. From each diagram a regression, graph was manually created and through parallel shifting along the x ‐axis until intersection with the stress–strain curve at 0.1% relative strain, the 0.1% offset yield strength was defined. Ultimate tensile stress was the highest stress value of each diagram (Figure ). To estimate the required sample size, we conducted a power analysis. Based on the previous literature, the minimum sample size to detect an effective size of 0.5 for all the properties tested, setting alpha to 0.05 and power to 0.80 was at least 15 segments of aligners for each period. The results from the different samples were compared using the two‐tailed Mann–Whitney test. For all the statistical tests, the level of significance was set at 95% ( p = 0.05).
Results The study sample consisted of four patients treated with Clear Aligner aligners. Each patient provided four sets of aligners and each aligner was divided into three segments, totalling to 96 samples that were tested for their mechanical properties. In addition, 16 samples of unprocessed material were included (six prepared by a sharp tool and 10 by a water jet machine). It is noted that 16.96% of samples had to be excluded from the analysis because they presented microcracks or flaws at the stage of preparation. The summary of the measurements is found in Tables , Table (for the processed material) and Table (for the unprocessed material). Based on our findings, the average elastic modulus of the segments (Figure ) presented statistically significant reduction from day 0 of use (911.66 ± 139.90 MPa) to day 14 (746.41 ± 145.41 MPa, p = 0.038). A reduction was observed for the intermediate time period, however this finding was not statistically significant. Comparison of the elastic modulus between anterior and posterior segments showed statistically significant difference among samples provided on day 14 of use (posterior segments [814.68 ± 118.64 MPa] versus anterior segments [609.87 ± 85.79 MPa], p = 0.003). Regarding the UTS, the maximum values were measured at day 0 of use and the lowest at day 14 (Figure ). Statistically significant decreases were measured for average UTS between day 0 (35.12 ± 3.83 MPa) and day 7 (30.85 ± 6.46 MPa, p = 0.01), and from day 7 to day 10 (23.43 ± 11.64 MPa, p = 0.05). No statistically significant differences were detected between anterior and posterior samples on any period assessed. Assessment of the yield stress (Figure ) showed a statistically significant reduction in the average yield stress during clinical use from day 0 of use (33.05 ± 3.76 MPa) to day 7 (28.90 ± 6.07 MPa, p = 0.0018). Anterior and posterior segments measurements did not show a statistically significant difference. Samples hardness evaluation showed, a decrease in the average hardness between day 0 and day 14 of use, which proved not statistically significant (Figure ). It was also found that the posterior segments (measured at the first molars) exhibited statistically significantly higher values of Shore D hardness in comparison to anterior segments (measured at central incisors) in all periods of treatment ( p < 0.01). Among the values measured for the anterior segments, statistically significant differences were observed from day 0 of use (57 ± 2 Shore D) to day 10 (54.38 ± 1.60 Shore D). The posterior segments demonstrated a statistically significant reduction in hardness from day 0 of use (64.06 ± 1.91 Shore D) to day 7 (61.38 ± 1.70 Shore D, p < 0.01). In regard to the measurements obtained for the unprocessed material samples prepared with a sharp tool, the mean elastic modulus was calculated at 1269.53 ± 131.56 MPa, the mean UTS at 51.76 ± 2.83 MPa, the mean yield stress at 50.87 ± 2.92 MPa and mean hardness at 60.33 ± 1.03 Shore D. These values were statistically significantly different from the corresponding values of the as‐received aligners (day 0 of use, p < 0.01). For the specimens prepared with the water jet machine, mean elastic modulus was measured at 1186.82 ± 127.60 MPa, mean ultimate tensile stress at 51.89 ± 1.01 MPa, mean yield stress at 49.44 ± 0.54 MPa and mean hardness at 60.3 ± 0.95 Shore D. These values were statistically significantly different from the corresponding values of the as‐received aligners (day 0 of use, p < 0.01), but were similar to the ones obtained from the specimens prepared with the sharp tool ( p > 0.05).
Discussion Aligners are becoming one of the most common methods of orthodontic treatment, preferred by patients mainly due to the aesthetic superiority, faster treatment and fewer emergencies compared with fixed orthodontic appliances . The daily use of aligners alters their mechanical properties; however, this alteration's effect on the clinical outcome of the orthodontic treatment is currently not known . In the present study, we investigated the mechanical properties of the orthodontic aligners—Clear Aligner—after intraoral use. Although other aligners have been studied, in the literature, there is paucity of data regarding the integrity of Clear Aligner over time. This is one of the first studies that examined this product, and the first in vivo study, and provides evidence of significant alteration of its mechanical properties over time during intraoral use. The results of our study indicate that the tensile properties and hardness of the aligners were decreased after intraoral use and therefore, the null hypothesis must be rejected. In particular, the elastic modulus was significantly decreased from day 0 of use to day 14, whereas yield stress and UTS were significantly decreased from day 0 of use to day 7. When we examined the anterior and posterior segments on day 14 of use, it was found that the posterior samples had significantly higher mean value of elastic modulus than the anterior, despite having similar values at day 0 of use. It is estimated that a potential reason for the observed difference are the higher occlusal forces which are exercised on the posterior segments during that prolonged duration of use and have possibly turned the material more brittle. According to the literature, ageing is expected to affect the structure and the mechanical properties of the orthodontic aligner materials to some extent. More specifically, in their study Schuster et al. who examined patients with Invisalign aligners, found that within 2 weeks of use, aligners may present increased hardness, calcification and general wear. Bradley et al. (2016) tested the mechanical properties of Invisalign aligners used for a mean period of 44 ± 15 days and concluded that they were adversely affected by intraoral ageing. In their study, the indentation modulus ( E IT ) in used aligners, was found to be significantly decreased compared with day 0 of use, whereas the elastic index value ( η IT ) was increased, indicating that the aged material had turned more brittle . In another study Papadopoulou et al. (2019) on Invisalign aligners after day 0, day 7 and day 14 of use, found that the aligners on day 7 and day 14 exhibited significantly inferior mechanical properties compared with day 0. However, no difference was observed between aligners on day 7 and day 14 of use . It is believed that the observed effects of intraoral ageing are due to several factors, such as relief of residual stresses induced by the manufacturing process, leaching of matrix plasticizers and polyurethane softening mechanism . On the other hand, Fang et al. (2020) examined the mechanical properties of Invisalign, on day 0 and day 14 of use and concluded that although surface morphology of the aligner may present wear and tear after use, its mechanical properties (elastic modulus) remained stable. However, they provided evidence that trace elements, such as aluminium, can be released during clinical use, which may be a consideration, especially for patients with allergy. Regarding Clear Aligner, Ihssen et al. (2019) studied the properties of the material used for aligner manufacturing in vitro. In their study, PETG was subjected to accelerated ageing by thermocycling after previous immersion in distilled water to simulate 14 days of intraoral use in two different temperatures, 22°C and 37°C. The estimated values for the unprocessed material ranged normally between 1.84 and 2.13 GPa for the elastic modulus, 32.2 and 41.51 MPa for the yield strength, and 40.75 and 50.26 MPa for the ultimate tensile strength. On the samples subjected to simulation of 14 days of intraoral stay, the corresponding values were 1.74–2.04 GPa for the elastic modulus, 31.93–39.80 MPa for the yield stress and 38.89–51.58 MPa for the UTS. The researchers concluded that the material's elastic modulus, yield strength and UTS were reduced compared with unused PETG specimens that were submitted to immersion. The specimens submitted to thermocycling showed a similar alteration, except for ultimate stress, which was increased . These changes could negatively impact treatment outcome as the lower elastic modulus may result to decreased orthodontic forces. However, at the same time, it could benefit treatment by exerting forces on a greater deflection range and thus decreasing the risk of root resorption . The results of our in vivo study are generally in agreement with those produced by Ihssen et al. (2019), although the magnitude of difference in properties of unprocessed material and after aligner intraoral ageing differ. In particular, the results of our research indicate for the unprocessed material prepared with water jet machine a mean value of 1186.82 ± 127.60 MPa elastic modulus, 51.89 ± 1.01 MPa for the UTS and 49.44 ± 0.54 MPa for the yield stress. For day 14 of intraoral ageing, the corresponding values calculated were 746.41 ± 145.41 MPa for elastic modulus, 21.29 ± 10.79 MPa for UTS and 19.94 ± 10.19 MPa for yield stress. It is hypothesised that this disparity could partly result from the different strain rate as in the present study the samples were stretched at a rate of 15 mm/min, whereas a rate of 300 mm/min was used in the other study. Further differences could stem from the material ageing process, due to different experiment design, as an in vivo study was performed in the present study, whereas for Ihssen et al. ageing was conducted in vitro. In addition, other studies have shown that thermoforming may negatively impact the mechanical properties of the aligners and may have a more dominant role than ageing alone . This is the first study that examines hardness in Clear Aligner aligners, and we generally observed a slight decrease with ageing. This decrease may translate to diminished wear resistance, thus making the aligner vulnerable to mastication forces . In particular, the posterior segments exhibited higher hardness values than the anterior ones at day 0 until day 14 of use. This may be related to the material morphology and the greater occlusal forces seen on molars in comparison to incisors . This difference in forces may lead to structural modifications resulting in increased plasticity and hardness . Bradley et al. (2016) found similar results with decreased hardness (measured by Martens Hardness) in Invisalign aligners used for a mean period of 44 ± 15 days, in comparison with unused. Similarly, another study concluded that Invisalign samples had significantly lower hardness (measured by Martens Hardness) after day 7 and day 14 of use compared with the unused one . Can et al. studied 3D printed aligners after 7 days of use and also demonstrated decreased, yet not statistically different hardness (measured by Martens Hardness) compared with unused aligners. Additional studies have confirmed decreased hardness after thermoforming and ageing, noting that changes in hardness can indicate variations in applied forces that may subsequently impact the efficacy of the aligner therapy . On the other hand, Schuster et al. found increased hardness (measured by Vickers Hardness) in Invisalign aligners after 14 days of use and attributed the result to the alteration of the polymer crystallinity due to cold work produced by the masticatory loads. Regarding the UTS and yield stress, in our study we found large standard deviation in the measurements at day 10 (SD: 11.64 [UTS], 10.98 [YS]) and day 14 (SD: 10.79 [UTS], 10.19 [YS]) of use. We believe that the observed differences between the tested samples are related to the increased intraoral stay and to staining and stress which turn the material brittle. In addition, imperfections may have been created by the long intraoral stay leading to earlier failures in some tests and increasing variation between the samples. Indication of the previous was found during the sample preparation stage as some samples broke after application of minimal force by the cutting tool. The latter was only observed for samples from longer intraoral stay aligners. The absence of any statistical difference between the values of unprocessed material samples that were prepared with a water jet machine and the samples prepared with the sharp tool, indicates that the sharp tool that was used for preparation of the aligners' segments should not compromise sample integrity. Further to the previous, and as a general comment regarding tensile testing, we should mention that due to the uneven morphology of the thermoformed material, matching the morphology of teeth, there is inherent non‐uniformity in terms of internal stress distribution and concentration. Thus, during the tensile testing some areas, such as the interdental areas, may be subjected to locally increased stresses because of reduced material area and intricate geometry with edges and curves, which can result to stress concentration. It is also noted that the thermoforming process has been confirmed to lead in uneven material thickness of the produced aligners . As the mean stress was measured, the numerical data presented may be in a degree underestimated, and the results should be evaluated qualitatively. This is the first research on orthodontic aligners that studies simultaneously multiple ageing periods and incorporates aligners that have been used intraorally, and also includes tests of samples produced by the unprocessed material. Another factor of originality is the separate study of the anterior and posterior segments of aligners. The results of our study indicate that the tensile properties of Clear Aligners potentially degrade after use, and statistically significant changes are observed after aligners' use. Greater deviations, however, are detected on the properties of the unprocessed material and the unused aligners (day 0), which indicates that the thermoforming process has significant effect on the properties of the final product. Based on the results of our study, thermoforming and ageing affect the mechanical properties of aligners' material. As the interest in orthodontic aligners is growing, it is deemed that further research on the ageing of aligner materials and their stress relaxation behaviour within the oral environment is warranted for optimal treatment design.
Conclusions The findings of our study indicate that thermoforming and ageing affect the mechanical properties of aligners. The unprocessed material displays statistically significant differences in every property tested in comparison to the material extracted from thermoformed aligners. These properties present further deterioration during clinical use on the basis of tests conducted for up to 14 days of intraoral stay.
The study was approved by the ethics committee of the National and Kapodistrian University of Athens (Athens, Greece) (protocol number: 39527).
The authors declare no conflicts of interest.
Figure S1. Specimen dimensions according to ISO 6892‐1:2019 specifications. Figure S2. Stress–strain curve example schematic; 1 = 0.1% offset yield strength; 2 = ultimate tensile strength; 3 = necking and failure region; 4 = slope parallel shifted to 0.1% strain. Table S1. p ‐Values of the tensile testing results on aligner segments of days 0, 7, 10 and 14.
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Posterior Dahl: A Minimally Invasive Method for the Treatment of Localized Posterior Tooth Wear | 2b645ed7-2f6c-4bff-9fec-c66b725c1b9f | 11734287 | Dentistry[mh] | General This study was conducted in full accordance with regulations by the World Medical Association Declaration of Helsinki, and ICH E6 (R2) Guidelines for Good Clinical Practice (GCP). Ethical approval was obtained from local Human Research Ethics Committee (case number 2022-15767). Patient Selection Patients were treated with the posterior Dahl concept, either at the Department of Dentistry of Radboud UMC in Nijmegen, or at the private dental practice of one of the authors (BL). The etiology of tooth wear in these patients was either mechanical and/or chemical . All patients who were selected needed treatment due to symptoms of sensitivity, threatening endodontic complications, or functional problems. The participants’ Tooth Wear Index (TWI) scores varied from 2 to 4. However, the primary reason for restoration was not the amount of tooth wear, but the reporting of pain and/or functional problems. Treatments were performed between October 2014 and April 2021. Retrospectively, the authors were able to retrieve complete intraoral scan-sets for nine patients. Intraoral scans were taken at the pre-treatment stage, immediately post-treatment and at successive follow-up appointments. Written informed consent to participate was obtained from all patients, as well as agreement for the use of anonymized intraoral scan data. Treatment: The Posterior Dahl Concept All patients were treated by one of the authors (BL or NO), both following the same clinical treatment protocol and both using the same materials. In the nine patients, the effects of localized posterior tooth wear – culminating in either functional-, sensitivity- or pain-related problems – were managed by the application of supra-occlusal, directly bonded resin composite restorations. The post-treatment vertical dimension of occlusion (VDO) was estimated by the amount of resin composite required for restoration of the anatomical proportions of the affected teeth. A minimally invasive procedure was applied, including minor roughening of the occlusal surfaces of the affected teeth using a diamond bur. In the event of an existing but insufficient restoration, this restoration was either replaced or repaired. Isolation of the operating field was achieved using rubber-dam, or the use of cotton rolls and suction devices. If required, appropriate matrix systems and wedges were also used. A 3-step etch-and-rinse adhesive was applied in accordance with the respective manufacturer’s instructions: 37% phosphoric acid (DMG; Hamburg, Germany), Clearfil SA Primer, and Clearfil Photobond (Kuraray Noritake; Tokyo, Japan). A microhybrid composite material (Clearfil AP-X, Kuraray Noritake) was used to restore the anatomical form of the treated teeth (approximately 1.0 – 1.5 mm supra-occlusal thickness). An LED polymerization unit with a minimal output of 1000 mW/cm 2 was used to light cure the restorations. Minimal occlusal morphology was applied in the restorations, and occlusal contacts were positioned in the center of the restoration(s). In the case of a unilaterally worn tooth, glass-ionomer cement (GIC, Fuji TRIAGE, GC; Tokyo, Japan) was placed on the contralateral molar, which was also elevated to the same occlusal plane as the definitively restored worn tooth. This was done to provide the patient with both bilateral occlusal stops on molars and also improved comfort during the procedure. All other teeth were left out of occlusion directly after treatment. Intraoral Scanning and Recalls Intraoral 3D scans were taken immediately pre- and post-treatment and at all subsequent recall visits. Recall visits differed in terms of the number of months after treatment . Scans were made using either a True Definition Scanner (3M Oral Care; St Paul, MN, USA) or a Medit i500 scanner (Medit; Seoul, South Korea), processed with open-source software MeshLab v2020.12 ( http://www.meshlab.net ). Patients sat in an upright position during scanning, with an OptraGate lip and cheek retractor inserted (Ivoclar Vivadent; Schaan, Liechtenstein). Qualitative Analysis All intraoral scans were analyzed by one author (PH). All pre- and post-treatment scans were scored for: Eruption of untreated (pre)molars (yes/no) Eruption from canine to canine (yes/no) Midline deviation (in mm) Angle classification of the first molars Any other remarks Questionnaire All patients received a short retrospective questionnaire to assess symptoms of discomfort, pain, functional problems, and their experience before and after treatment. The questionnaire items included: Presence of pain in the TMJs, musculature or teeth Presence of functional problems regarding frontal biting of food, posterior chewing of food, or speech Presence of discomfort regarding cheek biting or failure of restorations Did the treatment resolve the pre-treatment concerns? Overall (dis)satisfaction with this treatment method General remarks from patients All items were evaluated retrospectively (ie, after treatment and follow-up, merely for this study) by telephone interviews by one of the authors (PH) at the following timepoints: pre-treatment, immediately post-treatment, 1 week post-treatment and 2 months post-treatment. Except for the expression of “overall (dis)satisfaction” (scored using a 5-point verbal rating scale from very dissatisfied to very satisfied), all other answers were scored using a 3-point verbal rating scale (yes, no, or I can’t remember). This study was conducted in full accordance with regulations by the World Medical Association Declaration of Helsinki, and ICH E6 (R2) Guidelines for Good Clinical Practice (GCP). Ethical approval was obtained from local Human Research Ethics Committee (case number 2022-15767). Patients were treated with the posterior Dahl concept, either at the Department of Dentistry of Radboud UMC in Nijmegen, or at the private dental practice of one of the authors (BL). The etiology of tooth wear in these patients was either mechanical and/or chemical . All patients who were selected needed treatment due to symptoms of sensitivity, threatening endodontic complications, or functional problems. The participants’ Tooth Wear Index (TWI) scores varied from 2 to 4. However, the primary reason for restoration was not the amount of tooth wear, but the reporting of pain and/or functional problems. Treatments were performed between October 2014 and April 2021. Retrospectively, the authors were able to retrieve complete intraoral scan-sets for nine patients. Intraoral scans were taken at the pre-treatment stage, immediately post-treatment and at successive follow-up appointments. Written informed consent to participate was obtained from all patients, as well as agreement for the use of anonymized intraoral scan data. All patients were treated by one of the authors (BL or NO), both following the same clinical treatment protocol and both using the same materials. In the nine patients, the effects of localized posterior tooth wear – culminating in either functional-, sensitivity- or pain-related problems – were managed by the application of supra-occlusal, directly bonded resin composite restorations. The post-treatment vertical dimension of occlusion (VDO) was estimated by the amount of resin composite required for restoration of the anatomical proportions of the affected teeth. A minimally invasive procedure was applied, including minor roughening of the occlusal surfaces of the affected teeth using a diamond bur. In the event of an existing but insufficient restoration, this restoration was either replaced or repaired. Isolation of the operating field was achieved using rubber-dam, or the use of cotton rolls and suction devices. If required, appropriate matrix systems and wedges were also used. A 3-step etch-and-rinse adhesive was applied in accordance with the respective manufacturer’s instructions: 37% phosphoric acid (DMG; Hamburg, Germany), Clearfil SA Primer, and Clearfil Photobond (Kuraray Noritake; Tokyo, Japan). A microhybrid composite material (Clearfil AP-X, Kuraray Noritake) was used to restore the anatomical form of the treated teeth (approximately 1.0 – 1.5 mm supra-occlusal thickness). An LED polymerization unit with a minimal output of 1000 mW/cm 2 was used to light cure the restorations. Minimal occlusal morphology was applied in the restorations, and occlusal contacts were positioned in the center of the restoration(s). In the case of a unilaterally worn tooth, glass-ionomer cement (GIC, Fuji TRIAGE, GC; Tokyo, Japan) was placed on the contralateral molar, which was also elevated to the same occlusal plane as the definitively restored worn tooth. This was done to provide the patient with both bilateral occlusal stops on molars and also improved comfort during the procedure. All other teeth were left out of occlusion directly after treatment. Intraoral 3D scans were taken immediately pre- and post-treatment and at all subsequent recall visits. Recall visits differed in terms of the number of months after treatment . Scans were made using either a True Definition Scanner (3M Oral Care; St Paul, MN, USA) or a Medit i500 scanner (Medit; Seoul, South Korea), processed with open-source software MeshLab v2020.12 ( http://www.meshlab.net ). Patients sat in an upright position during scanning, with an OptraGate lip and cheek retractor inserted (Ivoclar Vivadent; Schaan, Liechtenstein). All intraoral scans were analyzed by one author (PH). All pre- and post-treatment scans were scored for: Eruption of untreated (pre)molars (yes/no) Eruption from canine to canine (yes/no) Midline deviation (in mm) Angle classification of the first molars Any other remarks All patients received a short retrospective questionnaire to assess symptoms of discomfort, pain, functional problems, and their experience before and after treatment. The questionnaire items included: Presence of pain in the TMJs, musculature or teeth Presence of functional problems regarding frontal biting of food, posterior chewing of food, or speech Presence of discomfort regarding cheek biting or failure of restorations Did the treatment resolve the pre-treatment concerns? Overall (dis)satisfaction with this treatment method General remarks from patients All items were evaluated retrospectively (ie, after treatment and follow-up, merely for this study) by telephone interviews by one of the authors (PH) at the following timepoints: pre-treatment, immediately post-treatment, 1 week post-treatment and 2 months post-treatment. Except for the expression of “overall (dis)satisfaction” (scored using a 5-point verbal rating scale from very dissatisfied to very satisfied), all other answers were scored using a 3-point verbal rating scale (yes, no, or I can’t remember). Patients and Treatments An overview of patient characteristics, intraoral scan recalls, and treatment details are presented in . Although no adverse events occurred during treatment, two restorations in one patient (number 7) had to be replaced after 4 months. This was due to partial fracture and wear (see “Questionnaire” below and ). Seven patients were treated bilaterally with Clearfil AP-X composite. Two patients presented with unilateral posterior localized tooth wear; the affected ipsilateral molars were restored using Clearfil AP-X composite, and GIC glass-ionomer cement was applied on the contralateral tooth where definitive restorative rehabilitation with resin composite was not indicated. The maximum follow-up period varied from 3 to 49 months, with a mean of 15 months . Qualitative Analysis Patient 1 is presented as a representative example in . Qualitative results of all patients are presented in , in which the clinical situation at the latest recall visit is given. In general, all patients showed an increase in the vertical dimension immediately after treatment, leading to slight distalization of the molar occlusion, and an open bite in the anterior area and the non-treated (pre)molars. In seven patients, these effects returned to the baseline situation during the observation period (ie, re-establishment of occlusion and closing of the open bite). Eight patients showed the same pattern of occlusal re-establishment, which appeared to progress from distal to mesial. One patient showed no closing of the open bite over time, either in the frontal area or in the non-treated (pre)molar areas (patient number 5). As a result, this patient was additionally provided with composite restorations on the non-treated (pre)molars 1.5 years after initial treatment. The open bite in the frontal area was left untreated, and did not close over time up until the last recall (49 months after initial treatment). As the patient did not have any complaints, it was decided to leave the open bite unchanged. In one other patient (number 2), the frontal open bite partially closed but did not fully return to the baseline situation. Two patients received contralateral GIC restorations. These GIC restorations showed progressive wear over time, with the subsequent re-establishment of occlusion of these molars . In five patients, mandibular midline deviation gradually occurred over time, with a maximum deviation of approximately 1 mm . Questionnaire shows the results of the pre-treatment questionnaire. Symptoms of pain in worn teeth were the primary concern for four patients, whereas restoration fracture was reported by two patients. The outcomes of the post-treatment questionnaire are given in . As for pain, the patient with pre-treatment TMJ complaints and chewing musculature pain indicated that these problems had resolved at 2 months post-treatment. Two out of the four patients who complained about pre-treatment pain in worn teeth mentioned resolution of the pain immediately post-treatment. In the other two patients with pain due to tooth wear, the symptoms of pain disappeared within one week or 2 months post-treatment, respectively. Three patients reported difficulty chewing at the one-day post-treatment evaluation; however, these symptoms were no longer present at the 1-week post-treatment recall. Two other patients described difficulty both with chewing and frontal biting as persisting up to one-week post-treatment. In one other patient, challenges with chewing and biting continued for approximately 6 months. Other functional problems, such as cheek biting and problems with speech, resolved at one-week post-treatment, except for one patient, in whom speech problems were still present one-week post-treatment. Although no patients mentioned failure of restorations of the treated molars within two months post-treatment, in one patient (number 7), two restored molars had to be re-treated due to a partial fracture of one restoration (tooth 37) and wear of the other restoration (tooth 47). The latter took place 4 months post-treatment . All except patient 7 mentioned that their pre-treatment problems were eventually resolved and gave patient satisfaction the score of “very satisfied”. Patient 7 answered “neutral” on patient satisfaction. An overview of patient characteristics, intraoral scan recalls, and treatment details are presented in . Although no adverse events occurred during treatment, two restorations in one patient (number 7) had to be replaced after 4 months. This was due to partial fracture and wear (see “Questionnaire” below and ). Seven patients were treated bilaterally with Clearfil AP-X composite. Two patients presented with unilateral posterior localized tooth wear; the affected ipsilateral molars were restored using Clearfil AP-X composite, and GIC glass-ionomer cement was applied on the contralateral tooth where definitive restorative rehabilitation with resin composite was not indicated. The maximum follow-up period varied from 3 to 49 months, with a mean of 15 months . Patient 1 is presented as a representative example in . Qualitative results of all patients are presented in , in which the clinical situation at the latest recall visit is given. In general, all patients showed an increase in the vertical dimension immediately after treatment, leading to slight distalization of the molar occlusion, and an open bite in the anterior area and the non-treated (pre)molars. In seven patients, these effects returned to the baseline situation during the observation period (ie, re-establishment of occlusion and closing of the open bite). Eight patients showed the same pattern of occlusal re-establishment, which appeared to progress from distal to mesial. One patient showed no closing of the open bite over time, either in the frontal area or in the non-treated (pre)molar areas (patient number 5). As a result, this patient was additionally provided with composite restorations on the non-treated (pre)molars 1.5 years after initial treatment. The open bite in the frontal area was left untreated, and did not close over time up until the last recall (49 months after initial treatment). As the patient did not have any complaints, it was decided to leave the open bite unchanged. In one other patient (number 2), the frontal open bite partially closed but did not fully return to the baseline situation. Two patients received contralateral GIC restorations. These GIC restorations showed progressive wear over time, with the subsequent re-establishment of occlusion of these molars . In five patients, mandibular midline deviation gradually occurred over time, with a maximum deviation of approximately 1 mm . shows the results of the pre-treatment questionnaire. Symptoms of pain in worn teeth were the primary concern for four patients, whereas restoration fracture was reported by two patients. The outcomes of the post-treatment questionnaire are given in . As for pain, the patient with pre-treatment TMJ complaints and chewing musculature pain indicated that these problems had resolved at 2 months post-treatment. Two out of the four patients who complained about pre-treatment pain in worn teeth mentioned resolution of the pain immediately post-treatment. In the other two patients with pain due to tooth wear, the symptoms of pain disappeared within one week or 2 months post-treatment, respectively. Three patients reported difficulty chewing at the one-day post-treatment evaluation; however, these symptoms were no longer present at the 1-week post-treatment recall. Two other patients described difficulty both with chewing and frontal biting as persisting up to one-week post-treatment. In one other patient, challenges with chewing and biting continued for approximately 6 months. Other functional problems, such as cheek biting and problems with speech, resolved at one-week post-treatment, except for one patient, in whom speech problems were still present one-week post-treatment. Although no patients mentioned failure of restorations of the treated molars within two months post-treatment, in one patient (number 7), two restored molars had to be re-treated due to a partial fracture of one restoration (tooth 37) and wear of the other restoration (tooth 47). The latter took place 4 months post-treatment . All except patient 7 mentioned that their pre-treatment problems were eventually resolved and gave patient satisfaction the score of “very satisfied”. Patient 7 answered “neutral” on patient satisfaction. For the restorative management of localized posterior tooth wear, limited treatment options are available. Occlusal reduction and subsequent restoration is perhaps the most obvious treatment method; however, this approach is rather invasive. Occlusal restoration without prior occlusal reduction as described in this study is quite unconventional. This treatment is a largely minimally invasive approach, and treatment with this experimental procedure by one of the authors (inspired by the work of Banerji et al ) showed good clinical results. The primary aims of this investigation were to describe the clinical outcomes (re-establishment of occlusion, mandibular midline deviation, occlusal changes, and restoration failure) and the levels of patient satisfaction (pain, chewing-, biting- and speech-related problems) following application of the posterior Dahl concept to manage localized posterior tooth wear in a sample of nine fully documented patients. A few studies have mentioned using the posterior Dahl concept, but their primary purpose was not to investigate this concept per se. Banerji et al and Chana et al used the posterior Dahl concept as a tool rather than the primary subject of their investigations. Recently, a paper with two case reports by Tew et al presented treatment of localized posterior tooth wear with the posterior Dahl concept to actually describe the concept itself. However, neither follow-up by means of intraoral scanning nor cast comparison were performed, nor were patients asked about their satisfaction with this approach. With these two elements included, our study adds value to research on the posterior Dahl concept. The results of our study are comparable to the most relevant study performed on this subject (Banerji et al), despite the fact that that they applied unilateral supra-occluding restorations instead of bilateral restorations. The main findings by Banerji et al were complete re-establishment of the occlusal contacts in the anterior teeth in 97.7% (n=128) of their cases at 3 months post-treatment. An overall success rate of the supra-occlusal restorations of 86.7% was reported after 3 months, including relief of the symptoms associated with CTS. This extra parameter for the definition of success clearly has a detrimental effect on the success rate. The restorations were reported to be well tolerated, with an overall acceptability of 97%. , With the conventional application of the anterior Dahl concept, the success rate for the re-establishment of occlusion on posterior teeth after placing supra-occluding anterior restorations to this end has been reported to range from 94% to 100%. , , Furthermore, survival of supra-occluding restorations has also been reported to be as high as 94%. The results of the studies involving the anterior Dahl concept are comparable with the results of the current investigation applying the posterior Dahl concept, in which only one patient did not display re-establishment of full occlusion on the non-treated teeth, and one restoration showed signs of partial fracture after 4 months. Failure to close open bite, as reported in the present study amongst two patients, warrants further discussion. One of these patients (no. 5) showed no closure of the open bite over time, either in the anterior area or in the non-treated (pre)molar areas. The vertical facial configuration of this patient was a long-face type, which is known to have a deviating occlusal force pattern compared to individuals with normal vertical facial dimensions. Since the re-establishment of occlusion is caused by relative and true axial tooth movement, , , , in which both intrusion of treated teeth and extrusion of non-treated teeth are involved, the intrusive component in this patient was probably not sufficient to achieve full occlusion in the non-treated areas. For the other patient (no. 2), the frontal open bite displayed partial closure; however, it did not fully return to the baseline situation. One patient showed fracture of one restoration after 4 months . This patient suffered from severe bruxism and showed multiple restoration fractures even before treatment with the posterior Dahl concept. Occlusal stress is a known risk factor for restoration failure by (chip) fracture, , and it may account for the restoration fracture observed in this patient. Van de Sande et al reported a 4-fold increased risk for restoration fracture amongst patients with bruxism compared to non-bruxing patients. The posterior teeth in this patient were severely worn and no space was available to place a restoration without endodontic treatment. Anticipating that the risk of failure would be higher compared to a “normal” situation, it was collectively decided with the patient to place a supra-occlusal restoration. As the goal of the posterior Dahl treatment in this case was to protect the underlying tooth material, it may be stated that the treatment goals were successfully met. In the case of a unilaterally worn tooth, a GIC restoration was placed on the contralateral non-worn molar at the same elevated level of occlusion. This was done in two cases to provide these patients with more comfort by delivering bilateral occlusal stops. The rationale behind the use of GIC is the higher wear rate compared to resin composite. The authors expected the GIC restorations to wear faster over time, causing compensatory eruption of the tooth, with eventual re-establishment of occlusion of the non-worn pre-treatment occlusal surface, and full disappearance of the GIC. These GIC restorations did indeed showed progressive wear over time, with the subsequent re-establishment of full occlusion of the pre-treatment occlusal surface of these molars. Patient satisfaction in this study was reported to be high (the maximum score “very satisfied” was given 8 times, and on one occasion, the middle score, “neutral” was documented). Only one study in the current literature could be found in which subjective patient satisfaction was evaluated after the application of the Dahl concept; however, it used the anterior Dahl method. In that case series, 5 of 6 patients reported no discomfort after treatment and 1 patient mentioned mild discomfort. Observational descriptive studies such as this case series are classified as providing a relatively low level of evidence. Nevertheless, they have great value in experimental treatment concepts, to generate hypotheses which can be tested more extensively with other types of studies. In future studies about the posterior Dahl concept, it would be worthwhile to compare active orthodontic bite-closure treatments with respect to treatment time, costs, and patient satisfaction. Furthermore, a prospective trial would make it possible to assess patient-reported outcome measures (PROMs), such as pain and other discomfort, with the use of a quantifiable visual analogue scale (VAS). The latter was not reliable enough in our study due to its retrospective nature. Also, the influence of patient-related factors such as gender, caries risk and activity, oral hygiene, facial type, and bruxism should be studied in the future, as these aspects are known to influence restoration survival. Application of the posterior Dahl concept appears to offer a promising minimally invasive and relatively simple yet effective approach for the treatment of localized posterior tooth wear. Although phenomena such as cheek biting, small fractures and midline deviation occurred, they were minor compared to the overall benefit for these patients, who gave “highly satisfied” scores. Re-establishment of occlusion was achieved in almost all cases and is comparable to the anterior Dahl treatment. A prospective trial on this treatment concept is needed to substantiate this method. |
Small RNA Detection by | be42123e-1e1a-482f-a4ce-5eff96ead53b | 4490494 | Pathology[mh] | There are several classes of small noncoding RNAs functioning in eukaryotic cells, which include, among others, 18–24 nt microRNAs (miRNAs), 21–22 nt short interfering RNAs (siRNAs), and 26–30 nt PIWI-associated RNAs (piRNAs). miRNAs are endogenous regulators of gene expression that primarily function at a posttranscriptional level, inhibiting mRNA translation with or without transcript degradation . They also function in the cell nucleus and take part in gene transcription regulation. Exogenous miRNA mimics are used to compensate for pathological miRNA deficiency, and anti-miRNAs are delivered to cells to downregulate overexpressed miRNAs. Endogenous siRNAs that are present in both mammalian and plant cells take advantage of the miRNA pathway for their functioning. The same pathway is used by exogenous siRNAs, which are principal reagents of RNA interference technology. Both endogenous and exogenous siRNAs, due to full sequence complementarity to their targets, induce transcript cleavage. piRNAs, which do not use the miRNA pathway, function in germ cells to regulate transposon activity. Many difficulties in reliably determining cellular levels, establishing intracellular localization, and demonstrating regulatory interactions of these RNAs are associated with their small size. Sensitive and highly specific small RNA detection is required not only for studying physiological processes regulated by these RNAs but also for better understanding relevant pathologies. Current research on small RNAs addresses numerous aspects of RNA biogenesis, localization, function and dysfunction. These aspects often need to be investigated in a single cell, with single molecule sensitivity, and spatial as well as temporal resolution. Physiologically, fluctuations of miRNA expression levels are observed during numerous cellular processes including cell division, maturation, and differentiation or after environment changes, e.g., drug treatment . With regard to pathogenesis, changes in cellular levels of small RNAs can either be a cause or result of developing pathogenic processes. miRNA levels have been demonstrated to have a strong correlation with disease progression in cancer, cardiovascular disease, neurodegeneration, and numerous other pathologies . Thus, miRNAs can be considered as diagnostic and prognostic disease markers. Many novel approaches have been proposed to gain better insight into cellular levels and localization of miRNAs, which can also be used for detecting other types of small RNAs. The methods that take advantage of PCR-free signal amplification, nanotechnology and capillary electrophoresis have been recently reviewed by Tian and colleagues . The only method that provides insight into both the level and localization in single cells is in situ hybridization (ISH) , which has increased considerably in importance in small RNA research over the last 10 years . Depending on the detection method, ISH can be divided into chromogenic enzyme-based in situ hybridization and fluorescent in situ hybridization (FISH). Multiple adaptations were included in standard ISH protocols for more efficient detection of small RNAs in cells, which we describe in detail in this review. We emphasize modifications at each step of the ISH protocols, probe design, cell fixation and permeabilization, hybridization, post-hybridization steps including washing, optional signal amplification and detection. Described protocol improvements provide better features, most of all higher sensitivity, specificity and resolution. We also present examples of small RNA ISH successful applications in different cell types and tissues, focusing on human and murine tissues.
miRNA ISH is exceptionally challenging because of miRNA features such as small size, sequence similarity among various miRNA family members and low tissue-specific or development-specific expression levels. The standard ISH protocol was modified to improve miRNA detection in various types of cell lines and tissues as well as whole embryos. Here, we describe these modifications with a focus on technical aspects and critically discuss these adaptations in the context of single molecule ISH and multi-miRNA detection. 2.1. Probes A very important step in an ISH experiment is probe design. Different probe types have different properties and detection options that enable selection of suitable solutions for many applications. These probes can be divided into two groups: linear probes directly labeled with fluorophore or ligand, and probes that enable sequence amplification. 2.1.1. Directly Labeled Probes In standard ISH, probes composed of DNA or RNA nucleotides are commonly used. Unmodified DNA and RNA probes have relatively poor binding affinity to target sequences , and therefore several modifications have been proposed to improve their properties . First and most commonly applied was Locked Nucleic Acid (LNA) modification, which remains the gold standard in RNA FISH not only in small RNA detection. LNA nucleotides, referred to as “locked” RNA, have an additional bridge connecting 4′C and 2′O atoms. LNA nucleotides are incorporated into DNA probes, which leads to the formation of hybrid LNA/DNA probes. LNA/DNA probes have been shown to be highly beneficial in miRNA detection because of a short hybridization time, high efficiency, discriminatory power and a high melting temperature of the miRNA:probe complex. The minimal length of the LNA/DNA probe was determined to be 12 nucleotides and these probes usually contain 30% LNA nucleotides. Besides their unquestionable advantages, these probes are expensive and can generate strong background signals resulting in a low signal-to-noise ratio for low abundant miRNA . Therefore, other modifications were also proposed. These modifications include 2′fluoro-modified RNA (2′F RNA), morpholino, Zip Nucleic Acids (ZNA) , N , N -diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN) and 2′O-Methyl (2′OMe) RNA modification. In comparison to DNA probes, 2′OMe RNA probes have faster hybridization kinetics and the ability to bind structured targets under standard conditions . The combination of 2′OMe RNA and LNA modifications (in a 2:1 ratio) resulted in improved specificity and stability of the probe:RNA duplex in comparison to the LNA/DNA probe . Specificity of the system may be further improved by shortening the probe length to 19 nt . As the LNA/2′OMe RNA probe binds more strongly to yeast RNA or salmon sperm RNA used in ISH as a standard blocking agent, better results were obtained without these RNA blockers in the hybridization step . 2′F RNA nucleotides incorporated in the DNA probes ensure increased binding to the target and better nuclease resistance . Morpholino modifications, often applied to inhibit translation, modify splicing patterns of the primary transcript, or block miRNAs, were also used to detect miRNAs because of their high stability . 2.1.2. Probes Used with the Sequence Amplification System Several types of probes are combined with sequence amplification techniques to increase signal strength obtained from single miRNA molecules ( A–E). One example that enables sequence amplification is the use of padlock probes. These probes are successfully used to detect not only miRNAs but also mRNAs and DNA sequences. Padlock probes guarantee high specificity and sensitivity with single nucleotide discrimination —what makes them applicable for allele-specific FISH . Briefly, linear DNA probe after annealing to the specific sequence with 5′ and 3′ arms is circularized by DNA ligase. Circularization enables further signal amplification by rolling circle amplification (RCA) ( A). RCA uses miRNA molecule as a primer and elongates the sequence using circular probe as a template. Detection of RCA product is possible with the use of probes complementary to the sequence amplified on the template of the padlock probe central sequence. Similar types of probes, which are used in miRNA ISH also in combination with RCA, are circular DNA probes ( B). Circular probes are obtained in vitro with the use of padlock probes, ligation probes, and DNA ligase, and are then hybridized to the target sequence in cells as circular molecules . Another type of probe used together with RCA is a seal probe with an adjustable toehold inside its loop . These probes have an ability to change their structure. The initial dumbbell shape of the probe is changed into a circular form when the target miRNAs bind into the toehold domain of the probe. As a result, a RCA reaction can be initiated. This new method, called toehold-initiated rolling circle amplification (TIRCA), is a combination of toehold-mediated strand displacement (TMSD) and RCA. The length of the toehold defines the stability of the probe and is the most important factor for detecting miRNAs with TIRCA. With increasing length of the toehold, the stability of the seal probe and selectivity of TIRCA decreases. There are many advantages in using TIRCA, such as reduced loss of miRNA molecules, because the detection process is conducted at a physiological temperature, short imaging time, and high sensitivity and specificity, which is even higher than in the case of a padlock probe based RCA reaction . 2.2. Fixation The first step in the ISH protocol is cell fixation, which on one hand should sufficiently preserve the number and localization of small RNA molecules, but, on the other hand, is mild enough to preserve cellular domains crucial for detection, which is especially important for ISH combined with protein labeling. Prevention of miRNA loss during fixation is essential, especially for detecting low abundant miRNAs. Highly abundant miRNAs can be successfully detected using standard fixation protocols . Significant improvement in miRNA recovery was shown by Tuschl and colleagues who used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to immobilize miRNA molecules via their 5′ end . EDC fixation needs to be used in combination with traditional formaldehyde fixation. Improvement in the signal to noise ratio by addition of an EDC-crosslinking step was confirmed in several studies of miRNAs . Importantly, standard formalin-fixation and paraffin-embedding (FFPE) as well as cryopreservation of tissues seems to preserve microRNAs sufficiently . 2.3. Permeabilization Permeabilization is used to improve cells and tissues penetration by the probe; however, permeabilization that is too strong can cause RNA loss from fixed material. Permeabilization is typically performed with the use of organic solvents including methanol or paraformaldehyde that are used also as fixatives, detergent like Saponin or Triton X-100, or proteinase. Briefly, organic solvents dissolve lipids from cell membranes. Some detergents, i.e. , Saponin, remove cholesterol from membranes in highly selective ways, but widely used Triton X-100 is not selective, which can lead to elimination of both proteins and lipids . To reduce cellular RNA diffusion, treatment with proteinases and detergents was either limited or eliminated in some miRNA ISH studies . The need for additional permeabilization decreases with the use of EDC fixation due to its auxiliary permeabilization activity. Long fixation of tissues also can decrease the need of permeabilization. Some differences are observed in permeabilization techniques used in ISH of fixed tissues and cell lines. Typically, tissues have to undergo stronger permeabilization because of their lower accessibility to the probe; however, permeabilization conditions differ among different types of tissues. Tissues are typically permeabilized with Triton X-100, but Proteinase K is also used. However, permeabilization conditions for specific types of tissues, e.g., embryos or brain tissues, still need to be optimized . Permeability of the tissue depends also on method of tissue preservation. Unfixed cryosections typically do not need permeabilization in contrary to fixed paraffin embedded sections. Cell lines are typically permeabilized with aceton, Triton X-100, Tween or overnight incubation with 70% ethanol. Fixation and permeabilization steps may be combined, e.g., by using a buffer containing paraformaldehyde and a short C-chain aliphatic carboxylic acid . There are also several commercial buffers used for permeabilization . Moreover, permeabilization conditions should be adjusted to the probe type and its length. Short linear probes used in miRNA ISH are able to easily enter cells and cellular compartments. Bigger, chemically modified probes or probes based on nanoshells would need stronger permeabilization treatment in comparison to short probes. 2.4. Hybridization The hybridization step proceeds as in a standard ISH protocol. During hybridization, the optimal time of incubation and the temperature need to be experimentally determined because a calculated temperature for probe hybridization does not always give the best results. Typically the used hybridization buffer (HB) is the same as in ISH used for mRNA labeling; however, in some miRNA ISH experiments, HB was slightly modified. Important change in HB was the use of less toxic urea instead of 50% formamide . In some specific experiments, it is desired to visualize more than one miRNA in a cell or tissue . Multicolor ISH of miRNA may be performed sequentially or simultaneously. When simultaneous hybridization is performed and optimal hybridization temperatures for the probes differ, a suboptimal temperature must be chosen. When sequential hybridization is performed, hybridization can be performed at the optimal temperature for each probe . 2.5. Post-Hybridization Washing The third step of an ISH protocol, which is crucial for specific detection of miRNA, is the washing step. Washing conditions need to be optimized to preserve the probe-target complex but at the same time eliminate off-target binding. One of the proposed improvements was changing the washing buffer composition to include tetramethylammonium chloride (TMAC). TMAC was used to compensate for the low specificity of RNA probes as highly stringent TMAC washing conditions decrease off-target binding . Moreover, TMAC washing allows probes for different miRNAs to be detected in unchanged conditions, which can be used for in situ analyses with multiple miRNA probes. TMAC is also used in combination with RNase A to prevent off-target hybridization of RNA probes with mismatches . In addition, it is important to adjust the washing temperature to provide specificity of binding while preserving the signal strength. 2.6. Sequence Amplification Methods With the objective of single molecule detection, different types of sequence and signal amplification techniques have been used to provide better resolution . Enzymatic signal amplification methods, described in the detection section, are used mainly to image low-abundant miRNAs in tissues. The most commonly used system for sequence amplification is RCA . The main advantages of RCA are better cellular resolution, super bright spots indicating miRNA and ultrahigh sensitivity . RCA uses the free 3′ terminus of miRNA acting as the primer itself. mRNA and miRNA precursors do not give a positive signal as the 3′ end of these molecules does not hybridize to the probe and cannot be elongated during the process. The RCA process of sequence amplification is facilitated by DNA polymerase, resulting in the generation of thousands of amplified segments, which contain sequences complementary to the detection probes. The product of the RCA reaction is visualized with many fluorescently labeled detection probes. The RCA method was combined with a proximity ligation assay (PLA) that led to development of a system named oligo-fluorescence in situ hybridization (O-FISH) ( D). The PLA technique was primarily developed to detect proteins with single molecule sensitivity. Briefly, biotin-labeled probe hybridizes to miRNA target and anti-biotin monoclonal antibodies detect the probe. The primary antibody is detected by two monoclonal antibodies conjugated with oligonucleotides. After secondary antibodies bind to the target, the antibody-conjugated oligonucleotides can hybridize to connector oligonucleotides due to their close proximity. Connector oligonucleotides are then ligated to circular DNA that can be used in RCA similar to the use of padlock probes. Oligonucleotides conjugated with antibodies serve as primers for RCA reaction . Sequence amplification is also achieved by RT in situ PCR. This method, called the ultramer extension method, is based on the use of longer probes and miRNA acting as a primer, as in the RCA method. The probe contains sequences complementary to miRNA and a series of 20 nt sequences at the 5′ end. During in situ PCR, digoxigenin (DIG)-labeled nucleotides are incorporated to the PCR product to be further detected with antibodies . 2.7. Detection Detection is the last, and a very important, step of miRNA ISH. In the first ISH experiments, radiolabeled probes were applied , but nonradioactive probes are currently used, giving information about the subcellular distribution of miRNA. In several experiments, probes directly labeled with fluorophores were used , but the obtained signal was not strong enough . Weak emission signals, rapid photobleaching, strong photoblinking and a lifetime close to cellular autofluorescence are major shortcomings of organic fluorophores. Several signal enhancement methods have been proposed to overcome these difficulties. In the commonly used miRNA ISH technique, enzyme-based detection is used. Briefly, nonradioactive haptens, combined with probes, are detected by histochemical enzymatic reactions after application of enzyme-conjugated anti-hapten antibodies. Alkaline phosphatase (AP) is most commonly used with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as a substrate . The tyramide signal amplification system (TSA), or, in other words, the catalyzed reporter deposition method (CARD), is a similar approach. Early versions of the commercially available TSA/CARD system used streptavidin-biotin affinity for initial signal detection . As endogenous biotin has been reported in many types of human tissue, currently LNA probes labeled with digoxigenin are mostly used in the hybridization step. In the TSA/CARD system, the horseradish peroxidase (HRP)-tagged anti-digoxigenin antibodies recognize digoxigenin moieties on probes. Next, HRP substrates, i.e. , cyanine 5 (Cy5), cyanine 3 (Cy3) or fluorescein-conjugated tyramides, are converted to highly reactive radicals by HRP and bind covalently to tyrosine residues located nearby . The radicals are extremely short-lived, which prevents them from diffusing away from the site of synthesis, which would decrease the signal-to-noise ratio . The TSA/CARD system improved the sensitivity up to 1000-fold compared to its early version . Another detection method is enzyme-labeled fluorescence signal amplification (ELF). In short, pro-luminescent substrate cleavage is performed by phosphatase, followed by precipitation of a bright, yellow-green fluorescent product. The obtained precipitate is highly photostable and gives a 40 times brighter signal in comparison to probes directly labeled with fluorophores . The dynamic range spans over three orders of magnitude, which means that 0 to 1000 copies of miRNA per cell can be quantified . Nanotechnology was recently introduced in the field of miRNA ISH to improve the detection of small RNAs. Metal nanoshells composed of silica spheres with encapsulated Ru(bpy) 3 2+ complexes as cores and thin silver layers as shells have been used for the detection of low-abundant miRNAs. The metal nanoshells are based on near-field interactions between organic fluorophores and metal nanoparticles. The use of nanoshells resulted in overcoming difficulties specific to organic fluorophores, including reduced photoblinking, increased photostability and intensity, as well as an increased lifetime of the organic fluorophores in comparison to the lifetime of cellular autofluorescence . However, the system needs to be optimized to provide better penetration of the nanoshells through the cell membrane, mobility in the cells as well as high specificity toward the miRNA target. Commercial assays for the detection of several miRNAs using in situ hybridization and fluorescence in situ hybridization are also available . A branched DNA system for signal amplification has been proposed to enable miRNA quantitative analysis . The system was also shown to be efficient in the detection of exogenous siRNA molecules. 2.8. Specificity Controls In RNA FISH, probes targeting different parts of mRNA are used to confirm probe specificity. This approach is not applicable in small RNA ISH due to miRNA short length. Thus, different controls need to be used to ensure probe specificity. In several experiments, probes complementary to miRNA analogs from different organism or even different kingdoms were used as negative control, e.g., for mammalian miRNA detection, probes against plant specific miRNA was used . This approach may be applied only if miRNA analogs sufficiently differ in sequence. Another type of negative control is the use of probes against miRNA that is not expressed in the analyzed tissue (according to microarray or deep sequencing results) or the use of scrambled probes . The scrambled probe should be checked not only for specificity against miRNA population but also against the whole transcriptome. In some experiments, probes similar in sequence to miRNA-specific probes but containing two or three mismatches with the target sequence was used . As a positive and negative control, miRNA-specific probes can be used to label different tissues that were shown to exhibit this miRNA expression or not, respectively . To exclude false positive results caused by cells or tissue autofluorescence, control experiments without any probes are used . Additionally, non-hybridization-based interactions are excluded by the treatment with unlabeled probes prior to hybridization with labeled probes . Hybridization with probes that were already successfully tested can serve as positive controls for adequate experimental conditions and good RNA quality, e.g., snRNA U6 as a target is commonly used . Interactions with DNA are excluded by DNase treatment and interactions with RNA are confirmed by RNase treatment prior to hybridization.
A very important step in an ISH experiment is probe design. Different probe types have different properties and detection options that enable selection of suitable solutions for many applications. These probes can be divided into two groups: linear probes directly labeled with fluorophore or ligand, and probes that enable sequence amplification. 2.1.1. Directly Labeled Probes In standard ISH, probes composed of DNA or RNA nucleotides are commonly used. Unmodified DNA and RNA probes have relatively poor binding affinity to target sequences , and therefore several modifications have been proposed to improve their properties . First and most commonly applied was Locked Nucleic Acid (LNA) modification, which remains the gold standard in RNA FISH not only in small RNA detection. LNA nucleotides, referred to as “locked” RNA, have an additional bridge connecting 4′C and 2′O atoms. LNA nucleotides are incorporated into DNA probes, which leads to the formation of hybrid LNA/DNA probes. LNA/DNA probes have been shown to be highly beneficial in miRNA detection because of a short hybridization time, high efficiency, discriminatory power and a high melting temperature of the miRNA:probe complex. The minimal length of the LNA/DNA probe was determined to be 12 nucleotides and these probes usually contain 30% LNA nucleotides. Besides their unquestionable advantages, these probes are expensive and can generate strong background signals resulting in a low signal-to-noise ratio for low abundant miRNA . Therefore, other modifications were also proposed. These modifications include 2′fluoro-modified RNA (2′F RNA), morpholino, Zip Nucleic Acids (ZNA) , N , N -diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN) and 2′O-Methyl (2′OMe) RNA modification. In comparison to DNA probes, 2′OMe RNA probes have faster hybridization kinetics and the ability to bind structured targets under standard conditions . The combination of 2′OMe RNA and LNA modifications (in a 2:1 ratio) resulted in improved specificity and stability of the probe:RNA duplex in comparison to the LNA/DNA probe . Specificity of the system may be further improved by shortening the probe length to 19 nt . As the LNA/2′OMe RNA probe binds more strongly to yeast RNA or salmon sperm RNA used in ISH as a standard blocking agent, better results were obtained without these RNA blockers in the hybridization step . 2′F RNA nucleotides incorporated in the DNA probes ensure increased binding to the target and better nuclease resistance . Morpholino modifications, often applied to inhibit translation, modify splicing patterns of the primary transcript, or block miRNAs, were also used to detect miRNAs because of their high stability . 2.1.2. Probes Used with the Sequence Amplification System Several types of probes are combined with sequence amplification techniques to increase signal strength obtained from single miRNA molecules ( A–E). One example that enables sequence amplification is the use of padlock probes. These probes are successfully used to detect not only miRNAs but also mRNAs and DNA sequences. Padlock probes guarantee high specificity and sensitivity with single nucleotide discrimination —what makes them applicable for allele-specific FISH . Briefly, linear DNA probe after annealing to the specific sequence with 5′ and 3′ arms is circularized by DNA ligase. Circularization enables further signal amplification by rolling circle amplification (RCA) ( A). RCA uses miRNA molecule as a primer and elongates the sequence using circular probe as a template. Detection of RCA product is possible with the use of probes complementary to the sequence amplified on the template of the padlock probe central sequence. Similar types of probes, which are used in miRNA ISH also in combination with RCA, are circular DNA probes ( B). Circular probes are obtained in vitro with the use of padlock probes, ligation probes, and DNA ligase, and are then hybridized to the target sequence in cells as circular molecules . Another type of probe used together with RCA is a seal probe with an adjustable toehold inside its loop . These probes have an ability to change their structure. The initial dumbbell shape of the probe is changed into a circular form when the target miRNAs bind into the toehold domain of the probe. As a result, a RCA reaction can be initiated. This new method, called toehold-initiated rolling circle amplification (TIRCA), is a combination of toehold-mediated strand displacement (TMSD) and RCA. The length of the toehold defines the stability of the probe and is the most important factor for detecting miRNAs with TIRCA. With increasing length of the toehold, the stability of the seal probe and selectivity of TIRCA decreases. There are many advantages in using TIRCA, such as reduced loss of miRNA molecules, because the detection process is conducted at a physiological temperature, short imaging time, and high sensitivity and specificity, which is even higher than in the case of a padlock probe based RCA reaction .
In standard ISH, probes composed of DNA or RNA nucleotides are commonly used. Unmodified DNA and RNA probes have relatively poor binding affinity to target sequences , and therefore several modifications have been proposed to improve their properties . First and most commonly applied was Locked Nucleic Acid (LNA) modification, which remains the gold standard in RNA FISH not only in small RNA detection. LNA nucleotides, referred to as “locked” RNA, have an additional bridge connecting 4′C and 2′O atoms. LNA nucleotides are incorporated into DNA probes, which leads to the formation of hybrid LNA/DNA probes. LNA/DNA probes have been shown to be highly beneficial in miRNA detection because of a short hybridization time, high efficiency, discriminatory power and a high melting temperature of the miRNA:probe complex. The minimal length of the LNA/DNA probe was determined to be 12 nucleotides and these probes usually contain 30% LNA nucleotides. Besides their unquestionable advantages, these probes are expensive and can generate strong background signals resulting in a low signal-to-noise ratio for low abundant miRNA . Therefore, other modifications were also proposed. These modifications include 2′fluoro-modified RNA (2′F RNA), morpholino, Zip Nucleic Acids (ZNA) , N , N -diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN) and 2′O-Methyl (2′OMe) RNA modification. In comparison to DNA probes, 2′OMe RNA probes have faster hybridization kinetics and the ability to bind structured targets under standard conditions . The combination of 2′OMe RNA and LNA modifications (in a 2:1 ratio) resulted in improved specificity and stability of the probe:RNA duplex in comparison to the LNA/DNA probe . Specificity of the system may be further improved by shortening the probe length to 19 nt . As the LNA/2′OMe RNA probe binds more strongly to yeast RNA or salmon sperm RNA used in ISH as a standard blocking agent, better results were obtained without these RNA blockers in the hybridization step . 2′F RNA nucleotides incorporated in the DNA probes ensure increased binding to the target and better nuclease resistance . Morpholino modifications, often applied to inhibit translation, modify splicing patterns of the primary transcript, or block miRNAs, were also used to detect miRNAs because of their high stability .
Several types of probes are combined with sequence amplification techniques to increase signal strength obtained from single miRNA molecules ( A–E). One example that enables sequence amplification is the use of padlock probes. These probes are successfully used to detect not only miRNAs but also mRNAs and DNA sequences. Padlock probes guarantee high specificity and sensitivity with single nucleotide discrimination —what makes them applicable for allele-specific FISH . Briefly, linear DNA probe after annealing to the specific sequence with 5′ and 3′ arms is circularized by DNA ligase. Circularization enables further signal amplification by rolling circle amplification (RCA) ( A). RCA uses miRNA molecule as a primer and elongates the sequence using circular probe as a template. Detection of RCA product is possible with the use of probes complementary to the sequence amplified on the template of the padlock probe central sequence. Similar types of probes, which are used in miRNA ISH also in combination with RCA, are circular DNA probes ( B). Circular probes are obtained in vitro with the use of padlock probes, ligation probes, and DNA ligase, and are then hybridized to the target sequence in cells as circular molecules . Another type of probe used together with RCA is a seal probe with an adjustable toehold inside its loop . These probes have an ability to change their structure. The initial dumbbell shape of the probe is changed into a circular form when the target miRNAs bind into the toehold domain of the probe. As a result, a RCA reaction can be initiated. This new method, called toehold-initiated rolling circle amplification (TIRCA), is a combination of toehold-mediated strand displacement (TMSD) and RCA. The length of the toehold defines the stability of the probe and is the most important factor for detecting miRNAs with TIRCA. With increasing length of the toehold, the stability of the seal probe and selectivity of TIRCA decreases. There are many advantages in using TIRCA, such as reduced loss of miRNA molecules, because the detection process is conducted at a physiological temperature, short imaging time, and high sensitivity and specificity, which is even higher than in the case of a padlock probe based RCA reaction .
The first step in the ISH protocol is cell fixation, which on one hand should sufficiently preserve the number and localization of small RNA molecules, but, on the other hand, is mild enough to preserve cellular domains crucial for detection, which is especially important for ISH combined with protein labeling. Prevention of miRNA loss during fixation is essential, especially for detecting low abundant miRNAs. Highly abundant miRNAs can be successfully detected using standard fixation protocols . Significant improvement in miRNA recovery was shown by Tuschl and colleagues who used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to immobilize miRNA molecules via their 5′ end . EDC fixation needs to be used in combination with traditional formaldehyde fixation. Improvement in the signal to noise ratio by addition of an EDC-crosslinking step was confirmed in several studies of miRNAs . Importantly, standard formalin-fixation and paraffin-embedding (FFPE) as well as cryopreservation of tissues seems to preserve microRNAs sufficiently .
Permeabilization is used to improve cells and tissues penetration by the probe; however, permeabilization that is too strong can cause RNA loss from fixed material. Permeabilization is typically performed with the use of organic solvents including methanol or paraformaldehyde that are used also as fixatives, detergent like Saponin or Triton X-100, or proteinase. Briefly, organic solvents dissolve lipids from cell membranes. Some detergents, i.e. , Saponin, remove cholesterol from membranes in highly selective ways, but widely used Triton X-100 is not selective, which can lead to elimination of both proteins and lipids . To reduce cellular RNA diffusion, treatment with proteinases and detergents was either limited or eliminated in some miRNA ISH studies . The need for additional permeabilization decreases with the use of EDC fixation due to its auxiliary permeabilization activity. Long fixation of tissues also can decrease the need of permeabilization. Some differences are observed in permeabilization techniques used in ISH of fixed tissues and cell lines. Typically, tissues have to undergo stronger permeabilization because of their lower accessibility to the probe; however, permeabilization conditions differ among different types of tissues. Tissues are typically permeabilized with Triton X-100, but Proteinase K is also used. However, permeabilization conditions for specific types of tissues, e.g., embryos or brain tissues, still need to be optimized . Permeability of the tissue depends also on method of tissue preservation. Unfixed cryosections typically do not need permeabilization in contrary to fixed paraffin embedded sections. Cell lines are typically permeabilized with aceton, Triton X-100, Tween or overnight incubation with 70% ethanol. Fixation and permeabilization steps may be combined, e.g., by using a buffer containing paraformaldehyde and a short C-chain aliphatic carboxylic acid . There are also several commercial buffers used for permeabilization . Moreover, permeabilization conditions should be adjusted to the probe type and its length. Short linear probes used in miRNA ISH are able to easily enter cells and cellular compartments. Bigger, chemically modified probes or probes based on nanoshells would need stronger permeabilization treatment in comparison to short probes.
The hybridization step proceeds as in a standard ISH protocol. During hybridization, the optimal time of incubation and the temperature need to be experimentally determined because a calculated temperature for probe hybridization does not always give the best results. Typically the used hybridization buffer (HB) is the same as in ISH used for mRNA labeling; however, in some miRNA ISH experiments, HB was slightly modified. Important change in HB was the use of less toxic urea instead of 50% formamide . In some specific experiments, it is desired to visualize more than one miRNA in a cell or tissue . Multicolor ISH of miRNA may be performed sequentially or simultaneously. When simultaneous hybridization is performed and optimal hybridization temperatures for the probes differ, a suboptimal temperature must be chosen. When sequential hybridization is performed, hybridization can be performed at the optimal temperature for each probe .
The third step of an ISH protocol, which is crucial for specific detection of miRNA, is the washing step. Washing conditions need to be optimized to preserve the probe-target complex but at the same time eliminate off-target binding. One of the proposed improvements was changing the washing buffer composition to include tetramethylammonium chloride (TMAC). TMAC was used to compensate for the low specificity of RNA probes as highly stringent TMAC washing conditions decrease off-target binding . Moreover, TMAC washing allows probes for different miRNAs to be detected in unchanged conditions, which can be used for in situ analyses with multiple miRNA probes. TMAC is also used in combination with RNase A to prevent off-target hybridization of RNA probes with mismatches . In addition, it is important to adjust the washing temperature to provide specificity of binding while preserving the signal strength.
With the objective of single molecule detection, different types of sequence and signal amplification techniques have been used to provide better resolution . Enzymatic signal amplification methods, described in the detection section, are used mainly to image low-abundant miRNAs in tissues. The most commonly used system for sequence amplification is RCA . The main advantages of RCA are better cellular resolution, super bright spots indicating miRNA and ultrahigh sensitivity . RCA uses the free 3′ terminus of miRNA acting as the primer itself. mRNA and miRNA precursors do not give a positive signal as the 3′ end of these molecules does not hybridize to the probe and cannot be elongated during the process. The RCA process of sequence amplification is facilitated by DNA polymerase, resulting in the generation of thousands of amplified segments, which contain sequences complementary to the detection probes. The product of the RCA reaction is visualized with many fluorescently labeled detection probes. The RCA method was combined with a proximity ligation assay (PLA) that led to development of a system named oligo-fluorescence in situ hybridization (O-FISH) ( D). The PLA technique was primarily developed to detect proteins with single molecule sensitivity. Briefly, biotin-labeled probe hybridizes to miRNA target and anti-biotin monoclonal antibodies detect the probe. The primary antibody is detected by two monoclonal antibodies conjugated with oligonucleotides. After secondary antibodies bind to the target, the antibody-conjugated oligonucleotides can hybridize to connector oligonucleotides due to their close proximity. Connector oligonucleotides are then ligated to circular DNA that can be used in RCA similar to the use of padlock probes. Oligonucleotides conjugated with antibodies serve as primers for RCA reaction . Sequence amplification is also achieved by RT in situ PCR. This method, called the ultramer extension method, is based on the use of longer probes and miRNA acting as a primer, as in the RCA method. The probe contains sequences complementary to miRNA and a series of 20 nt sequences at the 5′ end. During in situ PCR, digoxigenin (DIG)-labeled nucleotides are incorporated to the PCR product to be further detected with antibodies .
Detection is the last, and a very important, step of miRNA ISH. In the first ISH experiments, radiolabeled probes were applied , but nonradioactive probes are currently used, giving information about the subcellular distribution of miRNA. In several experiments, probes directly labeled with fluorophores were used , but the obtained signal was not strong enough . Weak emission signals, rapid photobleaching, strong photoblinking and a lifetime close to cellular autofluorescence are major shortcomings of organic fluorophores. Several signal enhancement methods have been proposed to overcome these difficulties. In the commonly used miRNA ISH technique, enzyme-based detection is used. Briefly, nonradioactive haptens, combined with probes, are detected by histochemical enzymatic reactions after application of enzyme-conjugated anti-hapten antibodies. Alkaline phosphatase (AP) is most commonly used with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as a substrate . The tyramide signal amplification system (TSA), or, in other words, the catalyzed reporter deposition method (CARD), is a similar approach. Early versions of the commercially available TSA/CARD system used streptavidin-biotin affinity for initial signal detection . As endogenous biotin has been reported in many types of human tissue, currently LNA probes labeled with digoxigenin are mostly used in the hybridization step. In the TSA/CARD system, the horseradish peroxidase (HRP)-tagged anti-digoxigenin antibodies recognize digoxigenin moieties on probes. Next, HRP substrates, i.e. , cyanine 5 (Cy5), cyanine 3 (Cy3) or fluorescein-conjugated tyramides, are converted to highly reactive radicals by HRP and bind covalently to tyrosine residues located nearby . The radicals are extremely short-lived, which prevents them from diffusing away from the site of synthesis, which would decrease the signal-to-noise ratio . The TSA/CARD system improved the sensitivity up to 1000-fold compared to its early version . Another detection method is enzyme-labeled fluorescence signal amplification (ELF). In short, pro-luminescent substrate cleavage is performed by phosphatase, followed by precipitation of a bright, yellow-green fluorescent product. The obtained precipitate is highly photostable and gives a 40 times brighter signal in comparison to probes directly labeled with fluorophores . The dynamic range spans over three orders of magnitude, which means that 0 to 1000 copies of miRNA per cell can be quantified . Nanotechnology was recently introduced in the field of miRNA ISH to improve the detection of small RNAs. Metal nanoshells composed of silica spheres with encapsulated Ru(bpy) 3 2+ complexes as cores and thin silver layers as shells have been used for the detection of low-abundant miRNAs. The metal nanoshells are based on near-field interactions between organic fluorophores and metal nanoparticles. The use of nanoshells resulted in overcoming difficulties specific to organic fluorophores, including reduced photoblinking, increased photostability and intensity, as well as an increased lifetime of the organic fluorophores in comparison to the lifetime of cellular autofluorescence . However, the system needs to be optimized to provide better penetration of the nanoshells through the cell membrane, mobility in the cells as well as high specificity toward the miRNA target. Commercial assays for the detection of several miRNAs using in situ hybridization and fluorescence in situ hybridization are also available . A branched DNA system for signal amplification has been proposed to enable miRNA quantitative analysis . The system was also shown to be efficient in the detection of exogenous siRNA molecules.
In RNA FISH, probes targeting different parts of mRNA are used to confirm probe specificity. This approach is not applicable in small RNA ISH due to miRNA short length. Thus, different controls need to be used to ensure probe specificity. In several experiments, probes complementary to miRNA analogs from different organism or even different kingdoms were used as negative control, e.g., for mammalian miRNA detection, probes against plant specific miRNA was used . This approach may be applied only if miRNA analogs sufficiently differ in sequence. Another type of negative control is the use of probes against miRNA that is not expressed in the analyzed tissue (according to microarray or deep sequencing results) or the use of scrambled probes . The scrambled probe should be checked not only for specificity against miRNA population but also against the whole transcriptome. In some experiments, probes similar in sequence to miRNA-specific probes but containing two or three mismatches with the target sequence was used . As a positive and negative control, miRNA-specific probes can be used to label different tissues that were shown to exhibit this miRNA expression or not, respectively . To exclude false positive results caused by cells or tissue autofluorescence, control experiments without any probes are used . Additionally, non-hybridization-based interactions are excluded by the treatment with unlabeled probes prior to hybridization with labeled probes . Hybridization with probes that were already successfully tested can serve as positive controls for adequate experimental conditions and good RNA quality, e.g., snRNA U6 as a target is commonly used . Interactions with DNA are excluded by DNase treatment and interactions with RNA are confirmed by RNase treatment prior to hybridization.
Different ISH techniques are used to detect small RNAs depending on the required resolution . Less precise enzymatic methods are typically used when low resolution is sufficient. When higher resolution is required, fluorescence-based techniques are used, which additionally enable studies of small RNA subcellular localization and function. Below, we briefly refer to the results of selected studies that were aimed at determining small RNA expression, subcellular localization and interactions and were obtained with methods differing in resolution. Changes in miRNA expression levels have a significant impact on cells, tissues and whole organisms. These changes can be observed during physiological processes such as development , cell differentiation and pathologies, e.g., cancer , cardiovascular and neurodegenerative disorders . Changes in miRNA levels were also observed during cell growth in specific conditions or after drug treatment . Observed changes rarely refer to single or a few miRNAs but more often to tens of miRNAs, and, therefore, multiplex ISH analyses are performed. In both research on physiological processes and various pathologies, the same ISH methods are applicable . The observed signal resolution depends mainly on the detection technique used. However, the critical step is probe design that distinguishes between closely related small RNA sequences, which is a very common problem for this class of RNAs. Different miRNA ISH protocols are used in studies involving fixed tissues and cell lines. In tissue studies, typically LNA/DNA probes with a TSA or AP detection system is used (see ). In clinical diagnostics, where tissue samples are mainly used, the same methods apply. For example, in analysis of miRNA levels for diagnostic purposes in order to distinguish between various cancer types, LNA/DNA probes with TSA or AP detection systems were successfully used . In cell line studies, higher resolution and sensitivity of imaging is often required, therefore, fluorescent detection as well as sequence and signal amplification techniques are applied. However, most commonly used LNA/DNA probes with direct or indirect fluorescent labeling remain the method of choice for the majority of applications. 3.1. Small RNAs in Tissues: Presence and Expression Levels Enzyme-based methods, which are widely used in tissue research, have low resolution, which is compensated by high signal strength, low cost and simplicity of the methods. With use of enzyme-based techniques, changes in miRNA abundance are observed on a tissue level, where differences can be observed between different sections of the tissue. Mostly, LNA/DNA probes labeled with DIG or fluorescein are used and detected by AP or the TSA system. Co-localization with proteins is studied with the use of immunohistochemistry (IHC) or immunofluorescence (IF) to define the precise localization of RNAs in specific cell types . For example, ISH was performed on mouse embryos to observe the influence of physiological cell-specific miRNA expression on mouse development and differentiation during embryogenesis , and similar studies were performed using chicken embryos and zebrafish . Tissue-specific expression levels of miRNAs were also observed in the brain in the context of synapse functions and plasticity as well as drug addiction . Tissue levels of miRNAs and cell type-specific miRNA expression are also widely studied in cancer . Some miRNAs show a strong correlation with cancer progression , therefore, in situ analysis of miRNA abundance can be used as a diagnostic or progression marker . Multiplex analysis of different miRNAs enables distinguishing between different types of cancers . Examples of specific miRNAs that show high changes in cancerous cells that were imaged with ISH methods are miR-21 in colon adenocarcinomas and gliomas as well as pancreatic and breast cancer , miR-10b in pancreatic ductal adenocarcinoma and miR-221 in breast cancer . Methods enabling the localization of RNAs in specific cells in tissue samples were used also to image other types of small RNAs besides miRNAs. Most of the experiments used DIG-labeled LNA/DNA probes for hybridization to a target sequence, but various detection methods including AP and fluorophore-conjugated antibodies were used. Trans-acting siRNAs (ta-siRNAs) , different piRNA molecules and piRNA like small RNAs (pilRNAs) were also localized successfully with the use of LNA/DNA probes. The same system was used to image miRNAs to confirm system reliability to detect all small RNAs . Endogenous siRNAs present in plant cells were imaged simultaneously with miRNAs in plants using two-color labeling . miRNA ISH based on enzymatic detection was also implemented in molecular diagnostics using tissue microarrays . This technique enables fast detection of specific miRNAs in many tissue samples simultaneously. This high throughput analysis was performed with LNA/DNA probes and TSA labeling . Automation in multiplex miRNA detection was also described with the use of the same detection system . 3.2. Small RNAs in Cells: Subcellular Localization With increased resolution of in situ hybridization methods, it became possible to analyze subcellular localization of small RNAs. Because of the need for higher resolution, a number of studies on miRNA localization were performed with FISH methods. miRNAs have been found dispersed in the cytoplasm, nucleoplasm or localized in specific parts of cells. The ISH technique was used to observe miRNAs in specific parts of the nucleolus and mitochondria . To precisely localize small RNAs in the cell, protein markers of cellular compartments are usually labeled with IF or with chimeric proteins fused to GFP . Subcellular localization of small RNAs was investigated with the use of directly labeled Cy3-LNA/DNA probes , indirect labeling with fluorophore-conjugated antibody , or even double indirect antibody detection . With the use of ISH techniques, miRNA biogenesis was also analyzed. Designing probes specific for the loop of miRNA precursors and probes specific for mature miRNAs enabled the comparison of levels and localization of both types of molecules . RT in situ PCR and ultramer probes were used to discriminate between mature miRNAs and their precursors . Padlock and circular probes with RCA detect only mature small RNAs; however, traditional probes are also used with the assumption that they are specific for mature miRNAs because of the secondary structure of their precursors. Detection of RNA localization can also be useful to follow the cellular localization and fate of exogenous small RNAs. For example, miRNA ISH is used to monitor the degree of siRNA incorporation into cells and observe the cellular localization of siRNAs. Alternatively, siRNAs can be labeled directly to observe their cellular uptake; however, the label attachment can influence their localization and activity. It was shown that siRNAs can be efficiently detected with the use of LNA/DNA probes labeled with DIG. The detection systems used for this purpose include TSA and AP . siRNAs were also detected using 2′OMe RNA probes directly labeled with Cy3 . The results of ISH were comparable with those obtained with direct labeling of small RNAs. 3.3. Small RNAs in Complexes: Co-Localization, Correlations and Interactions with DNA, RNA and Proteins The combination of small RNA ISH with labeling of other RNAs, DNA and proteins can provide additional insight into small RNA functions. When such interactions are analyzed with in situ techniques, the obtained information is the existence or lack of co-localization, which does not necessarily mean direct interaction. Low resolution ISH combined with IF and IHC can show the correlation of cellular levels of miRNAs and proteins that are regulated by them. This functional dependence is observed as a negative feedback loop between miRNA and protein levels. Such correlations were observed in research concerning pluripotency, proliferation and differentiation and in various cancers . Co-localization of several miRNAs with other classes of biomolecules was performed with the use of fluorescent detection methods. For example, co-localization with U3 snoRNA, a marker of the granular component of the nucleolus, was conducted with the use of DIG-labeled LNA probes and fluorescently labeled antibodies . Protein:miRNA co-localization was demonstrated for GW182 and miR-let7 in neurons with the use of DIG-labeled LNA probes . To increase the resolution of such analyses, an improved fluorescent signal amplification system, called ELF, was used, which is claimed to be able to detect miRNAs on a single molecule level. The problem occurs when miRNAs are localized close to each other because they can be mistakenly interpreted as a single molecule. ELF is the only system that enabled single molecule detection and counting miRNA molecules while testing against Cy5-labeled, Texas red-labeled and quantum dot labeled secondary antibodies . Several approaches based on RCA, which were also aimed at labeling single molecules, faced the same problem as ELF. These methods can be successfully applied to count small RNA granules in cells, however, without certainty that these granules contain single small RNA molecules.
Enzyme-based methods, which are widely used in tissue research, have low resolution, which is compensated by high signal strength, low cost and simplicity of the methods. With use of enzyme-based techniques, changes in miRNA abundance are observed on a tissue level, where differences can be observed between different sections of the tissue. Mostly, LNA/DNA probes labeled with DIG or fluorescein are used and detected by AP or the TSA system. Co-localization with proteins is studied with the use of immunohistochemistry (IHC) or immunofluorescence (IF) to define the precise localization of RNAs in specific cell types . For example, ISH was performed on mouse embryos to observe the influence of physiological cell-specific miRNA expression on mouse development and differentiation during embryogenesis , and similar studies were performed using chicken embryos and zebrafish . Tissue-specific expression levels of miRNAs were also observed in the brain in the context of synapse functions and plasticity as well as drug addiction . Tissue levels of miRNAs and cell type-specific miRNA expression are also widely studied in cancer . Some miRNAs show a strong correlation with cancer progression , therefore, in situ analysis of miRNA abundance can be used as a diagnostic or progression marker . Multiplex analysis of different miRNAs enables distinguishing between different types of cancers . Examples of specific miRNAs that show high changes in cancerous cells that were imaged with ISH methods are miR-21 in colon adenocarcinomas and gliomas as well as pancreatic and breast cancer , miR-10b in pancreatic ductal adenocarcinoma and miR-221 in breast cancer . Methods enabling the localization of RNAs in specific cells in tissue samples were used also to image other types of small RNAs besides miRNAs. Most of the experiments used DIG-labeled LNA/DNA probes for hybridization to a target sequence, but various detection methods including AP and fluorophore-conjugated antibodies were used. Trans-acting siRNAs (ta-siRNAs) , different piRNA molecules and piRNA like small RNAs (pilRNAs) were also localized successfully with the use of LNA/DNA probes. The same system was used to image miRNAs to confirm system reliability to detect all small RNAs . Endogenous siRNAs present in plant cells were imaged simultaneously with miRNAs in plants using two-color labeling . miRNA ISH based on enzymatic detection was also implemented in molecular diagnostics using tissue microarrays . This technique enables fast detection of specific miRNAs in many tissue samples simultaneously. This high throughput analysis was performed with LNA/DNA probes and TSA labeling . Automation in multiplex miRNA detection was also described with the use of the same detection system .
With increased resolution of in situ hybridization methods, it became possible to analyze subcellular localization of small RNAs. Because of the need for higher resolution, a number of studies on miRNA localization were performed with FISH methods. miRNAs have been found dispersed in the cytoplasm, nucleoplasm or localized in specific parts of cells. The ISH technique was used to observe miRNAs in specific parts of the nucleolus and mitochondria . To precisely localize small RNAs in the cell, protein markers of cellular compartments are usually labeled with IF or with chimeric proteins fused to GFP . Subcellular localization of small RNAs was investigated with the use of directly labeled Cy3-LNA/DNA probes , indirect labeling with fluorophore-conjugated antibody , or even double indirect antibody detection . With the use of ISH techniques, miRNA biogenesis was also analyzed. Designing probes specific for the loop of miRNA precursors and probes specific for mature miRNAs enabled the comparison of levels and localization of both types of molecules . RT in situ PCR and ultramer probes were used to discriminate between mature miRNAs and their precursors . Padlock and circular probes with RCA detect only mature small RNAs; however, traditional probes are also used with the assumption that they are specific for mature miRNAs because of the secondary structure of their precursors. Detection of RNA localization can also be useful to follow the cellular localization and fate of exogenous small RNAs. For example, miRNA ISH is used to monitor the degree of siRNA incorporation into cells and observe the cellular localization of siRNAs. Alternatively, siRNAs can be labeled directly to observe their cellular uptake; however, the label attachment can influence their localization and activity. It was shown that siRNAs can be efficiently detected with the use of LNA/DNA probes labeled with DIG. The detection systems used for this purpose include TSA and AP . siRNAs were also detected using 2′OMe RNA probes directly labeled with Cy3 . The results of ISH were comparable with those obtained with direct labeling of small RNAs.
The combination of small RNA ISH with labeling of other RNAs, DNA and proteins can provide additional insight into small RNA functions. When such interactions are analyzed with in situ techniques, the obtained information is the existence or lack of co-localization, which does not necessarily mean direct interaction. Low resolution ISH combined with IF and IHC can show the correlation of cellular levels of miRNAs and proteins that are regulated by them. This functional dependence is observed as a negative feedback loop between miRNA and protein levels. Such correlations were observed in research concerning pluripotency, proliferation and differentiation and in various cancers . Co-localization of several miRNAs with other classes of biomolecules was performed with the use of fluorescent detection methods. For example, co-localization with U3 snoRNA, a marker of the granular component of the nucleolus, was conducted with the use of DIG-labeled LNA probes and fluorescently labeled antibodies . Protein:miRNA co-localization was demonstrated for GW182 and miR-let7 in neurons with the use of DIG-labeled LNA probes . To increase the resolution of such analyses, an improved fluorescent signal amplification system, called ELF, was used, which is claimed to be able to detect miRNAs on a single molecule level. The problem occurs when miRNAs are localized close to each other because they can be mistakenly interpreted as a single molecule. ELF is the only system that enabled single molecule detection and counting miRNA molecules while testing against Cy5-labeled, Texas red-labeled and quantum dot labeled secondary antibodies . Several approaches based on RCA, which were also aimed at labeling single molecules, faced the same problem as ELF. These methods can be successfully applied to count small RNA granules in cells, however, without certainty that these granules contain single small RNA molecules.
It is apparent from this review that a handful of in situ hybridization methods are now available for researchers, and the choice of the right method to use depends on the specific questions asked. Global miRNA expression analysis in tissues is typically performed with the help of chromogenic enzyme-based detection methods, but, for more precise small RNA localization studies, fluorescent imaging is more suitable. Sequence and signal amplification methods significantly increase the signal-to-noise ratio and enable the detection of low abundant RNAs. However, neither the RCA-based techniques nor ELF signal amplification methods are recommended when quantitative determination of small RNA levels are required. Therefore, choosing the right specific ISH variation is very important, and the intention of this review was to facilitate making such choices by providing some helpful comments on the existing methods and showing representative examples of their applications. Despite the unquestionable success of the ISH adaptation for small RNA detection and subcellular localization, the various in situ hybridization techniques have their limitations. Their major shortcomings are the fact that they capture small RNAs in the cell at one point of time and are unable to distinguish between RNA functional and nonfunctional states. The latter may include RNA that is awaiting its cellular function or RNA stored for degradation. The only difference between the functionally active and inactive small RNAs might be the type of proteins or transcripts with which they interact while active or stored. The co-localization of miRNA with target mRNAs or Ago proteins would solve the problem partially. An important further insight could be provided by an analysis that captures the downstream effect of target transcript cleavage or degradation. Furthermore, there is no system available for live RNA imaging capable of labeling small RNAs . The MS2 system was adapted for imaging miRNA primary precursors, called pri-miRNA but not their processing products, mature miRNAs. Recently, the first attempt to monitor miRNAs in living cells was described . Another important challenge is the ability of the ISH method for single molecule detection. Methods developed for single mRNA transcript detection cannot be used for small RNA imaging because these methods are typically based on the use of multiple probes that target different regions of a single mRNA. Signal amplification techniques, which were adapted for small RNA analysis, enable the visualization of low abundant miRNAs; however, it has not yet been established whether these methods are capable of precise single molecule detection. To summarize, several new approaches for the detection and analysis of small RNAs were proposed, enabling insight into the subcellular localization and co-localization of these molecules. The methods, in addition to their strong impact on the progress of basic miRNA research, also enabled the use of miRNAs as biomarkers for monitoring the progression of human diseases and following the outcomes of therapeutic treatments. With the available methods, the level and localization of small RNAs can be determined in single cells; however, temporal context and single molecule resolution is still needed, which remains a challenge for the development of future methods.
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Antifungal and antibiofilm effects of probiotic | e73da2eb-f207-4a97-ab99-8b0e5ced36ca | 11183309 | Microbiology[mh] | Introduction Candida albicans ( C. albicans ) is a dimorphic fungus that causes a significant portion (50–90%) of candidiasis cases in humans . It colonizes the mucosal surfaces and skin of healthy humans, animals, and fish . Clinical manifestations of candidiasis range from localized to invasive and systemic disease, depending on individual’s immune status . C. albicans can cause life-threatening infections, especially infections caused by drug-resistant strains, in immunocompromised patients , resulting in significant morbidity, high mortality, and increased healthcare costs . Other Candida spp., such as C. tropicalis , C. glabrata and C. parapsilosis , cause 30 to 54% of candidiasis cases . Candida spp. can also cause acute diseases when contaminating potable water and are increasingly recognized as an emerging cause of chronic water quality problems as they are natural inhabitants of water and can thrive in low circulation areas of water systems . Several factors, including the host’s immune status, virulence-associated genes, and biofilm formation ability, contribute to the transition of Candida spp. from commensal to pathogenic form . The pathogenicity of Candida spp. is primarily linked to specific virulence genes, such as agglutinin-like sequence genes ( ALS3 ), secretory aspartate protease ( SAP4 ), hyphal wall protein 1 ( HWP1 ), and a hyphal-regulated gene ( HYR1 ), which facilitate the invasion of host tissues . These genes are essential for mycelial formation, adhesion, and the development of C. albicans biofilm . C. albicans biofilm consist of diverse three-dimensional combinations of hyphae and yeast embedded extracellularly by large numbers of matrix units . C. albicans can form biofilms on the surfaces of implantable medical instruments and is associated with device-related nosocomial infections. These Candida biofilms exhibit resistance levels 30 to 2000 times higher than those of planktonic cells to various antifungal agents, such as fluconazole, ketoconazole, itraconazole and amphotericin , as well as reduced susceptibility to host immune reactions . The widespread and prolonged use of conventional antimicrobial agents has led to the emergence of fungal drug fights . Researchers are now exploring innovative strategies to combat multidrug-resistant microbial strains, including the use of nanomaterials and probiotics , as the development of new antibiotics is challenging. Nanomaterials composed of various substances, including lipids, metals, and natural or synthetic polymers, serve as efficient drug carriers . Their rapid and effective biological absorption compared to larger macromolecules makes them excellent materials for delivery systems . In recent years, zinc nanoparticles (ZnNPs) have garnered significant attention as one of the most extensively studied nanostructures produced through nanotechnology, known for their antimicrobial properties against bacteria and fungi. ZnNPs disrupt fungal membranes, leading to cell death by compromising cell structure and function . On the other hand, probiotics, which are diverse antimicrobial substances produced by lactic acid bacteria (LAB) strains, can inhibit C. albicans proliferation, adhesion, and biofilm formation . Several studies have shown that Lactobacillus rhamnosus , Lactobacillus reuteri , and Lactobacillus salivarius ( L. salivarius ) possess potent antifungal activity against C. albicans infection . Supernatants of L. salivarius demonstrate antifungal effects against Candida spp., with lactic acid accumulation in the medium . Additionally, organic acids produced by Lactobacillus spp. enhance the efficacy of antifungal agents by increasing fungal plasma membrane permeability, facilitating azole uptake. However, lactic acid, low pH, and other secreted metabolites are environmental signals sensed by C. albicans , triggering changes in gene expression and the transition to hyphal growth. Another potential mechanism for the probiotic effect could involve competition for available niches and reduced adhesion . Therefore, the present study aimed to (i) detect the occurrence of Candida spp. in tilapia fish, fish wash water, and fish sellers in Sharkia Governorate, Egypt, (ii) identify the virotypes and genotypes of the isolated C. albicans , (iii) assess the susceptibility to antifungal drugs and biofilm forming abilities of the identified C. albicans , and (iv) investigate the antifungal and antibiofilm effects of L. salivarius and ZnNPs and nanocomposites (ZnNCs) on isolates of C. albicans .
Materials and methods 2.1 Study design and sampling A cross-sectional study collected 300 samples from Nile tilapia ( Oreochromis niloticus ), fish wash water, and human fish sellers (100 from each) at various retail fish markets in Sharkia Governorate, Egypt, between April 2022 and January 2023. Sterile cotton swabs were used to collect samples from fish and sellers. The swabs gently rubbed the fish surface and the human seller’s hand before being immersed in tubes containing buffered peptone water (BPW). Additionally, 100 mL of fish wash water was collected in sterile screw-capped glass bottles. The specimens were labeled with the sample identifier, sample type, and date of sampling, and then transported to the laboratory for later testing. 2.2 Isolation and biochemical identification All samples were cultured on Sabouraud Dextrose Agar (SDA; CONDA, Spain) slopes supplemented with 0.05 µg/mL of chloramphenicol and incubated for 24-72 h at 37°C . Any visible growth on the SDA slopes was further identified through microscopic examination, Gram staining, germ tube testing, and urea hydrolysis testing . Pasty and creamy colonies displaying Gram-positive staining and a negative urea hydrolysis test were purified and inoculated into CHROM agar for 48 h at 37°C . Colonies exhibiting a light green color were presumptively identified as C. albicans and preserved on SDA slopes at 4°C for further characterization. 2.3 Molecular characterization of C. albicans Suspected C. albicans colonies were confirmed using PCR targeting the ITS1 region of the C. albicans genome. DNA was isolated with a QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s guidelines. The PCR master mix (25 μL) included 12.5 μL of 2X Dream Taq Green master mix kit, 5.5 μL of PCR grade water, 1 μL of both forward and reverse primers (20 pmoL, each), and 5 μL of DNA template. The primers used were obtained from Metabion (Germany) and the PCR conditions are described in . Molecularly confirmed C. albicans isolates were subjected to virotyping by targeting the ALS3 gene , HWP1 gene , RAS1 gene and SAP4 gene . Positive controls ( C. albicans ATCC 90028) were included in the PCR assay with the tested strains. Genotyping of C. albicans was performed using RAPD-PCR fingerprinting with the primer OPA-18 following a previously described method . The RAPD-PCR fingerprinting information was presented as a binary code based on the absence or presence of every band. The discriminatory power of RAPD-PCR was assessed using Simpson’s index of diversity (D), as previously described . 2.4 Antifungal susceptibility test The antifungal susceptibility of C. albicans strains was assessed through Kirby-Bauer Disc Diffusion procedure with Mueller Hinton agar. Inhibition zones were determined and interpreted using the Clinical and Laboratory Standards Institute instructions . Eight antifungal agents, including amphotericin (AMB; 20 μg), nystatin (NYS; 100 I.U.); fluconazole (FLZ; 25 μg), itraconazole (ITR; 10 μg), flucytosine (FUS; 1 μg), caspofungin (CAS; 5 μg), micafungin (MIC; 1 μg) and terbinafine (TER; 30 μg), were tested. C. albicans ATCC 90028 was used as a quality control. The multiple antifungal resistance (MAR) index was calculated . Multidrug resistance (MDR) was also determined . 2.5 Biofilm formation and quantification Biofilm production of the C. albicans strains was assessed using the microtiter plate method . A loopful of colonies from fresh agar plates was cultured in SDB media (Oxoid Ltd., Cambridge, UK), overnight at 30°C at 150 rpm in an orbital shaker (Lab-line Incubator Shaker; Elliott Bay Laboratory Services Inc., Seattle, WA, USA). The yeast cells were harvested by centrifugation at 3,000 rpm for 10 min, washed twice in sterile phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO., USA), resuspended in RPMI 1640, counted using a hemocytometer, and concentration-adjusted to 1 × 10 6 cells/mL. A 100 μL aliquot of cell suspension ( C. albicans + SDB) was inoculated into individual well of the 96 well-flat bottom microtiter plates in triplicate. Wells of every plate are containing SDB that were recognized as negative controls. The plates were incubated for a day at 37°C, before being washed three times with sterile 200 µL PBS. The plates were inverted, and PBS was removed. The biofilms were subsequently fixed with 150 μL of 95% ethanol for 20 min. Finally, the biofilm was stained with 0.1% (w/v) crystal violet and left at room temperature for 15 min, then washed twice with PBS and left for 1 h to dry. The optical density (OD) of stained adherent C. albicans biofilm was measured at wavelength of 590 nm by an ELISA reader subsequent to the negative control (OD NC ) at zero. The means and standard deviations of OD values were recorded for all C. albicans isolates, positive ( C. albicans , ATCC 90028) and negative controls. The isolates were then considered to be non-biofilm formers (OD 590 ≤ OD NC ), weak (OD NC < OD 590 ≤ 2 x OD NC ), moderate (2 × OD NC < OD 590 ≤ 4 × OD NC ), or strong (4 × OD NC < OD 590 ) biofilm formers. 2.6 DNA sequencing and phylogenetic analysis The DNA products amplified from two MDR and strong biofilm-forming C. albicans strains were isolated with a QIAquick PCR Kit (QIAGEN, Valencia, CA, USA), following the manufacturer’s guidelines. ITS gene sequencing was carried out with primers and an automated sequencer, as previously described . The obtained sequences were resolved with the Basic Local Alignment Search Tool (BLAST ® analysis) of Informative Biotechnology website on the National Center. Gene sequences of the C. albicans isolates were submitted to the GenBank sequence database under accession numbers OQ150021 and OQ150022. Initial BLAST ® analysis was performed to establish sequence identity with GenBank accessions . These sequences were then aligned with others available in the GenBank sequence database. A phylogenetic tree was constructed using MEGA 6 . 2.7 Effects of probiotic L. salivarius , ZnNPs and ZnNCs on C. albicans 2.7.1 Biosynthesis of ZnNPs and ZnNCs ZnNPs biosynthesis followed the protocol previously described by . It involved using a 1 mM salt of ZnO 2 metal (Nanotech for Photo-Electronic Co., Dreamland, 6-October, Egypt) with enzyme extract from Aspergillus fumigatus . The mixture was then incubated at 28°C until ZnNPs biosynthesis was complete. The probiotic strain L. salivarius DSM 20555 from the Belgian Co-ordinated Collection of Microorganisms (BCCM) was cultivated in Mane Rogosae Sharp (MRS) broth (Oxoid, Roskilde, Denmark). Probiotic solution (1 mL) was applied to MRS broth (9 mL) and incubated for 24 h at 37°C. Probiotic cultures were centrifuged after incubation for 10 min at 4000 rpm, and the supernatant was collected and purify-sterilized by a 0.2 mm membrane syringe filter. The synthesis of ZnNCs was carried out through a sono-chemical method, in which 1 g of ZnNPs mixed in 200 mL of deionized distilled water was added to a 250 mL beaker with 1 mL of probiotic L. salivarius and subjected to sonication under conditions of 0.5 cycle and 50% amplitude for 2 h until a homogeneous mixture was obtained . 2.7.2 Characterization of ZnNPs and ZnNCs ZnNPs and ZnNCs were characterized using dynamic light scattering (DLS) to determine the particle diameter . Additionally, transmission electron microscopy (TEM) was performed on both ZnNPs and ZnNCs. In a solution of double-deionized water, ZnNPs and ZnNCs were added and then sonicated for 50 min at a frequency of 50 kHz with a cycle length of 0.65 and amplitude of 85% (UP400S, Hielscher, Germany). The resulting slurry was aliquoted (5 µL) and applied to a copper grid with carbon coating. TEM analysis was conducted using a TEM-2100 (JEOL, Tokyo 196-8558, Japan) . X-ray diffraction (XRD) test was performed using a Bruker D8 Discovers Set (Billerica, MA, USA) to determine the colloidal nature and evaluate the homogeneity and reunify of the synthesis methods for ZnNPs, probiotic L. salivarius , and ZnNCs. The probiotic L. salivarius and ZnNCs were subjected to assessment via Energy Dispersive-X-ray Spectroscopy (EDS) . 2.7.3 Antibiofilm effect A suspension (100 μL) of C. albicans (1 × 10 6 cells/mL) was plated in 96-well microtiter dishes as described earlier and incubated with agitation. The effects of ZnNPs, probiotic L. salivarius , ZnNCs, and antifungal (AMB) on hyphal development and the early phases of biofilm consistence (preventative) were assessed. Free floating cells were removed 1.5 h after adhesion by thoroughly washing each well with 200 μL of PBS. Subsequently, after another 24 h at 37°C with agitation, adhesive cells were exposed to 200 μL of various doses of the test drugs that had been diluted on a separate microtiter dish. The produced biofilm biomass was quantified for ZnNPs, probiotic L. salivarius , ZnNCs, and AMB in comparison to untreated controls. The crystal violet assay was used to quantify the resulting biofilm biomass compared with untreated controls. An ELISA reader (Sunrise R4, Tecan) calculated the OD of the biofilm at 590 nm after the negative control (OD NC ) was set at zero. The negative and positive controls used were sanitized medium only and the working solution, respectively . The rate of biofilm inhibition was measured through the subsequent equation: B i o f i l m i n h i b i t i o n r a t e = 1 − O D 590 o f t r e a t e d c e l l s O D 590 o f n o n t r e a t e d c o n t r o l × 100 2.7.4 Antifungal effect The antifungal efficacy of ZnNPs, probiotic L. salivarius , and ZnNCs was assessed against C. albicans isolates using the agar well diffusion method . In brief, 25 mL of SDA agar was individually inoculated with 100 µL (0.5 × 10 ^3 CFU/mL) of C. albicans isolates. The mixture was then added into a Petri dish and allowed to settle at room temperature for 30 min. Subsequently, a 10 mm diameter well was created in the agar using a sterile corkborer. The wells were supplied with 100 µg of each ZnNPs, probiotic L. salivarius , and ZnNCs. Wells filled with (20 µg) and broth media served as positive and negative controls, respectively. The plates were adjusted for 48 h at 37°C, and the diameters of the inhibition zones were measured using a ruler. The minimum inhibitory concentration (MIC) values of ZnNPs, probiotic L. salivarius , and ZnNCs against C. albicans were calculated by agar well diffusion technique as mentioned above. Different concentrations ranging from 5 to 100 µg/mL of each ZnNPs, probiotic L. salivarius , and ZnNCs were applied to the wells, and the plates were adjusted for 2 days at 37°C. The lowest agent concentration that inhibited germination parallels to the positive control after 48 h of incubation at 37°C was considered the MIC. The test was conducted in triplicate. 2.7.5 Quantitative analysis of biofilm gene expression Transcript expression was used to assess the effect of the tested factors on C. albicans adhesion ( ALS3 ), filamentation ( HWP1 ), the hyphal regulator ( RAS1 ), and hyphal formation and virulence ( SAP4 ), as previously described . A 1 mL suspension of each C. albicans strain, OQ150021 and OQ150022, was transferred to the wells of pre-sterilized 24-well microtiter plates with a flat bottom and incubated at 37°C for 1.5 h under agitation. The wells were washed with PBS. Fresh RPMI 1640 medium (1 mL) containing MIC-50 of ZnNPs, probiotic L. salivarius , ZnNCs, and AMB was added to each well, and the plate was further incubated for 24 h at 37°C. The control wells were used for comparison. The wells were washed twice with PBS and 600 mL of RLT buffer was added to the wells. The plate was incubated for 10 min, and then transferred to 1.5 mL microcentrifuge tubes. For sample homogenization, a 2-min high-speed (30-Hz) tissue lyser was used. For each harvested fungal culture, including untreated fungal culture (control) and the treated fungal culture (ZnNPs, probiotic L. salivarius , ZnNCs, and AMB-treated C. albicans ), one volume was added to one volume of RNA protection reagent at each sampling time (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA isolation was carried out using the QIAamp RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s guidelines. SYBR Green I-based real-time PCR with specific primers for ALS3 , HWP1 , RAS1 , and SAP4 virulence genes was used as previously described . The ITS gene was employed as a housekeeping gene . The PCR reaction contained 12.5 µL of SYBR Green PCR Master Mix (QIAGEN), 0.25 µL of reverse transcriptase (200 U/µL), 0.5 µL of each primer (20 pmoL), 8.25 µL of nuclease-free water, and 3 µL of RNA template. The amplification curves and cycle threshold (Ct) values were determined by Stratagene MX3005P software. The ΔΔCt method was performed, according to . 2.7.6 Scanning electron microscopy analysis The impact of ZnNCs on the morphology and structural integrity of C. albicans biofilm was examined using scanning electron microscopy (SEM) (Jeol 2100, Tokyo, Japan), following established protocols . Initially, 2 mL suspension of C. albicans OQ150021 cells at a concentration of 1 × 10 6 cells/mL in RPMI 1640 was incubated at 37°C for 24 h. Subsequently, the resulting biofilm was formed on sterile plastic coverslips (Nalge Nunc International) placed in 24-well tissue culture plates (Costar, Corning Inc., USA). The biofilm was treated with ZnNCs, washed with PBS, and then fixed in a solution of 0.1 M cacodylate buffer and 2.5% (vol/vol) glutaraldehyde (pH 7.2) overnight. After fixation, the cells were subjected to a series of ethanol washes for dehydration and air-dried in a desiccator. SEM analysis was conducted on the gold-coated samples using a Baltec SDC 050 sputter coater. 2.8 Data analysis All statistical tests and data visualization were performed with R software (version 4.2.0). A dendrogram was generated with the “ hclust ” function from the stats package. One-way analysis of variance compared the effects of AMB, ZnNPs, probiotic L. salivarius , and ZnNCs on C. albicans biofilm formation and biofilm gene expression. P -values <0.05 were considered statistically significant.
Study design and sampling A cross-sectional study collected 300 samples from Nile tilapia ( Oreochromis niloticus ), fish wash water, and human fish sellers (100 from each) at various retail fish markets in Sharkia Governorate, Egypt, between April 2022 and January 2023. Sterile cotton swabs were used to collect samples from fish and sellers. The swabs gently rubbed the fish surface and the human seller’s hand before being immersed in tubes containing buffered peptone water (BPW). Additionally, 100 mL of fish wash water was collected in sterile screw-capped glass bottles. The specimens were labeled with the sample identifier, sample type, and date of sampling, and then transported to the laboratory for later testing.
Isolation and biochemical identification All samples were cultured on Sabouraud Dextrose Agar (SDA; CONDA, Spain) slopes supplemented with 0.05 µg/mL of chloramphenicol and incubated for 24-72 h at 37°C . Any visible growth on the SDA slopes was further identified through microscopic examination, Gram staining, germ tube testing, and urea hydrolysis testing . Pasty and creamy colonies displaying Gram-positive staining and a negative urea hydrolysis test were purified and inoculated into CHROM agar for 48 h at 37°C . Colonies exhibiting a light green color were presumptively identified as C. albicans and preserved on SDA slopes at 4°C for further characterization.
Molecular characterization of C. albicans Suspected C. albicans colonies were confirmed using PCR targeting the ITS1 region of the C. albicans genome. DNA was isolated with a QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s guidelines. The PCR master mix (25 μL) included 12.5 μL of 2X Dream Taq Green master mix kit, 5.5 μL of PCR grade water, 1 μL of both forward and reverse primers (20 pmoL, each), and 5 μL of DNA template. The primers used were obtained from Metabion (Germany) and the PCR conditions are described in . Molecularly confirmed C. albicans isolates were subjected to virotyping by targeting the ALS3 gene , HWP1 gene , RAS1 gene and SAP4 gene . Positive controls ( C. albicans ATCC 90028) were included in the PCR assay with the tested strains. Genotyping of C. albicans was performed using RAPD-PCR fingerprinting with the primer OPA-18 following a previously described method . The RAPD-PCR fingerprinting information was presented as a binary code based on the absence or presence of every band. The discriminatory power of RAPD-PCR was assessed using Simpson’s index of diversity (D), as previously described .
Antifungal susceptibility test The antifungal susceptibility of C. albicans strains was assessed through Kirby-Bauer Disc Diffusion procedure with Mueller Hinton agar. Inhibition zones were determined and interpreted using the Clinical and Laboratory Standards Institute instructions . Eight antifungal agents, including amphotericin (AMB; 20 μg), nystatin (NYS; 100 I.U.); fluconazole (FLZ; 25 μg), itraconazole (ITR; 10 μg), flucytosine (FUS; 1 μg), caspofungin (CAS; 5 μg), micafungin (MIC; 1 μg) and terbinafine (TER; 30 μg), were tested. C. albicans ATCC 90028 was used as a quality control. The multiple antifungal resistance (MAR) index was calculated . Multidrug resistance (MDR) was also determined .
Biofilm formation and quantification Biofilm production of the C. albicans strains was assessed using the microtiter plate method . A loopful of colonies from fresh agar plates was cultured in SDB media (Oxoid Ltd., Cambridge, UK), overnight at 30°C at 150 rpm in an orbital shaker (Lab-line Incubator Shaker; Elliott Bay Laboratory Services Inc., Seattle, WA, USA). The yeast cells were harvested by centrifugation at 3,000 rpm for 10 min, washed twice in sterile phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO., USA), resuspended in RPMI 1640, counted using a hemocytometer, and concentration-adjusted to 1 × 10 6 cells/mL. A 100 μL aliquot of cell suspension ( C. albicans + SDB) was inoculated into individual well of the 96 well-flat bottom microtiter plates in triplicate. Wells of every plate are containing SDB that were recognized as negative controls. The plates were incubated for a day at 37°C, before being washed three times with sterile 200 µL PBS. The plates were inverted, and PBS was removed. The biofilms were subsequently fixed with 150 μL of 95% ethanol for 20 min. Finally, the biofilm was stained with 0.1% (w/v) crystal violet and left at room temperature for 15 min, then washed twice with PBS and left for 1 h to dry. The optical density (OD) of stained adherent C. albicans biofilm was measured at wavelength of 590 nm by an ELISA reader subsequent to the negative control (OD NC ) at zero. The means and standard deviations of OD values were recorded for all C. albicans isolates, positive ( C. albicans , ATCC 90028) and negative controls. The isolates were then considered to be non-biofilm formers (OD 590 ≤ OD NC ), weak (OD NC < OD 590 ≤ 2 x OD NC ), moderate (2 × OD NC < OD 590 ≤ 4 × OD NC ), or strong (4 × OD NC < OD 590 ) biofilm formers.
DNA sequencing and phylogenetic analysis The DNA products amplified from two MDR and strong biofilm-forming C. albicans strains were isolated with a QIAquick PCR Kit (QIAGEN, Valencia, CA, USA), following the manufacturer’s guidelines. ITS gene sequencing was carried out with primers and an automated sequencer, as previously described . The obtained sequences were resolved with the Basic Local Alignment Search Tool (BLAST ® analysis) of Informative Biotechnology website on the National Center. Gene sequences of the C. albicans isolates were submitted to the GenBank sequence database under accession numbers OQ150021 and OQ150022. Initial BLAST ® analysis was performed to establish sequence identity with GenBank accessions . These sequences were then aligned with others available in the GenBank sequence database. A phylogenetic tree was constructed using MEGA 6 .
Effects of probiotic L. salivarius , ZnNPs and ZnNCs on C. albicans 2.7.1 Biosynthesis of ZnNPs and ZnNCs ZnNPs biosynthesis followed the protocol previously described by . It involved using a 1 mM salt of ZnO 2 metal (Nanotech for Photo-Electronic Co., Dreamland, 6-October, Egypt) with enzyme extract from Aspergillus fumigatus . The mixture was then incubated at 28°C until ZnNPs biosynthesis was complete. The probiotic strain L. salivarius DSM 20555 from the Belgian Co-ordinated Collection of Microorganisms (BCCM) was cultivated in Mane Rogosae Sharp (MRS) broth (Oxoid, Roskilde, Denmark). Probiotic solution (1 mL) was applied to MRS broth (9 mL) and incubated for 24 h at 37°C. Probiotic cultures were centrifuged after incubation for 10 min at 4000 rpm, and the supernatant was collected and purify-sterilized by a 0.2 mm membrane syringe filter. The synthesis of ZnNCs was carried out through a sono-chemical method, in which 1 g of ZnNPs mixed in 200 mL of deionized distilled water was added to a 250 mL beaker with 1 mL of probiotic L. salivarius and subjected to sonication under conditions of 0.5 cycle and 50% amplitude for 2 h until a homogeneous mixture was obtained . 2.7.2 Characterization of ZnNPs and ZnNCs ZnNPs and ZnNCs were characterized using dynamic light scattering (DLS) to determine the particle diameter . Additionally, transmission electron microscopy (TEM) was performed on both ZnNPs and ZnNCs. In a solution of double-deionized water, ZnNPs and ZnNCs were added and then sonicated for 50 min at a frequency of 50 kHz with a cycle length of 0.65 and amplitude of 85% (UP400S, Hielscher, Germany). The resulting slurry was aliquoted (5 µL) and applied to a copper grid with carbon coating. TEM analysis was conducted using a TEM-2100 (JEOL, Tokyo 196-8558, Japan) . X-ray diffraction (XRD) test was performed using a Bruker D8 Discovers Set (Billerica, MA, USA) to determine the colloidal nature and evaluate the homogeneity and reunify of the synthesis methods for ZnNPs, probiotic L. salivarius , and ZnNCs. The probiotic L. salivarius and ZnNCs were subjected to assessment via Energy Dispersive-X-ray Spectroscopy (EDS) . 2.7.3 Antibiofilm effect A suspension (100 μL) of C. albicans (1 × 10 6 cells/mL) was plated in 96-well microtiter dishes as described earlier and incubated with agitation. The effects of ZnNPs, probiotic L. salivarius , ZnNCs, and antifungal (AMB) on hyphal development and the early phases of biofilm consistence (preventative) were assessed. Free floating cells were removed 1.5 h after adhesion by thoroughly washing each well with 200 μL of PBS. Subsequently, after another 24 h at 37°C with agitation, adhesive cells were exposed to 200 μL of various doses of the test drugs that had been diluted on a separate microtiter dish. The produced biofilm biomass was quantified for ZnNPs, probiotic L. salivarius , ZnNCs, and AMB in comparison to untreated controls. The crystal violet assay was used to quantify the resulting biofilm biomass compared with untreated controls. An ELISA reader (Sunrise R4, Tecan) calculated the OD of the biofilm at 590 nm after the negative control (OD NC ) was set at zero. The negative and positive controls used were sanitized medium only and the working solution, respectively . The rate of biofilm inhibition was measured through the subsequent equation: B i o f i l m i n h i b i t i o n r a t e = 1 − O D 590 o f t r e a t e d c e l l s O D 590 o f n o n t r e a t e d c o n t r o l × 100 2.7.4 Antifungal effect The antifungal efficacy of ZnNPs, probiotic L. salivarius , and ZnNCs was assessed against C. albicans isolates using the agar well diffusion method . In brief, 25 mL of SDA agar was individually inoculated with 100 µL (0.5 × 10 ^3 CFU/mL) of C. albicans isolates. The mixture was then added into a Petri dish and allowed to settle at room temperature for 30 min. Subsequently, a 10 mm diameter well was created in the agar using a sterile corkborer. The wells were supplied with 100 µg of each ZnNPs, probiotic L. salivarius , and ZnNCs. Wells filled with (20 µg) and broth media served as positive and negative controls, respectively. The plates were adjusted for 48 h at 37°C, and the diameters of the inhibition zones were measured using a ruler. The minimum inhibitory concentration (MIC) values of ZnNPs, probiotic L. salivarius , and ZnNCs against C. albicans were calculated by agar well diffusion technique as mentioned above. Different concentrations ranging from 5 to 100 µg/mL of each ZnNPs, probiotic L. salivarius , and ZnNCs were applied to the wells, and the plates were adjusted for 2 days at 37°C. The lowest agent concentration that inhibited germination parallels to the positive control after 48 h of incubation at 37°C was considered the MIC. The test was conducted in triplicate. 2.7.5 Quantitative analysis of biofilm gene expression Transcript expression was used to assess the effect of the tested factors on C. albicans adhesion ( ALS3 ), filamentation ( HWP1 ), the hyphal regulator ( RAS1 ), and hyphal formation and virulence ( SAP4 ), as previously described . A 1 mL suspension of each C. albicans strain, OQ150021 and OQ150022, was transferred to the wells of pre-sterilized 24-well microtiter plates with a flat bottom and incubated at 37°C for 1.5 h under agitation. The wells were washed with PBS. Fresh RPMI 1640 medium (1 mL) containing MIC-50 of ZnNPs, probiotic L. salivarius , ZnNCs, and AMB was added to each well, and the plate was further incubated for 24 h at 37°C. The control wells were used for comparison. The wells were washed twice with PBS and 600 mL of RLT buffer was added to the wells. The plate was incubated for 10 min, and then transferred to 1.5 mL microcentrifuge tubes. For sample homogenization, a 2-min high-speed (30-Hz) tissue lyser was used. For each harvested fungal culture, including untreated fungal culture (control) and the treated fungal culture (ZnNPs, probiotic L. salivarius , ZnNCs, and AMB-treated C. albicans ), one volume was added to one volume of RNA protection reagent at each sampling time (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA isolation was carried out using the QIAamp RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s guidelines. SYBR Green I-based real-time PCR with specific primers for ALS3 , HWP1 , RAS1 , and SAP4 virulence genes was used as previously described . The ITS gene was employed as a housekeeping gene . The PCR reaction contained 12.5 µL of SYBR Green PCR Master Mix (QIAGEN), 0.25 µL of reverse transcriptase (200 U/µL), 0.5 µL of each primer (20 pmoL), 8.25 µL of nuclease-free water, and 3 µL of RNA template. The amplification curves and cycle threshold (Ct) values were determined by Stratagene MX3005P software. The ΔΔCt method was performed, according to . 2.7.6 Scanning electron microscopy analysis The impact of ZnNCs on the morphology and structural integrity of C. albicans biofilm was examined using scanning electron microscopy (SEM) (Jeol 2100, Tokyo, Japan), following established protocols . Initially, 2 mL suspension of C. albicans OQ150021 cells at a concentration of 1 × 10 6 cells/mL in RPMI 1640 was incubated at 37°C for 24 h. Subsequently, the resulting biofilm was formed on sterile plastic coverslips (Nalge Nunc International) placed in 24-well tissue culture plates (Costar, Corning Inc., USA). The biofilm was treated with ZnNCs, washed with PBS, and then fixed in a solution of 0.1 M cacodylate buffer and 2.5% (vol/vol) glutaraldehyde (pH 7.2) overnight. After fixation, the cells were subjected to a series of ethanol washes for dehydration and air-dried in a desiccator. SEM analysis was conducted on the gold-coated samples using a Baltec SDC 050 sputter coater.
Biosynthesis of ZnNPs and ZnNCs ZnNPs biosynthesis followed the protocol previously described by . It involved using a 1 mM salt of ZnO 2 metal (Nanotech for Photo-Electronic Co., Dreamland, 6-October, Egypt) with enzyme extract from Aspergillus fumigatus . The mixture was then incubated at 28°C until ZnNPs biosynthesis was complete. The probiotic strain L. salivarius DSM 20555 from the Belgian Co-ordinated Collection of Microorganisms (BCCM) was cultivated in Mane Rogosae Sharp (MRS) broth (Oxoid, Roskilde, Denmark). Probiotic solution (1 mL) was applied to MRS broth (9 mL) and incubated for 24 h at 37°C. Probiotic cultures were centrifuged after incubation for 10 min at 4000 rpm, and the supernatant was collected and purify-sterilized by a 0.2 mm membrane syringe filter. The synthesis of ZnNCs was carried out through a sono-chemical method, in which 1 g of ZnNPs mixed in 200 mL of deionized distilled water was added to a 250 mL beaker with 1 mL of probiotic L. salivarius and subjected to sonication under conditions of 0.5 cycle and 50% amplitude for 2 h until a homogeneous mixture was obtained .
Characterization of ZnNPs and ZnNCs ZnNPs and ZnNCs were characterized using dynamic light scattering (DLS) to determine the particle diameter . Additionally, transmission electron microscopy (TEM) was performed on both ZnNPs and ZnNCs. In a solution of double-deionized water, ZnNPs and ZnNCs were added and then sonicated for 50 min at a frequency of 50 kHz with a cycle length of 0.65 and amplitude of 85% (UP400S, Hielscher, Germany). The resulting slurry was aliquoted (5 µL) and applied to a copper grid with carbon coating. TEM analysis was conducted using a TEM-2100 (JEOL, Tokyo 196-8558, Japan) . X-ray diffraction (XRD) test was performed using a Bruker D8 Discovers Set (Billerica, MA, USA) to determine the colloidal nature and evaluate the homogeneity and reunify of the synthesis methods for ZnNPs, probiotic L. salivarius , and ZnNCs. The probiotic L. salivarius and ZnNCs were subjected to assessment via Energy Dispersive-X-ray Spectroscopy (EDS) .
Antibiofilm effect A suspension (100 μL) of C. albicans (1 × 10 6 cells/mL) was plated in 96-well microtiter dishes as described earlier and incubated with agitation. The effects of ZnNPs, probiotic L. salivarius , ZnNCs, and antifungal (AMB) on hyphal development and the early phases of biofilm consistence (preventative) were assessed. Free floating cells were removed 1.5 h after adhesion by thoroughly washing each well with 200 μL of PBS. Subsequently, after another 24 h at 37°C with agitation, adhesive cells were exposed to 200 μL of various doses of the test drugs that had been diluted on a separate microtiter dish. The produced biofilm biomass was quantified for ZnNPs, probiotic L. salivarius , ZnNCs, and AMB in comparison to untreated controls. The crystal violet assay was used to quantify the resulting biofilm biomass compared with untreated controls. An ELISA reader (Sunrise R4, Tecan) calculated the OD of the biofilm at 590 nm after the negative control (OD NC ) was set at zero. The negative and positive controls used were sanitized medium only and the working solution, respectively . The rate of biofilm inhibition was measured through the subsequent equation: B i o f i l m i n h i b i t i o n r a t e = 1 − O D 590 o f t r e a t e d c e l l s O D 590 o f n o n t r e a t e d c o n t r o l × 100
Antifungal effect The antifungal efficacy of ZnNPs, probiotic L. salivarius , and ZnNCs was assessed against C. albicans isolates using the agar well diffusion method . In brief, 25 mL of SDA agar was individually inoculated with 100 µL (0.5 × 10 ^3 CFU/mL) of C. albicans isolates. The mixture was then added into a Petri dish and allowed to settle at room temperature for 30 min. Subsequently, a 10 mm diameter well was created in the agar using a sterile corkborer. The wells were supplied with 100 µg of each ZnNPs, probiotic L. salivarius , and ZnNCs. Wells filled with (20 µg) and broth media served as positive and negative controls, respectively. The plates were adjusted for 48 h at 37°C, and the diameters of the inhibition zones were measured using a ruler. The minimum inhibitory concentration (MIC) values of ZnNPs, probiotic L. salivarius , and ZnNCs against C. albicans were calculated by agar well diffusion technique as mentioned above. Different concentrations ranging from 5 to 100 µg/mL of each ZnNPs, probiotic L. salivarius , and ZnNCs were applied to the wells, and the plates were adjusted for 2 days at 37°C. The lowest agent concentration that inhibited germination parallels to the positive control after 48 h of incubation at 37°C was considered the MIC. The test was conducted in triplicate.
Quantitative analysis of biofilm gene expression Transcript expression was used to assess the effect of the tested factors on C. albicans adhesion ( ALS3 ), filamentation ( HWP1 ), the hyphal regulator ( RAS1 ), and hyphal formation and virulence ( SAP4 ), as previously described . A 1 mL suspension of each C. albicans strain, OQ150021 and OQ150022, was transferred to the wells of pre-sterilized 24-well microtiter plates with a flat bottom and incubated at 37°C for 1.5 h under agitation. The wells were washed with PBS. Fresh RPMI 1640 medium (1 mL) containing MIC-50 of ZnNPs, probiotic L. salivarius , ZnNCs, and AMB was added to each well, and the plate was further incubated for 24 h at 37°C. The control wells were used for comparison. The wells were washed twice with PBS and 600 mL of RLT buffer was added to the wells. The plate was incubated for 10 min, and then transferred to 1.5 mL microcentrifuge tubes. For sample homogenization, a 2-min high-speed (30-Hz) tissue lyser was used. For each harvested fungal culture, including untreated fungal culture (control) and the treated fungal culture (ZnNPs, probiotic L. salivarius , ZnNCs, and AMB-treated C. albicans ), one volume was added to one volume of RNA protection reagent at each sampling time (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA isolation was carried out using the QIAamp RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s guidelines. SYBR Green I-based real-time PCR with specific primers for ALS3 , HWP1 , RAS1 , and SAP4 virulence genes was used as previously described . The ITS gene was employed as a housekeeping gene . The PCR reaction contained 12.5 µL of SYBR Green PCR Master Mix (QIAGEN), 0.25 µL of reverse transcriptase (200 U/µL), 0.5 µL of each primer (20 pmoL), 8.25 µL of nuclease-free water, and 3 µL of RNA template. The amplification curves and cycle threshold (Ct) values were determined by Stratagene MX3005P software. The ΔΔCt method was performed, according to .
Scanning electron microscopy analysis The impact of ZnNCs on the morphology and structural integrity of C. albicans biofilm was examined using scanning electron microscopy (SEM) (Jeol 2100, Tokyo, Japan), following established protocols . Initially, 2 mL suspension of C. albicans OQ150021 cells at a concentration of 1 × 10 6 cells/mL in RPMI 1640 was incubated at 37°C for 24 h. Subsequently, the resulting biofilm was formed on sterile plastic coverslips (Nalge Nunc International) placed in 24-well tissue culture plates (Costar, Corning Inc., USA). The biofilm was treated with ZnNCs, washed with PBS, and then fixed in a solution of 0.1 M cacodylate buffer and 2.5% (vol/vol) glutaraldehyde (pH 7.2) overnight. After fixation, the cells were subjected to a series of ethanol washes for dehydration and air-dried in a desiccator. SEM analysis was conducted on the gold-coated samples using a Baltec SDC 050 sputter coater.
Data analysis All statistical tests and data visualization were performed with R software (version 4.2.0). A dendrogram was generated with the “ hclust ” function from the stats package. One-way analysis of variance compared the effects of AMB, ZnNPs, probiotic L. salivarius , and ZnNCs on C. albicans biofilm formation and biofilm gene expression. P -values <0.05 were considered statistically significant.
Results 3.1 Candida spp. isolation and identification Candida spp. were detected in 38 (12.7%) of the specimens, comprising 18 (18%) from Nile tilapia, 7 (7%) from fish wash water, and 13 (13%) from human sellers . Four Candida spp. were isolated as C. krusei , C. glabrata , C. parapsilosis and with C. albicans (42.1%) being the most frequently recovered. Notably, when focusing on hand swabs from human sellers, C. albicans accounted for a high percentage, specifically 42.9%. 3.2 Molecular characterization of C. albicans isolates Biochemically identified C. albicans were molecularly confirmed by detecting the ITS1 gene. C. albicans virulence-associated genes, including RAS1 , HWP1 , ALS3 , and SAP4 , were identified . The most frequently identified gene was ALS3 (81.3%), followed by HWP1 (56.3%), RAS1 (50%), and SAP4 (31.3%). RAPD-PCR analysis of the 16 C . albicans isolates revealed 10 distinct profiles, denoted as R1 to R10 . The power of RAPD-PCR was high, with a D value equal to 0.95. Cluster I included isolates obtained from tilapia, fish wash water, and human sellers . The similarity between the isolates from sellers and those from tilapia and water in clusters I and II was 100%, as determined by the Jaccard coefficient. The ITS region of two representative virulent biofilm-forming C. albicans , obtained from tilapia and human sellers, was sequenced and subsequently listed under accession numbers OQ150021 to OQ150022. Phylogenetic tree analysis revealed that the ITS sequences of C. albicans strains of fish and human sellers were associated with the lineage of other C. albicans from GenBank and were distinctly separated from other Candida spp. . 3.3 Antifungal susceptibility of C. albicans The antifungal resistance patterns of C. albicans isolates to the 8 antifungal drugs are presented in and . Among the isolates, 62.5% exhibited resistance to at least one antifungal agent. Notably, the isolates demonstrated an increase in the degree of resistance to NYS (62.5%), followed by FLZ and ITR (50% each). Conversely, the isolates displayed a high level of susceptibility to AMB (75%). The MAR index ranged from 0.25 to 0.87, with an average of 0.56 . 3.4 C. albicans biofilm formation ability All C. albicans isolates exhibited biofilm-forming capabilities, with 4 (25%) isolates displaying strong biofilm formation . However, the remaining 12 (75%) isolates showed moderate biofilm formation abilities. 3.5 Effects of probiotic L. salivarius , ZnNPs and ZnNCs on C. albicans 3.5.1 Characterization of ZnNPs and ZnNCs ZnNPs were biosynthesized by Aspergillus fumigatus and have a characteristic peak at 45.82 nm , however, ZnNCs had a characteristic peak at 85 nm . Under TEM, ZnNPs exhibited spherical and cubic shapes , while the ZnCNs have sub-rectangular shapes . X-ray diffraction patterns for ZnNPs, probiotic L. salivarius , and ZnNCs are presented in . ZnNPs displayed characteristic peaks at 2θ angles of 35.862°, 39.044°, 43.176°, 54.659°, 69.507°, and 76.012° . Probiotic L. salivarius exhibited characteristic peaks at 2θ angles of 9.145°, 18.398°, 28.305°, 41.484°, 49.805°, 58.560°, 60.231°, 68.803°, and 79.697° . On the other hand, ZnNCs displayed sharp characteristic peaks at 2θ angles of 9.124°, 14.558°, 18.734°, 20.637°, 28.284°, 33.442°, 40.194°, 40.662°, 54.659°, 59.291°, and 68.802°, indicating a cubic lattice of nanocomposites . The elemental composition of the surface of the probiotic L. salivarius and ZnNCs was analyzed using EDS. In the spectrum of probiotic L. salivarius , carbon (55.10%), nitrogen (1.8%), oxygen (31.9%), calcium (9.4%), and silicon (1.79%) were found . In the spectrum of ZnNCs, carbon (49.10%), nitrogen (1.75%), oxygen (27.79%), calcium (9.3%), silicon (1.89%), and zinc (9.35%) were identified . 3.5.2 Antibiofilm effect displays the biofilm inhibition rates of AMB, ZnNPs, probiotic L . salivarius , and ZnNCs on C. albicans isolates recovered from tilapia, fish wash water, and fish sellers at 37°C. ZnNCs exhibited the highest biofilm inhibition rate at 84.4%, followed by ZnNPs. C. albicans treated with AMB displayed a significantly lower biofilm inhibition rate compared to those treated with ZnNPs, probiotic L. salivarius , and ZnNCs. Moreover, a significant difference in biofilm inhibition rate was observed when C. albicans was treated with probiotic L. salivarius and ZnNPs ( P = 0.012) and ZnNCs ( P = 0.0003). On the other hand, no significant variation was found within ZnNPs and ZnNCs ( P = 0.685). 3.5.3 Antifungal effect The antifungal effects of ZnNPs, probiotic L. salivarius , and ZnNCs against C. albicans isolates recovered from tilapia and fish sellers were assessed using the disc diffusion method . AMB, which was sensitive to most of the isolates (75%), was used as a positive control. ZnNPs, probiotic L. salivarius , and ZnNCs exhibited significantly larger inhibition zones than AMB (20 μg) for all C. albicans isolates. Furthermore, the MIC for inhibition of C. albicans growth was 10 µg/mL for ZnNCs and 20 µg/mL for both ZnNPs and probiotic L. salivarius . 3.5.4 Effect on biofilm gene expression displays the particular gene expression patterns of four biofilm-forming genes ( ALS3 , HWP1 , RAS1 , and SAP4 ) in two C. albicans resistant isolates with strong biofilm-forming abilities that were subsequent tested with AMB, ZnNPs, probiotic L. salivarius , and ZnNCs. Treatment with ZnNPs significantly decreased the expression of the four biofilm genes compared to non-tested control isolates. However, ZnNCs showed a complete reduction in biofilm gene expression. 3.5.5 SEM analysis SEM images of biofilm formation by C. albicans (control) and those treated with ZnNCs . Untreated cells displayed a typical mature biofilm with multiple layers of pseudohyphae, blastoconidia and an extracellular matrix forming a dense and heterogeneous polysaccharide network in which cells, pseudohyphae, and hyphae were embedded. Yeast germ tubes, hyphae, bud scars and pseudohyphae are encased in matrix material. The yeast layer and hyphal conglomerate development embedded in the matrix material are not visible and upper layer mainly the hyphal layer is indicated. Conversely, C. albicans treated with 10 µg/mL ZnNCs exhibited the biofilm structure and prevented the formation of matrix material, resulting in an unusual biofilm architecture consisting of a single layer of loosely arranged yeast cells with noticeable morphological alterations, including small shrinkage of blastoconidia, absence of pseudohyphae, and reduction of the matrix layer.
Candida spp. isolation and identification Candida spp. were detected in 38 (12.7%) of the specimens, comprising 18 (18%) from Nile tilapia, 7 (7%) from fish wash water, and 13 (13%) from human sellers . Four Candida spp. were isolated as C. krusei , C. glabrata , C. parapsilosis and with C. albicans (42.1%) being the most frequently recovered. Notably, when focusing on hand swabs from human sellers, C. albicans accounted for a high percentage, specifically 42.9%.
Molecular characterization of C. albicans isolates Biochemically identified C. albicans were molecularly confirmed by detecting the ITS1 gene. C. albicans virulence-associated genes, including RAS1 , HWP1 , ALS3 , and SAP4 , were identified . The most frequently identified gene was ALS3 (81.3%), followed by HWP1 (56.3%), RAS1 (50%), and SAP4 (31.3%). RAPD-PCR analysis of the 16 C . albicans isolates revealed 10 distinct profiles, denoted as R1 to R10 . The power of RAPD-PCR was high, with a D value equal to 0.95. Cluster I included isolates obtained from tilapia, fish wash water, and human sellers . The similarity between the isolates from sellers and those from tilapia and water in clusters I and II was 100%, as determined by the Jaccard coefficient. The ITS region of two representative virulent biofilm-forming C. albicans , obtained from tilapia and human sellers, was sequenced and subsequently listed under accession numbers OQ150021 to OQ150022. Phylogenetic tree analysis revealed that the ITS sequences of C. albicans strains of fish and human sellers were associated with the lineage of other C. albicans from GenBank and were distinctly separated from other Candida spp. .
Antifungal susceptibility of C. albicans The antifungal resistance patterns of C. albicans isolates to the 8 antifungal drugs are presented in and . Among the isolates, 62.5% exhibited resistance to at least one antifungal agent. Notably, the isolates demonstrated an increase in the degree of resistance to NYS (62.5%), followed by FLZ and ITR (50% each). Conversely, the isolates displayed a high level of susceptibility to AMB (75%). The MAR index ranged from 0.25 to 0.87, with an average of 0.56 .
C. albicans biofilm formation ability All C. albicans isolates exhibited biofilm-forming capabilities, with 4 (25%) isolates displaying strong biofilm formation . However, the remaining 12 (75%) isolates showed moderate biofilm formation abilities.
Effects of probiotic L. salivarius , ZnNPs and ZnNCs on C. albicans 3.5.1 Characterization of ZnNPs and ZnNCs ZnNPs were biosynthesized by Aspergillus fumigatus and have a characteristic peak at 45.82 nm , however, ZnNCs had a characteristic peak at 85 nm . Under TEM, ZnNPs exhibited spherical and cubic shapes , while the ZnCNs have sub-rectangular shapes . X-ray diffraction patterns for ZnNPs, probiotic L. salivarius , and ZnNCs are presented in . ZnNPs displayed characteristic peaks at 2θ angles of 35.862°, 39.044°, 43.176°, 54.659°, 69.507°, and 76.012° . Probiotic L. salivarius exhibited characteristic peaks at 2θ angles of 9.145°, 18.398°, 28.305°, 41.484°, 49.805°, 58.560°, 60.231°, 68.803°, and 79.697° . On the other hand, ZnNCs displayed sharp characteristic peaks at 2θ angles of 9.124°, 14.558°, 18.734°, 20.637°, 28.284°, 33.442°, 40.194°, 40.662°, 54.659°, 59.291°, and 68.802°, indicating a cubic lattice of nanocomposites . The elemental composition of the surface of the probiotic L. salivarius and ZnNCs was analyzed using EDS. In the spectrum of probiotic L. salivarius , carbon (55.10%), nitrogen (1.8%), oxygen (31.9%), calcium (9.4%), and silicon (1.79%) were found . In the spectrum of ZnNCs, carbon (49.10%), nitrogen (1.75%), oxygen (27.79%), calcium (9.3%), silicon (1.89%), and zinc (9.35%) were identified . 3.5.2 Antibiofilm effect displays the biofilm inhibition rates of AMB, ZnNPs, probiotic L . salivarius , and ZnNCs on C. albicans isolates recovered from tilapia, fish wash water, and fish sellers at 37°C. ZnNCs exhibited the highest biofilm inhibition rate at 84.4%, followed by ZnNPs. C. albicans treated with AMB displayed a significantly lower biofilm inhibition rate compared to those treated with ZnNPs, probiotic L. salivarius , and ZnNCs. Moreover, a significant difference in biofilm inhibition rate was observed when C. albicans was treated with probiotic L. salivarius and ZnNPs ( P = 0.012) and ZnNCs ( P = 0.0003). On the other hand, no significant variation was found within ZnNPs and ZnNCs ( P = 0.685). 3.5.3 Antifungal effect The antifungal effects of ZnNPs, probiotic L. salivarius , and ZnNCs against C. albicans isolates recovered from tilapia and fish sellers were assessed using the disc diffusion method . AMB, which was sensitive to most of the isolates (75%), was used as a positive control. ZnNPs, probiotic L. salivarius , and ZnNCs exhibited significantly larger inhibition zones than AMB (20 μg) for all C. albicans isolates. Furthermore, the MIC for inhibition of C. albicans growth was 10 µg/mL for ZnNCs and 20 µg/mL for both ZnNPs and probiotic L. salivarius . 3.5.4 Effect on biofilm gene expression displays the particular gene expression patterns of four biofilm-forming genes ( ALS3 , HWP1 , RAS1 , and SAP4 ) in two C. albicans resistant isolates with strong biofilm-forming abilities that were subsequent tested with AMB, ZnNPs, probiotic L. salivarius , and ZnNCs. Treatment with ZnNPs significantly decreased the expression of the four biofilm genes compared to non-tested control isolates. However, ZnNCs showed a complete reduction in biofilm gene expression. 3.5.5 SEM analysis SEM images of biofilm formation by C. albicans (control) and those treated with ZnNCs . Untreated cells displayed a typical mature biofilm with multiple layers of pseudohyphae, blastoconidia and an extracellular matrix forming a dense and heterogeneous polysaccharide network in which cells, pseudohyphae, and hyphae were embedded. Yeast germ tubes, hyphae, bud scars and pseudohyphae are encased in matrix material. The yeast layer and hyphal conglomerate development embedded in the matrix material are not visible and upper layer mainly the hyphal layer is indicated. Conversely, C. albicans treated with 10 µg/mL ZnNCs exhibited the biofilm structure and prevented the formation of matrix material, resulting in an unusual biofilm architecture consisting of a single layer of loosely arranged yeast cells with noticeable morphological alterations, including small shrinkage of blastoconidia, absence of pseudohyphae, and reduction of the matrix layer.
Characterization of ZnNPs and ZnNCs ZnNPs were biosynthesized by Aspergillus fumigatus and have a characteristic peak at 45.82 nm , however, ZnNCs had a characteristic peak at 85 nm . Under TEM, ZnNPs exhibited spherical and cubic shapes , while the ZnCNs have sub-rectangular shapes . X-ray diffraction patterns for ZnNPs, probiotic L. salivarius , and ZnNCs are presented in . ZnNPs displayed characteristic peaks at 2θ angles of 35.862°, 39.044°, 43.176°, 54.659°, 69.507°, and 76.012° . Probiotic L. salivarius exhibited characteristic peaks at 2θ angles of 9.145°, 18.398°, 28.305°, 41.484°, 49.805°, 58.560°, 60.231°, 68.803°, and 79.697° . On the other hand, ZnNCs displayed sharp characteristic peaks at 2θ angles of 9.124°, 14.558°, 18.734°, 20.637°, 28.284°, 33.442°, 40.194°, 40.662°, 54.659°, 59.291°, and 68.802°, indicating a cubic lattice of nanocomposites . The elemental composition of the surface of the probiotic L. salivarius and ZnNCs was analyzed using EDS. In the spectrum of probiotic L. salivarius , carbon (55.10%), nitrogen (1.8%), oxygen (31.9%), calcium (9.4%), and silicon (1.79%) were found . In the spectrum of ZnNCs, carbon (49.10%), nitrogen (1.75%), oxygen (27.79%), calcium (9.3%), silicon (1.89%), and zinc (9.35%) were identified .
Antibiofilm effect displays the biofilm inhibition rates of AMB, ZnNPs, probiotic L . salivarius , and ZnNCs on C. albicans isolates recovered from tilapia, fish wash water, and fish sellers at 37°C. ZnNCs exhibited the highest biofilm inhibition rate at 84.4%, followed by ZnNPs. C. albicans treated with AMB displayed a significantly lower biofilm inhibition rate compared to those treated with ZnNPs, probiotic L. salivarius , and ZnNCs. Moreover, a significant difference in biofilm inhibition rate was observed when C. albicans was treated with probiotic L. salivarius and ZnNPs ( P = 0.012) and ZnNCs ( P = 0.0003). On the other hand, no significant variation was found within ZnNPs and ZnNCs ( P = 0.685).
Antifungal effect The antifungal effects of ZnNPs, probiotic L. salivarius , and ZnNCs against C. albicans isolates recovered from tilapia and fish sellers were assessed using the disc diffusion method . AMB, which was sensitive to most of the isolates (75%), was used as a positive control. ZnNPs, probiotic L. salivarius , and ZnNCs exhibited significantly larger inhibition zones than AMB (20 μg) for all C. albicans isolates. Furthermore, the MIC for inhibition of C. albicans growth was 10 µg/mL for ZnNCs and 20 µg/mL for both ZnNPs and probiotic L. salivarius .
Effect on biofilm gene expression displays the particular gene expression patterns of four biofilm-forming genes ( ALS3 , HWP1 , RAS1 , and SAP4 ) in two C. albicans resistant isolates with strong biofilm-forming abilities that were subsequent tested with AMB, ZnNPs, probiotic L. salivarius , and ZnNCs. Treatment with ZnNPs significantly decreased the expression of the four biofilm genes compared to non-tested control isolates. However, ZnNCs showed a complete reduction in biofilm gene expression.
SEM analysis SEM images of biofilm formation by C. albicans (control) and those treated with ZnNCs . Untreated cells displayed a typical mature biofilm with multiple layers of pseudohyphae, blastoconidia and an extracellular matrix forming a dense and heterogeneous polysaccharide network in which cells, pseudohyphae, and hyphae were embedded. Yeast germ tubes, hyphae, bud scars and pseudohyphae are encased in matrix material. The yeast layer and hyphal conglomerate development embedded in the matrix material are not visible and upper layer mainly the hyphal layer is indicated. Conversely, C. albicans treated with 10 µg/mL ZnNCs exhibited the biofilm structure and prevented the formation of matrix material, resulting in an unusual biofilm architecture consisting of a single layer of loosely arranged yeast cells with noticeable morphological alterations, including small shrinkage of blastoconidia, absence of pseudohyphae, and reduction of the matrix layer.
Discussion Candida spp. are commensal yeasts found on the gastrointestinal tract, skin, and other mucosal surfaces in healthy humans. These opportunistic pathogens are generally harmless to healthy individuals but can potentially lead to invasive diseases in immunocompromised individuals . C. albicans is typically present in the human microbiota and is commonly associated with organic matter. However, this opportunistic disease is known to increase the prevalence of pathogenicity in immunocompromised persons . In our study, Candida spp. were identified in 12.7% of the samples, with 18% originating from tilapia, which aligns with the finding of a previous Egyptian study in which Candida spp. were isolated from tilapia, catfish, and gray mullet . Among the identified Candida spp., C. albicans was the prevalent strain, consistent with findings in previous studies . The high isolation rate of C. albicans can be attributed to its remarkable adaptability, as C. albicans can thrive in vastly different environments characterized by varying nutrient availability, temperature fluctuations, pH levels, osmolarity, and oxygen concentrations . In the current study, at least one of the virulence-associated genes ( ALS3 , HWP1 , RAS1 , and SAP4 ) was identified in all C. albicans isolates. Previous studies in Egypt have reported a higher prevalence of HWP1 and ALS1 genes in C. albicans isolates from patients . Additionally, another study revealed that RAS1 and ALS1 were present in all Candida isolates, while HWP1 and SAP4 were found in half of the isolates . In addition, reported that 53.9% of recognized C. albicans strains had the ALS1 gene and only 5.3% of recognized strains had the HWP1 gene. The variation in the frequency of virulence genes among these studies may be attributed to differences in sample size, source of C. albicans isolates, the host, geographic location, the number of isolates studied, the site from which C. albicans was isolated, sample types, and diagnostic technique used . Antifungal susceptibility testing is a crucial tool for determining clinical responses, facilitating the selection of appropriate antifungal agents, predicting treatment outcomes and studying the epidemiology of drug-resistant C. albicans . Monitoring the emergence of resistant isolates is essential for providing valuable information to clinicians for effective therapeutic decisions . In our current study, AMB is the preferred drug for treating candidiasis in animals, poultry, and humans in Egypt. However, most of the isolates in our study exhibited resistance to NYS, FLZ, and ITR, which consistent with the findings of previous studies . In contrast, reported an increase in resistance (100%) to FLZ, AMB, and FUS, with high susceptibility to ketoconazole. observed a significant increase in FLZ susceptibility in 2005 from 87.5% to 97.4% in 2007. found that all C. albicans isolates were susceptible to NYS, while 94.9% and 97.4% were resistant to FLZ and ketoconazole, respectively. noted that resistance to antifungal agents, ITR, FLZ, and CAS, was 8% for each and 9% for AMB. Furthermore, reported that more than 50% of C. albicans isolates from South Africa and Cameroon exhibited resistance to FLZ. On the other hand, an earlier South African investigation revealed 100% susceptibility of C. albicans to FLZ. Biofilm formation is essential for long-range colonization in host tissues and resistance to external stressors, such as oxidative stress and antifungal factors . Once a biofilm forms, candidal cells are encased in an extracellular matrix that confers resistance to fungicides and various sanitizing techniques . Innovative antibiofilm therapies are urgently needed to address these challenging conditions. In our study, all C. albicans isolates demonstrated biofilm-forming abilities, consistent with the presence of biofilm-associated genes. Biofilm associated genes such as ALS3 , ALS1 , and HWP1 within the ALS family are integral to the adhesion and biofilm formation processes of Candida spp. . These genes encode proteins that are essential for cell wall integrity and play a role in the initiation of hyphal development. ALS3 and HWP1 are cell wall proteins that play roles in intercellular adherence and cell-substrate interactions for proper formation of three-dimensional biofilm architectures . Previous studies in Egypt have reported that 84.6% of C. albicans isolates are capable of forming biofilms , while 58% of C. albicans isolated from intensive care unit patients with nosocomial infections exhibit biofilm-forming abilities . The ability of C. albicans to form biofilms is a major part of most C. albicans infections , necessitating the development of alternative treatment strategies to combat biofilm-associated candidiasis. Recently, there has been an interest in the use of nanoparticles and probiotics to counteract microbial pathogenicity, particularly related to biofilm formation . Various studies have shown the broad-spectrum actions of different probiotics, which can inhibit C. albicans proliferation, adhesion, and biofilm formation, making them valuable antimicrobial agents . L. salivarius exhibits an antifungal effect against Candida spp. through the production of lactic acid and organic acids that positively influence the efficacy of antifungal agents by increasing the permeability of the fungal plasma membrane structure followed by changes in gene expression and the transition to hyphal growth . In addition, L. salivarius showed a decrease in both the number of colonies and the biofilm biomass, as did the cross-linking of the biofilm structure . Therefore, the development of safe antimicrobial probiotics with nanoparticle drug delivery capabilities is essential. Where, we investigated the inhibitory roles of ZnNPs, probiotic L. salivarius , and ZnNCs against C. albicans biofilm formation. Zn-NPs have a long history of use in treating various ailments and have garnered significant interest from the scientific community . These agents have already demonstrated their potency as antiseptic and antimicrobial agents, making them particularly attractive for developing a new class of antimicrobial agents based on nanomaterials . The DLS data showed a single peak at 45.82 nm for ZnNPs. TEM revealed an even distribution without agglomeration and a variety of shapes of ZnNPs in solution. This information may help explain the diversity of molecules involved in ZnNPs production, as these compounds can act as capping and agglomeration-preventing agents . X-ray analysis confirmed the appearance of a distinctive cubic lattice and peaks corresponding to the ZnNPs in a novel probiotic nanocomposite. The nanocomposite of probiotics with ZnNPs was further investigated by EDS surface area analysis, which supported previous findings . The inhibitory effects of nanoparticles can vary depending on their size and concentration . In the present study, ZnNCs demonstrated significantly higher antifungal activity than ZnNPs, probiotics, or AMB. This finding aligns with , who demonstrated a synergistic interaction when two agents are combined, resulting in an inhibitory effect greater than the sum of their individual effects. Another previous study revealed that the combination of FLZ or voriconazole with silver nanoparticles effectively treated drug-resistant C. albicans . The relationship between nanoparticle concentration and fungicidal action depends on the type of fungus . Biofilms are microbial colonization of tightly adherent cells to a surface that is embedded in a polymeric extracellular matrix. ZnNCs disrupted the ultrastructure of C. albicans biofilms due to their strong antifungal activity. Prior research has shown that nanoparticles alter fungal cell walls and membranes . In this study, ZnNCs prevented C. albicans from budding and forming germ tubes, similar to findings with silver nanocomposites . ZnNPs easily bind to biofilms and improve probiotic penetration, disrupting the lipidome. The antibiofilm activity of ZnNPs included inhibiting blastospores and hyphae and preventing Candida biofilm formation . The inhibitory efficacy of probiotics is attributed to bacteriocins and organic acids. Bacteriocins’ positive charged sediment of amino acids creates pores and electrostatic difference in cell membranes, combined with cell lysis. Organic acids in probiotics reduce pH, creating an acidic environment where pathogenic Candida spp. cannot thrive . C. albicans ’ virulence depends on the ability of C. albicans to transition from yeast to hyphae, promoting adherence and biofilm formation. Inhibiting this transition can prevent infection . A gene expression experiment showed that ZnNCs significantly decreased the expression of C. albicans genes, which are implicated in the yeast-to-hypha transition. This downregulation was observed in previous studies, and was associated with delayed or halted infection ( ; A ). In addition, previous studies have shown that the saturation of adhesion sites and coaggregation of Lactobacillus spp. prevent adherence of C. albicans . Gene expression in C. albicans changes due to the presence of Lactobacillus . The expression of genes responsible for adherence and yeast hyphal formation is reduced. The presence of Lactobacillus can alter the host immune response during Candida colonization to attract granulocytes and promote immune defense . Thus, ZnNCs have the potential to target C. albicans ’ virulence factors, suggesting that ZnNCs could be a novel therapeutic approach for treating candidiasis.
Conclusion This study isolated Candida spp. from Nile tilapia, fish wash water, and human fish sellers in retail markets in Sharika Government, Egypt. C. albicans was the most frequently isolated Candida spp., with all isolates having at least one virulence gene, resistance to one antifungal agent, and moderate to strong biofilm-forming abilities, thus posing a significant risk to food safety and public health. The findings demonstrated that ZnNPs, probiotic L. salivarius , and ZnNCs have potential antifungal and antibiofilm activities against C. albicans . Our results suggested that ZnNCs could be promising, cost-effective antifungal drugs for inhibiting key virulence factors and preventing pathogenesis in C. albicans . In addition, ZnNCs demonstrated significantly higher antifungal activity than ZnNPs, probiotics, or AMB. However, further research is essential to determine the full potential of ZnNCs as robust antifungal and antibiofilm agents for managing critical C. albicans infections.
The original contributions presented in the study are included in the article/ . Further inquiries can be directed to the corresponding authors.
The studies involving humans were approved by Institutional Review Board under the number ZU-IRB #11274 -29/11-2023. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. The animal study was approved by Institutional Animal Care and Use Committee (IACUC) of Zagazig University (Ref. No.: IACUC/2/F/234/2023). The study was conducted in accordance with the local legislation and institutional requirements.
NE: Conceptualization, Data curation, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. RA: Conceptualization, Data curation, Investigation, Methodology, Resources, Validation, Writing – original draft, Writing – review & editing. AA: Conceptualization, Investigation, Methodology, Resources, Writing – review & editing. AA: Data curation, Funding acquisition, Resources, Validation, Writing – review & editing. MD: Methodology, Data curation, Investigation, Resources, Software, Validation, Visualization, Writing – review & editing. EY: Funding acquisition, Resources, Validation, Writing – review & editing. RE: Data curation, Investigation, Methodology, Resources, Validation, Visualization, Writing – review & editing. KM: Investigation, Methodology, Resources, Validation, Writing – review & editing. SD: Funding acquisition, Resources, Validation, Writing – review & editing. IE: Data curation, Formal analysis, Software, Validation, Visualization, Writing – original draft, Writing – review & editing.
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Impact of educational intervention on understanding health recommendations after liver transplantation | 6dcaf3c3-ebc1-4886-aeea-2d7fd9fbe012 | 11290744 | Patient Education as Topic[mh] | Health education is considered one of the paths that favors health promotion, through educational interventions that help in the development of individual responsibility and disease prevention. These actions are outlined by health professionals in order to enable the patient not only to understand their health status, but also to enable them to make decisions related to their health care . The nurse can be considered a catalyst for change to promote health, when carrying out educational interventions with patients. Catalyzing changes means creating environments and configurations of educational interventions that strengthen the participation and ownership of individuals, acting as a leader of their own treatment . In nursing, these practices direct individual training, aim to facilitate and develop personal skills, which consequently result in self-care empowerment, in addition to strengthening the bond between patient and professional . Educational intervention, whether individual or in groups, provided in an interactive way promotes learning and represents great potential to promote adherence to the desired behavior in treatment. To achieve this, the chosen educational strategy must be based on empowering the person, in order to make them responsible for managing their health situation, supporting adherence as a focus for the practice of self-care . Learning is defined as the process that allows individuals to modify their behavior . Liver transplantation is a complex surgical procedure that involves permanent care due to the risk of graft rejection. To achieve this, it is necessary to adhere to lifestyle changes, such as strict medication use and monitoring, hygienic habits, oral hydration and preventive measures for infection . After liver transplantation, the individual needs knowledge to change some habits and improve their quality of life. This behavioral change includes adherence to the proposed treatment after the transplant, which involves the new medication regimen, attending appointments with the health team, performing routine physical activity and paying attention to eating habits. It is crucial that patients take responsibility for their treatment. Lack of knowledge for the conduction of the recommendations to be practiced has a negative influence on the process of adherence to treatment, becoming a risk for the evolution of adequate graft functioning.
To evaluate the impact of an educational intervention on understanding health recommendations after liver transplantation. The following hypothesis was tested: Does an educational intervention have a positive impact on patient’s understanding health recommendations after liver transplantation?
Ethical aspects The present study was approved by the Research Ethics Committees from the Federal University of Minas Gerais and Santa Casa in Belo Horizonte, registered in the Brazilian Clinical Trials Registry (ReBEC), under the code RBR-454t52. Study Design This is a randomized and prospective clinical trial. This study followed the Consolidated Standards of Reporting Trials (CONSORT) guidelines for the presentation of clinical trials and was registered in the Brazilian Registry of Clinical Trials (REBEC) under the registration code RBR-454t52. Period, setting, and population The study was carried out, from January 2016 to July 2019, in two general and public hospitals in Belo Horizonte that have a liver transplant program registered in the National Transplant System. Patients aged 18 years old or over, hospitalized for their first liver transplant, with clinical conditions to receive health recommendations for home self-care after hospital discharge, were included. Sample selection and criteria Randomization occurred on the day of transplantation through a draw. Recruitment was carried out by the researcher after the transplant, one day after transferring the patient from the intensive care unit to the hospitalization ward. In the initial approach, patients who agreed to participate in the educational action signed the informed consent form and were included in the study. Informed Consent was obtained from all individuals involved in the study in writing. The sample size, determined using a significance level of 5% and an estimated proportion of 0.5, indicated the need for at least 31 patients/group. In total, 118 patients underwent liver transplantation in these healthcare institutions during the study period. Of these transplant recipients, 68 met the inclusion criteria and were distributed to one of the two groups of the study: those who received health recommendations plus specific recommendations on immunosuppressive drugs, using the patient’s visual, auditory and tactile resources in the educational action, were allocated to the intervention group (IG; n=34), and the control group (CG; n=34) was composed of those who received health recommendations using the patient’s auditory and visual resources in the conventional educational action, consolidated in the institution. shows the CONSORT diagram, with the flow of participants during the study phases. Instruments In the conventional educational action of the CG, an explanatory and validated form was used in health institutions, with consolidated use for hospital discharge of liver transplant patients. For the IG, material prepared by the author was used (educational folder and spiral binding of 15 illustrative figures in JPEG format printed on A3 sheets), not revalidated as it was the reorganization of information from the institutional printout added with attractive colors, figures and details. Intervention The educational action took place at the time of hospital discharge, when each of the transplant patients received the same specific health recommendations for self-care. These were provided in different ways for the IG and the CC. The health recommendations included nutrition, taking medications at the correct times and doses, and not taking the dose before carrying out laboratory serum tests to control the dosage of immunosuppressants. Behaviors to be avoided were also recommended, such as crowding of people, contact with animals, contact with newborns who were immunized with the Sabin vaccine and with sick people. Furthermore, wearing a mask in the first 2 months post-transplant, practicing light physical exercise such as walking, drinking water frequently, applying moisturizing cream daily and attending outpatient appointments on the stipulated dates were also emphasized. The educational action was carried out as follows in the groups: The CG received recommendations for hospital discharge from the nurses who were members of the Nursing Team of the hospitalization ward of the health institutions under study. The material used was an explanatory printout specific to health institutions, containing a page printed on both sides. On the front there were instructions for the medication map, illustrated with medication taking times, and on the back there were written health recommendations, as shown in . The operationalization of the CG’s educational action began with the nurse transcribing the medications prescribed by the medical team into the medication map. Then, the professional went to the patient’s room and carried out the educational action in two moments, as shown in . In the first moment, the patient’s auditory resource was used, with the nurse carrying out a dialogued reading of the recommendations written on the back of the printed form. In the second moment, the patient’s visual aid was used with the delivery of a printed form with a transcribed medication map. The nurse, together with the patient, checked the medications patients’ had in their hands with those prescribed, identified them on the map of transcribed medications and showed the doses to be administered by the patient at home. The IG received the recommendations from the research nurse, with material used that included a printed folder plus a medication map and bound material with illustrative figures corresponding to all health recommendations and specific immunosuppressive medications, according to . The operationalization of the IG’s educational action took place in three moments using the patient’s auditory, visual and tactile resources, according to . The first and second moments took place concomitantly, using the patient’s auditory resources with dialogued reading of the educational folder and visual resource for the patient with presentation of bound illustrative figures referring to the recommendations in the folder. In the third moment, the patient’s tactile resources were used to manipulate the medications in order to recognize them (shape and texture) followed by the creation of the map contained in the folder. With the help of the nurse, the patient transcribed the medications prescribed by the doctor, as well as marking the times and doses to be administered. The professional also helped the patient identify the medications in hand and their location on the map transcribed by him. Data collection Data collection took place 15 days after hospital discharge, in the outpatient clinic, where the patient was interviewed. Two questionnaires prepared by the author were used, the first with information on the sociodemographic and clinical variables described in the literature as relevant (age, gender, city of residence, educational background, years of study, indication for transplant, date of registration on the waiting list, date of transplant and date of hospital discharge), completed by the researcher through an interview and consultation of medical records. The second questionnaire aimed to estimate understanding of the recommendations and was composed of 13 multiple-choice questions, referring to the recommendations received during hospital discharge. This questionnaire was given to the participant to be completed (self-administered) by an external evaluator, who had no knowledge about the educational actions and objectives of the study. For each statement, the interviewee indicated only one answer, such as: totally true, partially true, can’t say, partially false and totally false. The script assessed the patient’s level of understanding from health recommendations received during hospital discharge and experienced for 15 days at home. The total score indicates the number of items that were answered correctly (1 point for each correct answer), making it possible to obtain points from 0 to 13. After summing up the points obtained, an analysis of agreement was carried out between the interviewee’s response and the guidance provided during hospital discharge. The level of understanding was then classified as follows: Excellent, for interviewees who obtained 10 to 13 points, reasonable, for those who obtained 6 to 9 points, poor, for those who obtained 5 to 3 points, and very poor, for those below 2 points. In addition to classifying the interviewee’s level of understanding, there was also an investigation, at the end of the questionnaire, in relation to their opinion on the impact of the recommendations received during hospital discharge, in meeting their needs at home. This occurred through the answer to a perceptual multiple-choice question that classified recommendations received as insufficient, adequate or excellent. Each interviewee received a code to guarantee their anonymity in the study. Data processing and analysis Statistical analysis was performed using the Software Package for Social Science (SPSS) version 20.0 for Mac (SPSS Inc., Chicago, Illinois, USA). Initially, a descriptive analysis of the data was carried out using frequency tables for nominal variables and those related to quantitative variables, which were summarized in the form of mean, median, standard deviation, interquartile and percentages. Then, this Kolmorov-Smirnov was applied to the distribution of quantitative variables. In the second moment, the Chi-square test was performed to compare CG and IG for nominal variables. The Chi-square test compared understanding and detected significant differences between nominal variables. The Student’s T test was performed for data with normal distribution and the Mann-Whitney test for data with non-normal distribution. The results were presented with mean plus or minus (±) standard deviation for numerical variables with normal distribution, and median and interquartile range for numerical variables with non-normal distribution. The data was distributed and related in tables.
The present study was approved by the Research Ethics Committees from the Federal University of Minas Gerais and Santa Casa in Belo Horizonte, registered in the Brazilian Clinical Trials Registry (ReBEC), under the code RBR-454t52.
This is a randomized and prospective clinical trial. This study followed the Consolidated Standards of Reporting Trials (CONSORT) guidelines for the presentation of clinical trials and was registered in the Brazilian Registry of Clinical Trials (REBEC) under the registration code RBR-454t52.
The study was carried out, from January 2016 to July 2019, in two general and public hospitals in Belo Horizonte that have a liver transplant program registered in the National Transplant System. Patients aged 18 years old or over, hospitalized for their first liver transplant, with clinical conditions to receive health recommendations for home self-care after hospital discharge, were included.
Randomization occurred on the day of transplantation through a draw. Recruitment was carried out by the researcher after the transplant, one day after transferring the patient from the intensive care unit to the hospitalization ward. In the initial approach, patients who agreed to participate in the educational action signed the informed consent form and were included in the study. Informed Consent was obtained from all individuals involved in the study in writing. The sample size, determined using a significance level of 5% and an estimated proportion of 0.5, indicated the need for at least 31 patients/group. In total, 118 patients underwent liver transplantation in these healthcare institutions during the study period. Of these transplant recipients, 68 met the inclusion criteria and were distributed to one of the two groups of the study: those who received health recommendations plus specific recommendations on immunosuppressive drugs, using the patient’s visual, auditory and tactile resources in the educational action, were allocated to the intervention group (IG; n=34), and the control group (CG; n=34) was composed of those who received health recommendations using the patient’s auditory and visual resources in the conventional educational action, consolidated in the institution. shows the CONSORT diagram, with the flow of participants during the study phases.
In the conventional educational action of the CG, an explanatory and validated form was used in health institutions, with consolidated use for hospital discharge of liver transplant patients. For the IG, material prepared by the author was used (educational folder and spiral binding of 15 illustrative figures in JPEG format printed on A3 sheets), not revalidated as it was the reorganization of information from the institutional printout added with attractive colors, figures and details.
The educational action took place at the time of hospital discharge, when each of the transplant patients received the same specific health recommendations for self-care. These were provided in different ways for the IG and the CC. The health recommendations included nutrition, taking medications at the correct times and doses, and not taking the dose before carrying out laboratory serum tests to control the dosage of immunosuppressants. Behaviors to be avoided were also recommended, such as crowding of people, contact with animals, contact with newborns who were immunized with the Sabin vaccine and with sick people. Furthermore, wearing a mask in the first 2 months post-transplant, practicing light physical exercise such as walking, drinking water frequently, applying moisturizing cream daily and attending outpatient appointments on the stipulated dates were also emphasized. The educational action was carried out as follows in the groups: The CG received recommendations for hospital discharge from the nurses who were members of the Nursing Team of the hospitalization ward of the health institutions under study. The material used was an explanatory printout specific to health institutions, containing a page printed on both sides. On the front there were instructions for the medication map, illustrated with medication taking times, and on the back there were written health recommendations, as shown in . The operationalization of the CG’s educational action began with the nurse transcribing the medications prescribed by the medical team into the medication map. Then, the professional went to the patient’s room and carried out the educational action in two moments, as shown in . In the first moment, the patient’s auditory resource was used, with the nurse carrying out a dialogued reading of the recommendations written on the back of the printed form. In the second moment, the patient’s visual aid was used with the delivery of a printed form with a transcribed medication map. The nurse, together with the patient, checked the medications patients’ had in their hands with those prescribed, identified them on the map of transcribed medications and showed the doses to be administered by the patient at home. The IG received the recommendations from the research nurse, with material used that included a printed folder plus a medication map and bound material with illustrative figures corresponding to all health recommendations and specific immunosuppressive medications, according to . The operationalization of the IG’s educational action took place in three moments using the patient’s auditory, visual and tactile resources, according to . The first and second moments took place concomitantly, using the patient’s auditory resources with dialogued reading of the educational folder and visual resource for the patient with presentation of bound illustrative figures referring to the recommendations in the folder. In the third moment, the patient’s tactile resources were used to manipulate the medications in order to recognize them (shape and texture) followed by the creation of the map contained in the folder. With the help of the nurse, the patient transcribed the medications prescribed by the doctor, as well as marking the times and doses to be administered. The professional also helped the patient identify the medications in hand and their location on the map transcribed by him.
Data collection took place 15 days after hospital discharge, in the outpatient clinic, where the patient was interviewed. Two questionnaires prepared by the author were used, the first with information on the sociodemographic and clinical variables described in the literature as relevant (age, gender, city of residence, educational background, years of study, indication for transplant, date of registration on the waiting list, date of transplant and date of hospital discharge), completed by the researcher through an interview and consultation of medical records. The second questionnaire aimed to estimate understanding of the recommendations and was composed of 13 multiple-choice questions, referring to the recommendations received during hospital discharge. This questionnaire was given to the participant to be completed (self-administered) by an external evaluator, who had no knowledge about the educational actions and objectives of the study. For each statement, the interviewee indicated only one answer, such as: totally true, partially true, can’t say, partially false and totally false. The script assessed the patient’s level of understanding from health recommendations received during hospital discharge and experienced for 15 days at home. The total score indicates the number of items that were answered correctly (1 point for each correct answer), making it possible to obtain points from 0 to 13. After summing up the points obtained, an analysis of agreement was carried out between the interviewee’s response and the guidance provided during hospital discharge. The level of understanding was then classified as follows: Excellent, for interviewees who obtained 10 to 13 points, reasonable, for those who obtained 6 to 9 points, poor, for those who obtained 5 to 3 points, and very poor, for those below 2 points. In addition to classifying the interviewee’s level of understanding, there was also an investigation, at the end of the questionnaire, in relation to their opinion on the impact of the recommendations received during hospital discharge, in meeting their needs at home. This occurred through the answer to a perceptual multiple-choice question that classified recommendations received as insufficient, adequate or excellent. Each interviewee received a code to guarantee their anonymity in the study.
Statistical analysis was performed using the Software Package for Social Science (SPSS) version 20.0 for Mac (SPSS Inc., Chicago, Illinois, USA). Initially, a descriptive analysis of the data was carried out using frequency tables for nominal variables and those related to quantitative variables, which were summarized in the form of mean, median, standard deviation, interquartile and percentages. Then, this Kolmorov-Smirnov was applied to the distribution of quantitative variables. In the second moment, the Chi-square test was performed to compare CG and IG for nominal variables. The Chi-square test compared understanding and detected significant differences between nominal variables. The Student’s T test was performed for data with normal distribution and the Mann-Whitney test for data with non-normal distribution. The results were presented with mean plus or minus (±) standard deviation for numerical variables with normal distribution, and median and interquartile range for numerical variables with non-normal distribution. The data was distributed and related in tables.
Among the 68 patients who participated in the study, 46 (67.6%) were men and 22 (32.4%) were women. Their median age at transplantation was 56.5 years (IIQ = 63.75). Thirty-seven patients (54.4%) came from the metropolitan region of Belo Horizonte and 31 (45.6%) came from inner cities from the State of Minas Gerais. The average number of years of study was 8.5 (±4.0), with 34 (50.0%) having completed primary school and 34 (50.0%) having secondary education or more. The average hospitalization time (from the day of transplantation to the day of hospital discharge) was 20 days (±12.6), with the median waiting time for an organ being 63.0 days (IIQ = 120 ,0). The indications for transplantation were grouped, resulting in 32 (47.1%) parenchymal liver disease, 19 (27.9%) neoplasia, 5 (7.4%) disease of biliary origin, 3 (4.4%) liver disease, autoimmune origin, 1 (1.5%) fulminant hepatitis and 8 (11.8%) other diseases were combined due to lack of similarity with diseases from previously established groups, namely: 02 Caroli’s disease, 03 Budd Chiari Syndrome, 01 Familial amyloidosis, 01 Alpha 1 Antitrypsin Deficiency and 01 Hereditary hemochromatosis. Regarding the sample’s correct answer rates, the recommendations provided regarding: nutrition, correct use of immunosuppressant medication, functionality of immunosuppressants and recommendation of frequent oral hydration showed more than 80% of correct answers. Hand hygiene and behaviors to be avoided such as crowds and contact with animals expressed less than 10% of correct answers . In the comparative analysis of correct answers between the groups under study, participants from the CG and IG obtained more than 80% correct answers on questions relating to nutrition, correct usage of immunosuppressive medication, function of immunosuppressive medication and oral hydration. The IG participants obtained more correct answers, between 40% and 70%, in the recommendations regarding physical activity, not taking immunosuppressant medication before blood collection and behaviors to be avoided such as: contact with sick people and newborns vaccinated with Sabin, in addition to skin care. The skin care recommendation was answered incorrectly by 40 (58.8%) of the 68 study participants. In the comparison between the groups, only this recommendation showed a statistical difference (p = 0.014), with the most frequent correct answers being in the IG . Regarding the comparative analysis of incorrect answers between the groups, although the CG made more errors when questioning about hand hygiene recommendations and the avoidance of crowds of people, the IG was more frequently wrong about the avoidance of contact with animals, there was no significant difference in responses between the groups studied . Regarding the analysis of agreement between the interviewee’s response to the recommendations provided during hospital discharge of the 68 patients, 53 (77.9%) showed a reasonable level of understanding. 66 of them (97.0%) considered the information received to be excellent and adequate . There was no significant difference in the level of understanding when comparing the study groups (p=0.399) .
The educational action took place with teaching resources and presentation of the material in different ways. This intervention took place during the hospital discharge of patients who underwent liver transplantation, providing an enlightening moment for the patient and their families. The findings of this study draw attention to the varied impact on participants’ understanding, evidenced by responses with extreme rates of errors and successes. A positive impact can be seen from dietary recommendations, frequent oral hydration, functionality and use of immunosuppressants, which stood out due to the high rates of correct answers obtained. It is important to note that these recommendations were experienced by the patient during the hospital stay (which lasted an average of 20 days) and, consequently, were topics addressed daily by the healthcare team. Therefore, prior contact may have facilitated the understanding and maintenance of behaviors at home. However, the recommendation on hand hygiene did not have a relevant impact based on the responses obtained. Possibly, the frequent use of alcohol in the hospital environment may have caused some confusion in the patient about the correct way to wash their hands at home. Changes in the educational action regarding this recommendation during health education may be necessary, to reinforce the habit of washing hands with soap and water, both for the patient and also for family and visitors. During the hospitalization period, the desire for improved health may arise, which causes the patient to create expectations that directly influence their recovery . People are able to store much more information when they see and hear it. Repetition of information is cited as an important form of memorization, which leads us to rethink teaching-learning strategies currently used . Other recommendations, such as avoiding crowds of people and contact with animals (mainly cats, dogs and birds), had no impact, evidenced by lower accuracy rates among participants. It was observed that in these two topics it is also necessary to modify the way of teaching, reinforcing with examples of everyday situations that should be avoided, such as attending religious temples, stadiums, supermarkets, shopping malls, bank branches or sharing a bed or sofa with a domestic animal. It is also necessary to emphasize negative aspects of proximity to animals, such as the possibility of transmitting zoonoses through contact with the animal’s feces and saliva . In the present study, a relevant point was the positive impact on the group that received recommendations using the association of the patient’s auditory, visual and tactile resources, which reached the level of understanding of 82.3% of these participants. In this group, a better understanding of the recommendation for daily skin care with the application of moisturizing cream stands out (p=0.014). In Spain, a study showed that educational measures after liver transplantation through the association of verbal, written and audiovisual resources as a teaching-learning strategy about healthy lifestyle habits were beneficial, corroborating the present study. According to the authors, the association of verbal, written and audiovisual resources in educational action is more effective than just the use of verbal resources . However, the present study detected a deficiency in understanding the skin care routine, since 58.8% of patients answered incorrectly about the daily use of moisturizing cream on their skin. Dermatological care is recommended because organ transplant recipients are at higher risk for infections and skin cancer. The immunosuppressive regimen alleviates about 70% of serious skin infections that appear during the first 3 months after transplantation. It is recommended that solid organ transplant recipients play an active role in preventing skin complications. Educational interventions can result in better information for this specific population and favor immediate treatment, with better clinical results . Regarding the prevention of skin cancer, a study showed that there is a tendency for men not to apply sunscreen to their skin routinely and regularly as self-care. Women use sunscreen more frequently on a daily basis than men . This behavior reveals a problem to be combatted through elaborate campaigns for photo protection, with the aim of reaching the male public more efficiently and encouraging the habit of applying sunscreen . Men seem less concerned about the harmful effects of ultraviolet radiation than women, who wear sunscreen more frequently, probably due to a greater sense of care for their health and aesthetics . Thus, if on the one hand the results pointed to a better understanding of the dermatological self-care of the IG participants, on the other hand, this skin care habit, which differs between genders in our society, draws attention. Knowledge of such behavior relevant for formulating concise, robust interventions targeted at this identified demand. The evaluation of the educational action identified a reasonable level of understanding of the participants in the present study when using teaching material with explanatory and illustrated folders, explanatory printed material, medication map, in addition to the presence of the professional educator (nurse), who used auditory, visual and sensory senses of the patient to perform this action. The nurse plays a fundamental role in implementing these educational actions, which constitute relatively simple and low-cost, yet effective measures . On topics that did not reach adequate levels of understanding, the study was useful in identifying the need for different approaches. The literature suggests the incorporation of the use of technologies for educational intervention, which can be an option used by transplant centers in a useful and effective way . The use of applications for patient education is recent, however the use of messages, videos, images and animations are resources that present effective intervention results in terms of knowledge and care . As it was an educational intervention, it was expected that the educational level could compromise the patient’s understanding of the recommendations provided. The study showed that 77.9% of participants showed reasonable understanding, regardless of their level of education. Other studies corroborate this finding, also pointing out that individual perception and satisfaction are more influential in adherence to treatment than the level of education . This study showed that 69.1% of participants rated the recommendations given as excellent in meeting their needs. Even with the participants’ reasonable level of understanding, this finding reflects the interviewees’ state of satisfaction with the information that supported their home self-care. Study limitations However, it is important to highlight that the present study has as a limitation: the lack of longitudinal results. However, this limitation did not compromise the quality of the evaluation and the results obtained, which aimed to verify the early impact on compliance with the recommendations. Contributions to the field Our most relevant contribution was demonstrating that preparation for patient self-care can be carried out during the hospitalization period, with the participation of the assistant healthcare team, providing opportunities for the experience experienced by the patient and family. We believe that the greater the number of approaches the nurse implements in the patient and family educational process, in its different forms, the better the continuity of self-care will be. However, to more adequately measure this effect, other specific prospective studies must be carried out.
However, it is important to highlight that the present study has as a limitation: the lack of longitudinal results. However, this limitation did not compromise the quality of the evaluation and the results obtained, which aimed to verify the early impact on compliance with the recommendations.
Our most relevant contribution was demonstrating that preparation for patient self-care can be carried out during the hospitalization period, with the participation of the assistant healthcare team, providing opportunities for the experience experienced by the patient and family. We believe that the greater the number of approaches the nurse implements in the patient and family educational process, in its different forms, the better the continuity of self-care will be. However, to more adequately measure this effect, other specific prospective studies must be carried out.
The educational health teaching action, carried out at the time of hospital discharge and focusing on the practice of self-care, achieved a reasonable level of understanding among transplant patients. The educational intervention developed had a positive impact on understanding health recommendations after liver transplantation.
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A mobile revolution for healthcare? Setting the agenda for bioethics | 3b2c50b8-4aec-45eb-acc4-147f7637fd2c | 6173811 | Preventive Medicine[mh] | In April 2015, Apple officially launched the Apple Watch. Already before its take-off, many online fora and media published discussions on how the development of the Apple Watch confirmed the multinational company’s interest to enter the health and wellness market. So it was not a surprise that on the very day the Apple Watch began shipping, already 264 Apple Watch apps related to health or fitness were found in the App Store. These apps (mobile applications) included ‘fitness’ (or workout) programmes, systems for medication adherence, hydration, and fertility and pregnancy tracking systems. As this example suggests, the world of wearable devices has been connected to the world of health and well-being at its very onset. The Apple Watch is only one among many examples in the emerging market of wearable devices, apps and sensors offering health-related services, which are also described as mobile health (mHealth). mHealth is a broad label for a variety of services and technologies supported by mobile devices, such as smartphones, patient monitoring devices, personal digital assistants and other wireless devices to improve healthy behaviours, quality of life and well-being of individuals. An increasing number of websites showcase sleek wearable gadgets with apps continuously capturing body movements, producing graphs of daily calorie consumption or workouts, and providing health advice and self-management tools to chronic patients. Healthy individuals and chronic patients and healthcare professionals are expected targets of the mobile revolution. According to digital health proponents, like cardiologist Eric Topol or ‘medical futurist’ Bertalan Mesko, the healthcare of the future will be powered by digital tools ; market reports, user surveys and policy strategic documents by national and international organisations confirm that the mHealth revolution is on its way. According to a market report, 97 000 mHealth apps were released in 2013, and the expected revenue would be of $26 billion in 2017, while 485 million wearable devices can be expected to be sold in 2018. User uptake also seems to increase: as shown in a 2012 Pew Institute report, 31% of cell phone owners and 52% of smartphone owners have used their phones to look up health or medical information, and 19% of smartphone owners have downloaded an app specifically to track or manage health. More importantly, mHealth receives support from regulatory institutions such as the European Commission, arguing that it could be one of the tools to tackle the challenges faced by European healthcare systems, such as the ageing of the population and increased budgetary pressure. mHealth, as a subset of the broader field of ‘digital health’ (or digital medicine), comes with promises of revolutionising healthcare by increasing patients’ self-management and empowerment, fostering efficiency and disease prevention, and promoting accessibility to health around the globe. The rapid growth of this field has up until now only received little attention from bioethicists. In this paper, first we describe and critically assess the promises of mHealth and their ethical relevance. Subsequently, we offer some suggestions to promote further ethical reflection on this rapidly growing development. The expectation of a ‘mobile revolution’ in healthcare is based on the fact that mobile phones are always in our pockets, portable and increasingly cheap (eg, ref ). According to these narratives, the portability of mHealth systems and the ubiquity of the mobile network allow patients to freely move around while being checked remotely by healthcare providers and monitoring themselves outside the spaces traditionally dedicated to healthcare. This results in a wider range of places for healthcare and a change of roles for patients and for physicians. Mobility is considered revolutionary because it allows for people in rural areas to be as connected and cared for as people in more equipped urban places. Furthermore, although the relatively cheap and easy movement of health data from sites of collection to sites of analysis and decision-making allows for the inclusion of more data-rich and real-life data in care, it also requires to rethink roles and responsibilities in the data interpretation and therapeutic decisions. As it has happened in the past in the fields of nanotechnology, genetics and personalised medicine, mHealth claims to revolutionise healthcare and solve many pressing healthcare challenges. The narrative of a revolution is based on a certain techno-optimism, which rests on the promise that technological inventions are good in themselves. As it has been pointed out in other cases, such claims of healthcare revolutions are rhetorical devices and they need to be assessed carefully with respect to their plausibility before we can engage in a reflection on the new ethical challenges that these technologies arise. At the same time, however, mHealth does indeed initiate new practices in the medical domain which need careful assessment, whether they are novel ethical challenges or more familiar ones. Earlier debates around revolutionising technologies, in fact, have shown that we have to critically assess these far-fetched promises to steer the development of technologies in such a way that we can benefit from its positive effects, but also anticipate downsides and possible risks. In the following, we critically analyse three recurrent promises of mHealth—fostering efficiency and prevention in healthcare, increasing patient self-management and empowerment, and promoting global accessibility to health—showing their ‘rhetorical contradictions’ : not only expectations often do not resonate with their practical use in healthcare, also they are not necessarily as positive as they are presented, but instead raise several ethical issues that need consideration. Efficiency and prevention mHealth is argued to offer efficient and cost-effective solutions for disease prevention, monitoring and management. Take for example an app that uses smartphone cameras to check moles for skin cancer risk (eg, https://skinvision.com/ ): the user holds the device over a spot on her skin and takes a picture, and the app’s algorithm immediately analyses the spot and recommends an action to take (eg, whether the user should go to a specialist) or provides relevant information on skin cancer. This system allows users to archive their skin pictures, keep track of changes over time and share them with their doctor. These types of apps claim to prevent risks of skin cancer by offering a cheap tool for early self-assessment. To live up to these promises, mHealth systems, like other medical interventions, should not harm and should (preferably) benefit users. However, despite the hype around mHealth, there are still many uncertainties around the safety, reliability and accuracy of mHealth systems. Risks include security issues and harms that may derive from potential disclosure of sensitive information to third parties or identity theft, and risks of false results. For example, a skin screening app may not be accurate and might fail to recognise an early-stage melanoma, falsely reassuring rather than alerting the user. Other concerns have been raised concerning the efficiency of these devices: it has been argued that most sensors are unreliable in their signal detections, that it is unclear whether these devices can induce people to change their behaviour towards healthier lifestyles, and that clinical professionals are unaware of how to integrate these systems in their workflow. Moreover, when weighting the benefits and harms of mHealth, one needs to think of which benefits and harms are involved, and to consider who will actually benefit and who will experience the harms from such technologies. Some apps are primarily helpful for patients and may simultaneously help physicians, healthcare personnel or commercial parties to monitor patients, do research or sell products. The benefits and risks may not be evenly distributed among these stakeholders. It is important to ensure that the risks of harm will not predominantly lie at the user/patient side, because they are the more vulnerable actors and may have no understanding of or protection against such harms. In general, despite the promise of providing an effective and efficient tool for disease prevention, the actual benefits of mHealth are still unclear. Although mHealth systems are sometimes presented as a replacement of traditional care, it is unlikely that they will completely replace traditional healthcare. Empirical research has indicated that patients and physicians are happy about the quality of web consultations, but little is known about their safety, feasibility, cost-effectiveness and efficiency. It is not clear in what contexts and situations these tools are the best possible option to meet patients’ care needs: for example, it may not always be desirable, as face-to-face contact can be preferred, more efficient or simply irreplaceable, because of the need for physical examination, sensitivity of the problems, validation of mHealth data or for maintenance of the doctor–patient relationship. Also, more research is needed to understand which types of interventions and monitoring can be left to apps and wearables, and when personal, synchronous care relationship will need to be kept to ensure good care. Self-management and empowerment Another promise is that mHealth will empower patients. Many apps and wearable sensors provide self-management functions for patients or healthy citizens who want to engage in healthy lifestyles. Having access to one’s own health data, without the interference of health professionals, is argued to be a prerequisite to understanding, controlling and managing one’s behaviour, and therefore regarded as empowering patients and supporting their autonomy. An example is offered by the apps provided mymhealth.com for patients with asthma, chronic obstructive pulmonary disease, diabetes and heart disease that enable patients to ‘achieve their goals and self-management’ while being followed by a clinical team (see https://mymhealth.com/ ). The claim that these digital tools empower patients and support their autonomy through self-management should be critically assessed with regard to the degree and types of autonomy that these tools promote. The underlying idea of self-management of such apps induces patients to comply to a strict (medical) regimen rather than enhancing their self-determination. As these devices influence or direct the behaviour of the users, concerns have been uttered that these technologies may be disciplining the users to perform specific medical tasks, rather than truly empowering them. Moreover, as described in the context of governmental surveillance of internet use, the so-called ‘chilling effects’ can make users of mHealth adapt their behaviours when using wearables and apps because of the feeling they are being monitored, rather than because they are themselves motivated to behave in a certain way. This means that mHealth technologies indirectly hinder them to act in the way they would have otherwise, and raise questions concerning the true meaning of the promised patient empowerment. Not only expectations that apps and self-monitoring devices empower patients are deceptive because they promote a specific medical quantified regimen rather than supporting a plurality of self-determined patients’ value , they also seem to assign extra responsibilities to patients. In fact, these apps and wearables delegate some tasks to patients that are traditionally carried out by healthcare professionals (eg, monitoring vital signals, updating symptoms) and continuously demand patients to perform tasks within a certain time frame. This shift of tasks is the flip side of having direct access to health data and falls into the general neoliberal trend of shifting responsibilities from the state (or healthcare providers/experts) to citizens (or individual patients), a trend often referred to as ‘responsibilisation’ (see http://nuffieldbioethics.org/report/personalised-healthcare-2/what-is-personalised-healthcare ). Several concerns have been raised towards such trend and are likely to apply to the field of mHealth : first of all, it may charge patients with additional practical burdens and labour which would otherwise be taken up by healthcare professionals. Also, such burden may cause unduly emotional stress for patients who may feel left alone to their own care by the system. Furthermore, as several empirical studies indicate, merely having access to data does not imply that people have an increased control over their behaviour and health outcomes, as for behavioural change to happen the intervention of a health professional or counsellor is often necessary. Whether the expectations of empowering patients are indeed met or not, an aspect that needs close consideration is how these devices and practices influence the doctor–patient relationship. It is not only a matter of introducing more distance in the doctor–patient interaction via online tools, but also a matter of giving patients tools to control their health and rethinking the roles that doctors have in healthcare. By shifting tasks and responsibilities, proposing specific roles and offering tools to understand health and its correlation with lifestyle, these systems introduce new interactions between care providers and patients. This raises questions on how these relationships will take shape, whether valuable features of traditional relationships will get lost, whether medical training needs to be rethought or patient expectations redirected. Yet another critical aspect related to promises to support patients’ autonomy concerns the confidentiality and control of data. Data collected and processed by wearable sensors and apps may contain sensitive information that is made accessible to several actors, as once such data are obtained they can be reproduced and used endlessly. For example, data may be analysed by the manufacturers for improving the app performance or sold to third parties for research or marketing. Users of such wearables and apps often have limited control over who has access to their data behind the scenes. Third parties’ access to health-related data can be harmful for app users who could, for example, be profiled by marketing agencies and receive targeted unwanted or upsetting advertisements, or incur in discriminatory policies by insurance companies or employers. Although safeguards are in place and users have the right to know what data will be collected, how these will be processed and to whom these will be distributed, this appear to be problematic and complicated in practice in the age of increasing data collection and big data analytics . Just to mention two issues: first, in a context wherein credit card purchases reveal individuals’ lifestyle choices (eg, eating or working out habits) and enable correlations with health conditions through linkages with medical or health data (collected via apps, for example), the distinction between sensitive and non-sensitive data is ambiguous, and the definition of health-related data becomes too broad to be meaningful. Second, traditional safeguards in the clinical context, like practices of informed consent, are inapplicable in a consumer-focused domain where the mediation of healthcare professionals and researchers is shrinking. In an age of ambiguity between commercial and medical domain, confidentiality of health data is constantly challenged, and there is a need to discuss the moral basis of governance models that foster autonomous choice and encourage mHealth users to make an informed decision and control which data remain protected. Wide accessibility to health and social justice Finally, mHealth comes with the promise to make healthcare widely accessible. mHealth is argued to have the potential to bring healthcare to areas that are difficult to reach, or to people who have mobility problems and cannot easily go to the hospital to receive care. An example can be found in a campaign raising health awareness in Bangladesh via SMS (text messages) to mobilise citizens for National Immunisation Day. In the context of this campaign, messages were sent to encourage parents to bring their children to get vaccinated, along with the event’s date. A similar campaign involved pregnant women in remote villages who could register their mobile numbers to receive prenatal advice related to their gestation stage. With the spreading of mobile phone use in developing and rural areas, mHealth is considered a tremendous opportunity to improve healthcare in resource-poor countries in affordable ways. The promise of increasing accessibility to healthcare meets several challenges. For example, mHealth interventions like the ones described above, using SMS (text messages), may not reach those who are most in need of care, either because they are illiterate or local languages are not supported by mobile phones, or because apps require mobile phones with a fast internet connection and some abilities to interact with these phones, thereby excluding certain groups (eg, low-income groups or elderly people with less dexterity with smartphones). These issues raise the question whether the people who actually are in need of better care or improved access to care are currently being served by mHealth technologies. Furthermore, it also indicates a certain hidden normativity in the ways in which services are offered and the expected users. Accessibility-related concerns also play a role in Western countries as users of some of these technologies (eg, iPhones, Fitbit) are on average younger, more educated and wealthier than average. These aspects threaten the promises of accessibility to health and raise questions whether mHealth exacerbates rather than mitigates issues of social justice both at a global and local level by excluding groups from using these services. mHealth is argued to offer efficient and cost-effective solutions for disease prevention, monitoring and management. Take for example an app that uses smartphone cameras to check moles for skin cancer risk (eg, https://skinvision.com/ ): the user holds the device over a spot on her skin and takes a picture, and the app’s algorithm immediately analyses the spot and recommends an action to take (eg, whether the user should go to a specialist) or provides relevant information on skin cancer. This system allows users to archive their skin pictures, keep track of changes over time and share them with their doctor. These types of apps claim to prevent risks of skin cancer by offering a cheap tool for early self-assessment. To live up to these promises, mHealth systems, like other medical interventions, should not harm and should (preferably) benefit users. However, despite the hype around mHealth, there are still many uncertainties around the safety, reliability and accuracy of mHealth systems. Risks include security issues and harms that may derive from potential disclosure of sensitive information to third parties or identity theft, and risks of false results. For example, a skin screening app may not be accurate and might fail to recognise an early-stage melanoma, falsely reassuring rather than alerting the user. Other concerns have been raised concerning the efficiency of these devices: it has been argued that most sensors are unreliable in their signal detections, that it is unclear whether these devices can induce people to change their behaviour towards healthier lifestyles, and that clinical professionals are unaware of how to integrate these systems in their workflow. Moreover, when weighting the benefits and harms of mHealth, one needs to think of which benefits and harms are involved, and to consider who will actually benefit and who will experience the harms from such technologies. Some apps are primarily helpful for patients and may simultaneously help physicians, healthcare personnel or commercial parties to monitor patients, do research or sell products. The benefits and risks may not be evenly distributed among these stakeholders. It is important to ensure that the risks of harm will not predominantly lie at the user/patient side, because they are the more vulnerable actors and may have no understanding of or protection against such harms. In general, despite the promise of providing an effective and efficient tool for disease prevention, the actual benefits of mHealth are still unclear. Although mHealth systems are sometimes presented as a replacement of traditional care, it is unlikely that they will completely replace traditional healthcare. Empirical research has indicated that patients and physicians are happy about the quality of web consultations, but little is known about their safety, feasibility, cost-effectiveness and efficiency. It is not clear in what contexts and situations these tools are the best possible option to meet patients’ care needs: for example, it may not always be desirable, as face-to-face contact can be preferred, more efficient or simply irreplaceable, because of the need for physical examination, sensitivity of the problems, validation of mHealth data or for maintenance of the doctor–patient relationship. Also, more research is needed to understand which types of interventions and monitoring can be left to apps and wearables, and when personal, synchronous care relationship will need to be kept to ensure good care. Another promise is that mHealth will empower patients. Many apps and wearable sensors provide self-management functions for patients or healthy citizens who want to engage in healthy lifestyles. Having access to one’s own health data, without the interference of health professionals, is argued to be a prerequisite to understanding, controlling and managing one’s behaviour, and therefore regarded as empowering patients and supporting their autonomy. An example is offered by the apps provided mymhealth.com for patients with asthma, chronic obstructive pulmonary disease, diabetes and heart disease that enable patients to ‘achieve their goals and self-management’ while being followed by a clinical team (see https://mymhealth.com/ ). The claim that these digital tools empower patients and support their autonomy through self-management should be critically assessed with regard to the degree and types of autonomy that these tools promote. The underlying idea of self-management of such apps induces patients to comply to a strict (medical) regimen rather than enhancing their self-determination. As these devices influence or direct the behaviour of the users, concerns have been uttered that these technologies may be disciplining the users to perform specific medical tasks, rather than truly empowering them. Moreover, as described in the context of governmental surveillance of internet use, the so-called ‘chilling effects’ can make users of mHealth adapt their behaviours when using wearables and apps because of the feeling they are being monitored, rather than because they are themselves motivated to behave in a certain way. This means that mHealth technologies indirectly hinder them to act in the way they would have otherwise, and raise questions concerning the true meaning of the promised patient empowerment. Not only expectations that apps and self-monitoring devices empower patients are deceptive because they promote a specific medical quantified regimen rather than supporting a plurality of self-determined patients’ value , they also seem to assign extra responsibilities to patients. In fact, these apps and wearables delegate some tasks to patients that are traditionally carried out by healthcare professionals (eg, monitoring vital signals, updating symptoms) and continuously demand patients to perform tasks within a certain time frame. This shift of tasks is the flip side of having direct access to health data and falls into the general neoliberal trend of shifting responsibilities from the state (or healthcare providers/experts) to citizens (or individual patients), a trend often referred to as ‘responsibilisation’ (see http://nuffieldbioethics.org/report/personalised-healthcare-2/what-is-personalised-healthcare ). Several concerns have been raised towards such trend and are likely to apply to the field of mHealth : first of all, it may charge patients with additional practical burdens and labour which would otherwise be taken up by healthcare professionals. Also, such burden may cause unduly emotional stress for patients who may feel left alone to their own care by the system. Furthermore, as several empirical studies indicate, merely having access to data does not imply that people have an increased control over their behaviour and health outcomes, as for behavioural change to happen the intervention of a health professional or counsellor is often necessary. Whether the expectations of empowering patients are indeed met or not, an aspect that needs close consideration is how these devices and practices influence the doctor–patient relationship. It is not only a matter of introducing more distance in the doctor–patient interaction via online tools, but also a matter of giving patients tools to control their health and rethinking the roles that doctors have in healthcare. By shifting tasks and responsibilities, proposing specific roles and offering tools to understand health and its correlation with lifestyle, these systems introduce new interactions between care providers and patients. This raises questions on how these relationships will take shape, whether valuable features of traditional relationships will get lost, whether medical training needs to be rethought or patient expectations redirected. Yet another critical aspect related to promises to support patients’ autonomy concerns the confidentiality and control of data. Data collected and processed by wearable sensors and apps may contain sensitive information that is made accessible to several actors, as once such data are obtained they can be reproduced and used endlessly. For example, data may be analysed by the manufacturers for improving the app performance or sold to third parties for research or marketing. Users of such wearables and apps often have limited control over who has access to their data behind the scenes. Third parties’ access to health-related data can be harmful for app users who could, for example, be profiled by marketing agencies and receive targeted unwanted or upsetting advertisements, or incur in discriminatory policies by insurance companies or employers. Although safeguards are in place and users have the right to know what data will be collected, how these will be processed and to whom these will be distributed, this appear to be problematic and complicated in practice in the age of increasing data collection and big data analytics . Just to mention two issues: first, in a context wherein credit card purchases reveal individuals’ lifestyle choices (eg, eating or working out habits) and enable correlations with health conditions through linkages with medical or health data (collected via apps, for example), the distinction between sensitive and non-sensitive data is ambiguous, and the definition of health-related data becomes too broad to be meaningful. Second, traditional safeguards in the clinical context, like practices of informed consent, are inapplicable in a consumer-focused domain where the mediation of healthcare professionals and researchers is shrinking. In an age of ambiguity between commercial and medical domain, confidentiality of health data is constantly challenged, and there is a need to discuss the moral basis of governance models that foster autonomous choice and encourage mHealth users to make an informed decision and control which data remain protected. Finally, mHealth comes with the promise to make healthcare widely accessible. mHealth is argued to have the potential to bring healthcare to areas that are difficult to reach, or to people who have mobility problems and cannot easily go to the hospital to receive care. An example can be found in a campaign raising health awareness in Bangladesh via SMS (text messages) to mobilise citizens for National Immunisation Day. In the context of this campaign, messages were sent to encourage parents to bring their children to get vaccinated, along with the event’s date. A similar campaign involved pregnant women in remote villages who could register their mobile numbers to receive prenatal advice related to their gestation stage. With the spreading of mobile phone use in developing and rural areas, mHealth is considered a tremendous opportunity to improve healthcare in resource-poor countries in affordable ways. The promise of increasing accessibility to healthcare meets several challenges. For example, mHealth interventions like the ones described above, using SMS (text messages), may not reach those who are most in need of care, either because they are illiterate or local languages are not supported by mobile phones, or because apps require mobile phones with a fast internet connection and some abilities to interact with these phones, thereby excluding certain groups (eg, low-income groups or elderly people with less dexterity with smartphones). These issues raise the question whether the people who actually are in need of better care or improved access to care are currently being served by mHealth technologies. Furthermore, it also indicates a certain hidden normativity in the ways in which services are offered and the expected users. Accessibility-related concerns also play a role in Western countries as users of some of these technologies (eg, iPhones, Fitbit) are on average younger, more educated and wealthier than average. These aspects threaten the promises of accessibility to health and raise questions whether mHealth exacerbates rather than mitigates issues of social justice both at a global and local level by excluding groups from using these services. The preceding sections explicate why we need to be critical towards the promises of mHealth and discuss the moral concerns that are inherent to these very promises. But if an ethical reflection on mHealth is needed and if current promises are often misleading, how can bioethicists and medical ethicists approach the topic? In the following, we will outline two suggestions for approaching the ethics of mHealth. From promises to practices When exploring potential implications of mHealth systems, we need to be aware that developers’ websites, explanations or demos are only one side of the story describing the intended use. The way in which these technologies will actually be used in specific contexts by real people is a different matter, but both may be relevant for the ethical assessment of mHealth technologies. Literature on the history of technology and user studies show that there is often a gap between expected technology-driven scenarios and their actual realisation in specific societies. When used in practice technologies often ‘bite back’, having the exact opposite effect in practice to what they were supposed to do. For example, although computers were often introduced in working spaces with the promise of creating a ‘paperless office’, they had the opposite effect as people tended to print the same or even more than usual to store and distribute documents. Mismatches between designers’ expectations of users’ preferences and habits, on the one hand, and actual uses of technologies are very frequent as technologies introduce new burdens, labour and responsibilities that remain invisible at a first sight. An investigation of actual uses is not always feasible (eg, the system is at an early stage of development or only used in few pilot studies, or logistical constraints prevent ethicists from engaging in such fieldwork). However, bioethicists can play a role, already during the development of devices, by uncovering the hidden normativity in the intended use and users, and by exploring possibilities for abuse and unforeseen effects. This type of early analysis can result in an alteration of the design of the device, app or service. To this end, not only the end-product but the criteria that inform different design stages should be explored and critically assessed. Bioethicists can ask questions like: is this particular system configuration going to exclude some users? Shall users be enabled to choose how they want their clinicians to monitor them through an app? These questions can then be discussed with manufacturers and prospective users to design more desirable products. It is therefore important for bioethicists to go beyond the abstract promises and rhetoric of mHealth and situate the ethical analysis in real-world practices and specific contexts. As mHealth is often used as an ‘umbrella term’ to refer to a broad variety of devices with diverse functionalities, it is crucial to establish relevant moral differences among these objects and raise specific normative questions concerning the types of users, places where systems are used, the type of device and practices that develop around it. Beyond safety and effectiveness As the problem of securing sensitive health information and protecting individual privacy becomes crucial in the context of commercial gadgets collecting health data and the question of uploading these data on medical records is explored, most policy discussions around mHealth focus on issues of effectiveness and safety of these devices . While these topics are indeed crucial for the ethical assessment of mHealth as they refer to principles of beneficence and non-maleficence, these are not the only values and principles that are relevant from an ethical perspective. Issues of social justice, for example, should receive more attention. As discussed above, while mHealth has the potential to serve a wider range of people, it might as well exclude a large range of proportion of the population from care altogether. Questions with regard to who has access, who is excluded and whether those in need are actually served and get adequate care require careful analysis and justification, already during the development of such technologies. Some of these questions are not new as mHealth is perpetuating existing injustices of problems with new means. However, digital health, including mHealth, also brings about new ways of exploitation and commodification of data. Many mHealth services have an ambivalent status between lifestyle/well-being gadgets and health-related products, making it opaque how these services should be governed. Data shared on social media, online patient platforms or sold to third parties require ethical scrutiny, for privacy reasons and to protect vulnerable groups from harm and discrimination. Finally, as mentioned above while changing the ways in which healthcare is offered, mHealth affects the relationship between healthcare experts and patients, as well as people’s understandings of self-care and management. The meanings of mHealth services for individuals and collectives should be explored both empirically and conceptually. Bioethicists are exceptionally placed to bring these more conceptual and empirical issues into the public debate and extend the current discourse beyond questions of effectiveness and safety of mHealth. When exploring potential implications of mHealth systems, we need to be aware that developers’ websites, explanations or demos are only one side of the story describing the intended use. The way in which these technologies will actually be used in specific contexts by real people is a different matter, but both may be relevant for the ethical assessment of mHealth technologies. Literature on the history of technology and user studies show that there is often a gap between expected technology-driven scenarios and their actual realisation in specific societies. When used in practice technologies often ‘bite back’, having the exact opposite effect in practice to what they were supposed to do. For example, although computers were often introduced in working spaces with the promise of creating a ‘paperless office’, they had the opposite effect as people tended to print the same or even more than usual to store and distribute documents. Mismatches between designers’ expectations of users’ preferences and habits, on the one hand, and actual uses of technologies are very frequent as technologies introduce new burdens, labour and responsibilities that remain invisible at a first sight. An investigation of actual uses is not always feasible (eg, the system is at an early stage of development or only used in few pilot studies, or logistical constraints prevent ethicists from engaging in such fieldwork). However, bioethicists can play a role, already during the development of devices, by uncovering the hidden normativity in the intended use and users, and by exploring possibilities for abuse and unforeseen effects. This type of early analysis can result in an alteration of the design of the device, app or service. To this end, not only the end-product but the criteria that inform different design stages should be explored and critically assessed. Bioethicists can ask questions like: is this particular system configuration going to exclude some users? Shall users be enabled to choose how they want their clinicians to monitor them through an app? These questions can then be discussed with manufacturers and prospective users to design more desirable products. It is therefore important for bioethicists to go beyond the abstract promises and rhetoric of mHealth and situate the ethical analysis in real-world practices and specific contexts. As mHealth is often used as an ‘umbrella term’ to refer to a broad variety of devices with diverse functionalities, it is crucial to establish relevant moral differences among these objects and raise specific normative questions concerning the types of users, places where systems are used, the type of device and practices that develop around it. As the problem of securing sensitive health information and protecting individual privacy becomes crucial in the context of commercial gadgets collecting health data and the question of uploading these data on medical records is explored, most policy discussions around mHealth focus on issues of effectiveness and safety of these devices . While these topics are indeed crucial for the ethical assessment of mHealth as they refer to principles of beneficence and non-maleficence, these are not the only values and principles that are relevant from an ethical perspective. Issues of social justice, for example, should receive more attention. As discussed above, while mHealth has the potential to serve a wider range of people, it might as well exclude a large range of proportion of the population from care altogether. Questions with regard to who has access, who is excluded and whether those in need are actually served and get adequate care require careful analysis and justification, already during the development of such technologies. Some of these questions are not new as mHealth is perpetuating existing injustices of problems with new means. However, digital health, including mHealth, also brings about new ways of exploitation and commodification of data. Many mHealth services have an ambivalent status between lifestyle/well-being gadgets and health-related products, making it opaque how these services should be governed. Data shared on social media, online patient platforms or sold to third parties require ethical scrutiny, for privacy reasons and to protect vulnerable groups from harm and discrimination. Finally, as mentioned above while changing the ways in which healthcare is offered, mHealth affects the relationship between healthcare experts and patients, as well as people’s understandings of self-care and management. The meanings of mHealth services for individuals and collectives should be explored both empirically and conceptually. Bioethicists are exceptionally placed to bring these more conceptual and empirical issues into the public debate and extend the current discourse beyond questions of effectiveness and safety of mHealth. mHealth is rapidly entering institutional healthcare settings and our private spaces, and is surrounded with shiny promise of revolutionising medicine, but very little is known about their actual use and harms, which is important for the ethical assessment of such innovations. mHealth brings about new challenges and magnifies old challenges by its portability, the changing role of the patient and its market orientation. These changes have moral impact and have to be carefully scrutinised to develop suitable solutions. The visions, promises and the intended use of such technologies do provide an important basis for the ethical evaluation of such technologies, even when real-life evidence is scarce, as these often conceal hidden normativities. But to provide ethical guidance, these promises should also be compared with actual uses and practices. Because the enthusiastic promises may not all come true, and most likely, not for everyone, ethical reflection on mHealth development, promises and practices will help in providing orientation towards desirable pathways and required governance structures. Our paper has indicated that mHealth technologies pose challenges to our ethical reflection. Our analysis has pointed out that we need to carefully reflect on the possible benefits of mHealth of empowering users, being beneficial to patients, reaching rural areas and providing affordable care infrastructure, as these promises show only one side of the coin. mHealth is a container concept featuring many different objects, with a diverse set of functions and potential uses, by a variety of users in different contexts. Such diversity is extremely important for the ethical assessment of such devices. To develop the potential of mHealth to the fullest and implement it in an ethically sound way, ethical reflection should be involved from the early development stages of these devices. This would allow us to learn step by step from mistakes, inaccuracy, gaps between intended and actual use, and the situated use of such technologies. |
Acylcarnitines in Ophthalmology: Promising Emerging Biomarkers | a8ff5ceb-f48e-435e-9364-4f0f3db27d5e | 9784861 | Ophthalmology[mh] | It is well known that vision impairment and blindness have a significant negative effect on the quality of everyday life, with these disabilities posing a significant financial burden and becoming a global public concern . Recently, it has been reported by WHO that, globally, at least 2.2 billion people have a near or distant impairment , whereas two years earlier, the number was up to 1.3 billion . The majority of them are over 50 years of age, although vision loss can affect people of all ages. Fortunately, drug discovery for eye diseases has significantly advanced in the past decade. A variety of valuable ocular therapeutics comprising small molecular weight drugs, fixed dose combination (FDC), gene therapy, ocular sealants, and antibody fragment inhibitors of vascular endothelial growth factor (anti-VEGF) have been developed in the field of ophthalmology . However, despite this progress, there are problems that need to be addressed. Specifically, in early stages, some ocular diseases, for example, the majority of retinal diseases, have no clear symptoms, whereas during the progression of the disease, therapeutic options such as surgery, photocoagulation, or anti-VEGF agents have several limitations. Moreover, there are still pathophysiological mechanisms related to ocular diseases that have not been completely explained. In this context, there is a demand for a better understanding of ocular diseases and the development of novel biomarkers to enable earlier detection and diagnosis, treatment, progression, and prognosis. Metabolomics focuses on the identification and quantification of key metabolites involved in metabolic perturbations related to certain pathophysiological states in a biological system, and it is a powerful tool for investigating the pathogenesis of a disease and the discovery of new biomarkers . With the development of precision medicine, metabolomic analysis has been applied in ocular-derived matrices, and some interesting reviews have been recently published . According to the literature, the metabolic profile of eye biospecimens under normal or several pathological conditions has been studied, and several metabolites have been identified to be involved in these. For example, taurine has been found to exhibit a protective effect against mitochondria-related metabolic impairments in the retinal pigment epithelium , whereas nicotinamide has shown to have neuroprotection on glaucoma . Carnitines are a class of metabolites that have been recently shown to be related to ocular diseases . Nevertheless, most clinical studies involving metabolomics studies are still preliminary and show important limitations. Based on the above and on the fact that eyes are among the most metabolically active organs in the body and that several ocular diseases can be caused by metabolic dysfunction , and also given the important biological role of carnitines in numerous metabolic functions, we aimed to focus our study on the role of acylcarnitines in ocular diseases and investigate their potential as early diagnostic or prognostic biomarkers of pathological states leading to vision impairment. Thus, in this review, we present the progress that has been made regarding the application of carnitine derivatives as potential biomarkers in common ocular diseases. Herein the importance of the role of carnitines in human health are discussed and, thereafter, their connection with ocular diseases and the recent advances that have been made in the identification of carnitines as metabolites in common eye diseases. Finally, the conclusions and future directions are discussed.
A PubMed/MEDLINE, Google Scholar and SciFinder literature search for English language articles was performed using the terms “retinal disease”, “age-related macular degeneration”, “diabetic retinopathy”, “retinopathy of prematurity”, “glaucoma” AND “metabolomics” OR “carnitine metabolite” OR “acylcarnitines” OR “metabolic profiling”. The abstracts were reviewed and, among them, the relevant articles were retrieved for comprehensive evaluation. References cited in selected articles were also reviewed to identify additional relevant reports.
L-carnitine is a quaternary ammonium compound known as L-3-hydroxy-4-aminobutyrobetaine or, alternatively, as L-3-hydroxy-4- N- trimethylaminobutanoic acid . It is also known as levocarnitine and vitamin BT. L-carnitine plays important role in physiological reactions throughout the body, including sugar aerobic metabolism and oxidative phosphorylation. In addition, L-carnitine has antiapoptotic, antioxidative, and osmoregulatory properties, which may be useful in the treatment of ocular pathologies. Being an essential metabolite, carnitine is absorbed from the diet, but it is also synthesized in the kidney, liver, and brain. It transports fatty acids across the mitochondrial membrane to undergo β-oxidation and produce energy . In animal tissues, L-carnitine concentrations are relatively high, typically between 0.2 and 6 mmol/kg, with almost being all in the heart and skeletal muscle . While carnitine levels in the human ocular tissue are unknown, animal studies indicate that carnitine is differentially distributed within the eye with the highest concentrations are reported in the iris, ciliary body, and the choroid-retina . Carnitine acyl esters, known as acylcarnitines, are essential for the oxidative catabolism of fatty acids and, consequently, for maintaining energy homeostasis in the human body . Acylcarnitines are characterized as short, medium, and long chain depending on the size of the acyl group . Very recently, a useful and extensive review of acylcarnitines has been published by Damprova et al. . The authors provide a detailed description of acylcarnitines’ identity, nomenclature, classification, biochemistry, pathophysiology, supplementary use, potential drug targets, and clinical trials. They also summarized these updates in the Human Metabolome Database, which now includes information on the structures, chemical formulae, chemical/spectral properties, descriptions, and pathways for 1240 acylcarnitines. Carnitine and its derivatives have been found to be related to a great variety of diseases. For example, recently they have been studied as a therapy or protective agent for many neurological diseases and neurotoxicity , while it has also been shown that some analogues are related to cardiovascular events . Furthermore, a decreased level of hydroxydecanoylcarnitine and methylglutarylcarnitine has been suggested to be associated with the risk of metabolic syndrome , whereas lauroylcarnitine has been implicated as a mediator of obesity-induced inflammation . Moreover, the circulating levels of bile acids and carnitine are differentially altered in patients with primary biliary cirrhosis . Recently, the US Food and Drug Administration approved L-carnitine, along with short-chain acylcarnitines (acetylcarnitine and propionylcarnitine as a dietary supplement) .
Over the last decade, several metabolomic studies regarding ocular health and disease have appeared in the literature. These studies have highlighted several metabolite species playing a role in the onset or progression of such conditions. In these cases, either targeted or untargeted metabolic profiling methodologies have been followed to study blood samples and, in fewer cases, ocular-derived specimens, such as aqueous humor, vitreous humor, and tears. Based on an untargeted approach, several classes of molecules have been identified as important to these conditions, such as amino acids, carbohydrates, phospholipids, and nucleotides, but also L-carnitine and various short-, medium- and long-chain acylcarnitines. In the cases where targeted metabolic profiling is followed, a set of preselected acylcarnitine species are measured in the samples. The preferable analytical technique is mass spectroscopy (MS) or tandem mass spectrometry (MS/MS), which, in most cases, are applied in hyphenation to liquid chromatography (LC-MS or LC-MS/MS) . It is well known that MS identifies metabolite species on the basis of their mass/charge ratio ( m/z ), whereas MS/MS allows for the use of two or more stages of mass analysis to focus on the fragmentation of an ion within a mixture in order to enhance resolution and accuracy. Separation of the analyte via LC is used to facilitate the identification and quantification of the analyte. There are, however, cases where direct infusion to MS is applied (FIA-MS, or FIA-MS/MS) without prior chromatographic separation. Due to the fact that carnitines contain a hydrophobic chain and a charged moiety, both reverse phase (RP) and hydrophilic interaction liquid chromatography (HILIC) have been used . Nevertheless, the findings suggest that carnitine and its derivatives are highly implicated in common ocular dysfunctions. summarizes the studies that imply untargeted or targeted metabolic profiling and that have indicated carnitine and/or its derivatives as biomarker of ocular diseases, while these are discussed in the following sections. A comprehensive scheme illustrating all the ocular diseases that have been reported to be related to carnitine metabolism is given in . 4.1. Age-Related Macular Degeneration (AMD) AMD is a disease that affects central vision progressively and is the major cause of visual impairment in people over 60 years of age. It constitutes 8.7% of worldwide total blindness, affecting approximately 150 million globally, and this number is projected to incline to 288 million by 2040 . AMD is classified mainly as early stage (small-medium drusen deposits), intermediate (larger drusen deposits and/or retinal pigmentary changes), and late AMD. The latter is subdivided into two categories: (i) geographic atrophy or dry AMD, characterized by a gradual degeneration of retinal cells, and (ii) neovascular or exudative or wet AMD, in which new choroidal blood vessels are formed and proliferate, leading to exudation, retinal edema, hemorrhages, and central vision loss . A combination of risk factors, including age, genetics, and environmental factors, such as tobacco use, a low dietary consumption of antioxidants, and hypercholesterolemia, leads to AMD . Although several factors associated with the development of AMD have been identified, the etiology and the pathogenesis of the disease have not been fully delineated. Moreover, the interactions among cells and the exchange of metabolites between the retina, the retina pigment epithelium, and the choroid complex highlight the significance of finding metabolic local and systemic biomarkers which are indicative of the disease and improve the outcome. Therefore, AMD progression is related with metabolic dysfunction, and alterations in the level of metabolites, such as carnitines, might act as a diagnostic biomarker for its development. Recently, a metabolomic study conducted by Mitchell et al. compared metabolite levels in plasma samples obtained from patients with intermediate AMD (IAMD), neovascular AMD (NVAMD), and non-AMD controls . The aforementioned levels of metabolites among all groups were compared. By using a liquid chromatography-mass spectrometry (LC-MS) platform to analyze samples, and partial least-squares discriminant analysis (PLS-DA) and linear regression (LR) to identify discriminatory metabolic features, six acylcarnitines were found to meet the significance criteria. Among them, two carnitines (specifically linoleyl and linolenyl carnitine) were nearly 1.5-fold (1.63 and 1.49, respectively) higher in AMD patients compared to disease-free controls. Four carnitines (heptadecanoyl carnitine, 11Z-octadecenylcarnitine, glutaconylcarnitine, and stearoylcarnitine) were between 1.7- and 2.10-fold (2.08, 1.74, 1.75, 1.83, respectively) higher in NVAMD patients compared to IAMD patients. It should be mentioned that five of the acylcarnitines belong to medium- or long-chain acylcarnitine species. Furthermore, seven additional discriminatory features were identified with a medium or high confidence match with acylcarnitines, which presented higher levels along with more advance disease. Regarding metabolic pathway analysis, the carnitine shuttle pathway was the one that was significantly altered in all comparisons (AMD vs. control, IAMD vs. control, NVAMD vs. control, and NVAMD vs. IAMD). This study reveals the fact that plasma levels of certain acylcarnitines were not only higher in AMD patients and NVAMD patients compared with controls, but also in NVAMD patients compared with IAMD patients . Furthermore, Mitchel et al. previously reported a metabolomic analysis of plasma samples obtained from NVAMD patients and healthy controls. Among the plethora of metabolic features analyzed, 39 of them were annotated with confidence, from which multiple were carnitine species. Using LC-MS/MS analysis, the identity of five acylcarnitine intermediates was confirmed. The plasma levels of the aforementioned five long-chain acylcarnitines (9-hexadecenoylcarnitine, heptadecanoylcarnitine, 11Z-octadecenylcarnitine, L-palmitoylcarnitine, and stearoylcarnitine) were significantly higher, approximately 2-fold (2.17, 2.16, 2.03, 1.95, and 1.65, respectively), in NVAMD patients compared to healthy controls. Furthermore, metabolite pathway analysis showed that the carnitine shuttle pathway was significantly changed in NVAMD patients . Two more case–control studies have addressed altered plasma acylcarnitines in AMD patients. The first study, conducted by Luo et al., was an untargeted metabolomic study. It compared metabolomic features in the morning plasma of Chinese patients with wet AMD and healthy controls. By using ultra-high-pressure liquid chromatography and quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF MS) to detect metabolic differences and multidimensional statistical methods to analyze samples, 10 metabolites were identified to differ significantly between the two groups of patients. Among them, L-palmitoylcarnitine was found to be lower in the group of patients with wet AMD (fold change 0.79) (variable importance for projection (VIP) >1, p < 0.05) . The second case–control study by Chao de la Barca et al., which was based on a targeted metabolomics approach, compared plasma samples from patients with exudative/wet AMD and healthy controls. Among the 188 metabolites that were analyzed, only six remained statistically significantly altered after Benjamini–Hochberg correction, and two of them were carnitines, specifically free L-carnitine (C0) and valerylcarnitine (C5). The concentration of both carnitines was statistically increased in the plasma of AMD patients compared to controls and, in fact, valerylcarnitine was approximately 1.4 more concentrated in the blood of AMD individuals . In another study conducted by Han et al., metabolomic changes in the aqueous humor of patients with wet AMD, in comparison to controls without AMD that underwent cataract surgery, were investigated. The untargeted metabolomics study, performed with the use of UHPLC-MS/MS and univariate analysis, showed 18 statistically significantly altered metabolites. Among them, two carnitines were identified, namely free L-carnitine (C0), which was decreased in the wet AMD group (approximately 0.72-fold change), and deoxycarnitine, which was increased in the patient AMD group (almost 1.87-fold change) compared to the control group. In addition, since the aforementioned molecule is the precursor substrate for carnitine biosynthesis, it should be mentioned that 6-N-trimethyl-L-lysine (TML) was significantly higher in the aqueous humor of AMD patients (1.84-fold change) compared with non-AMD controls. Therefore, alterations in free L-carnitine, deoxycarnitine, and TML suggest a disturbance in the carnitine pathway associated with mitochondrial dysfunction and fatty acid metabolism, possibly contributing to the pathogenesis of AMD . 4.2. Diabetic Retinopathy (DR) DR, being a common complication of diabetes mellitus (DM), constitutes the main cause of blindness in the adult, working-age population . It affects approximately 103 million patients worldwide, and the number is estimated to rise to 160.5 million patients in 2045 . The disease leads to visual impairment, having a devastating impact on patients’ quality of life . DR is categorized mainly into two stages: non-proliferative DR (NPDR), characterized by microaneurysms, haemorrhages, cotton-wool spots, and intra-retinal microvascular anomalies (IRMAs); and proliferative DR (PDR), which is characterized by neovascularization and/or vitreous haemorrhage . The risk factors for DR include poor glycemic control and, thus, hyperglycemia, as well as hypertension and hyperlipidemia . Therapeutic strategies, such as anti-VEGF injections, are limited and the understanding of the DR pathogenesis is still incomplete. Therefore, further investigation should be undertaken and new metabolites, such as carnitines, could play an important role in the early detection of the disease and the optimization of patient care. In a recent study conducted by Sumarriva et al., metabolic profile differences of plasma samples between patients with type 2 DM, with DR and without DR (diabetic controls), and between patients with PDR and NPDR have been shown. LC-MS analysis was performed; features were selected using PLS-DA (VIP ≥ 1.5) and were significantly associated ( p < 0.05) in a Wilcoxon rank sum test. Several metabolic features were found to differ significantly between DR patients and diabetic controls, four of which constituted key contributors to the pathways analysis differences detected in the study; dehydroxycarnitine was among them and its concentration was found to be significantly increased in DR patients ( p = 0.0069). Regarding PDR, pathway analysis revealed alterations in the β-oxidation of saturated fatty acids ( p = 0.032), as well as fatty acid metabolism (0.038). Carnitine, being a key molecule in the metabolism and oxidation of fatty acids in mitochondria, was found to be statistically significantly increased in PDR patients in comparison with NPDR patients . Furthermore, Yun et al. performed a metabolomics analysis in serum samples obtained from patients with type 2 DM. Specifically, patients were divided into three groups, i.e., NDR, NPDR, and PDR. Metabolites were quantified with the use of LC-MS and FIA-MS, and statistical analysis was performed with R software. The concentrations of several carnitines were found to be significantly different among groups and they could act as potential disease markers. In fact, propionylcarnitine (C3) and butyrylcarnitine (C4) showed significantly higher concentrations in the DR patient group compared with the NDR group (a 1.16- and 1.31-fold change, respectively), while the concentrations of dodecanoylcarnitine (C12), tetradecenoylcarnitine (C14:1), tetradecadienylcarnitine (C14:2), hexadecanoylcarnitine (C16), octadecenoylcarnitine (C18:1), and octadecadienylcarnitine (C18:2) were lower in the DR group (0.89-, 0.87-, 0.87-, 0.86-, 0.87-, and 0.87-fold change, respectively). Additionally, comparing serum samples from NPDR and NDR patients, the concentrations of three acylcarnitines, namely L-carnitine (C0), tetradecenoylcarnitine (C14:1), and hexadecanoylcarnitine (C16), were found to be lower in the NPDR group (0.96-, 0.88-, and 0.87-fold change, respectively). Comparing the concentrations of metabolites between PDR and NDR patients, three short-chain acylcarnitines (propionylcarnitine (C3), butyrylcarnitine (C4), and valerycarnitine (C5)) showed statistically higher concentrations in PDR patients (1.25-, 1.63-, and 1.23-fold change, respectively), while five long-chain acylcarnitines, tetradecenoylcarnitine (C14:1), hexadecanoylcarnitine (C16), octadecanoylcarnitine (C18), octadecenoylcarnitine (C18:1), and octadecadienylcarnitine (C18:2), showed lower concentrations in the PDR group (0.84-, 0.82-, 0.83-, 0.81-, and 0.81-fold change, respectively). Moreover, among the metabolites that statistically differed between PDR and NDPR patients, the medium-chain pimelycarnitine (C7:DC) was found to be elevated in the PDR group, and its concentration was reported to be 1.10-fold higher. Finally, the concentrations of sixteen metabolites showed differences when comparing both PDR and NPDR patients to NDR patients. Two acylcarnitines, namely tetradecenoylcarnitine (C14:1) and hexadecanoylcarnitine (C16), were included among them, and their concentrations were lower in the PDR and NPDR groups than in the NDR group (0.87- and 0.86-fold, respectively) . Interestingly, in a prior study by Paris et al., the content of metabolites in vitreous humor samples from patients with PDR and non-diabetic controls were compared. Initial analysis was performed in a first set of patients, and afterwards, the findings were validated through the analysis of a second set of patient samples. The concentrations of two medium-chain carnitines, specifically decanoylcarnitine (C10) and octanoylcarnitine (C8), were increased in the group of PDR patients compared to non-diabetic controls in both sets of patients (C10 with 1.7- and 1.4-fold changes and C8 with 2.2- and 1.7-fold changes in the first and second set of patients, respectively) . 4.3. Retinopathy of Prematurity (ROP) ROP is a disease characterized by immature retinal vascular development and the presence of retinal ischemia and neovascularization, which can lead to retinal detachment and vision loss. Both prematurity and low birth weight are factors strongly related to a high risk of the disease . It should be mentioned that ROP affects almost 100,000 children globally, according to an epidemiological study . Indeed, the incidence and the severity of ROP is still high, and the pathogenesis is not yet fully understood. Therefore, metabolites, such as carnitines, could act as potential biomarkers and contribute to the early diagnosis and better understanding of the underlying mechanisms of the disease. Recently, Yang et al. conducted a study based on a targeted metabolomics analysis of blood from premature infants, comprising two groups of cases: ROP patients and non-ROP controls . UPLC-MS targeted analysis was applied for the measurement of malonylcarnitine. Subsequently, standard multivariate and univariate analysis was performed for interpretation of the results. Malonyl carnitine (C3DC) was found and confirmed to be statistically significantly different ( p < 0.001) between ROP and non-ROP infants. C3DC constituted an independent strong risk factor for ROP, since the higher the value of the specific acylcarnitine, the higher the risk of ROP. Although a probable association is revealed between C3DC and the risk of ROP, this is not the case for the severity of the disease. In addition, the predictive ability of C3DC for ROP diagnosis was the best among the other discriminant metabolites (area under the curve (AUC) = 0.914, sensitivity = 97.5%, and specificity = 68.3%), showing that C3DC could be a potential biomarker for the diagnosis of ROP . 4.4. Central Retinal Vein Occlusion (CRVO) CRVO is a common disease of the retinal vasculature which may lead to visual loss, usually unilateral . It is estimated to affect approximately 0.1–0.5% of the population. Its pathogenesis is associated with atherosclerosis of the central retinal artery, which is situated adjacent to the vein within the same adventitia, compressing and inducing stasis and thrombosis in the vein lumen, and leading to CRVO . In this condition, there is an increase in intravenous hydrostatic pressure and a nonperfusion of the retinal capillaries, leading to a decrease in oxygen supply. The aforementioned alterations in the retinal blood flow result in ischemia and hypoxia-induced metabolic dysregulation . Therefore, changes in the concentrations of metabolic substances and dysfunction of several pathways, among which is the carnitine shuttle pathway, could reveal the pathological mechanisms and the progression of the disease. A study conducted by Wei et al. performed a metabolomic analysis of the aqueous humor obtained from fifteen patients with CRVO and twenty patients who underwent cataract surgery (controls). UHPLC-MS/MS was used to identify the involved metabolites . Butyrylcarnitine ( p = 0.003) and deoxycarnitine ( p value not reported) were found to be elevated and among the statistically significant metabolites in CRVO group. Furthermore, 6-N-trimethyllysine (TML), being the compound from which the biosynthesis of carnitines starts, was significantly altered in CRVO patients as well. A speculation that was suggested is that the dysregulated carnitine metabolism pathway might be involved in mitochondrial dysfunction in CRVO patients; however, regarding the pathogenesis of CRVO, any exact involvement of such metabolomic pathways is still unknown. 4.5. Primary Open-Angle Glaucoma (POAG) POAG represents the most common form of glaucoma and it is a group of chronic optic degenerative neuropathies characterized by progressive impairment of retinal ganglion cells and visual field loss without an identifiable cause . The risk of developing POAG is related to many factors, among which the most prominent one is elevated intraocular pressure (IOP) . POAG affects approximately 57.5 million people and is the leading cause of irreversible blindness globally; it is projected to reach 111.8 million cases in 2040 . Due to the slow and asymptomatic progression of the disease in the early stages, the diagnosis of POAG often does not occur until later when severe field damage and loss of central fixation are established . Therefore, the asymptomatic nature of POAG, combined with the worldwide prevalence of the disease and its estimated dramatic increase in the ageing population in the following years, necessitate the need of discovering new strategies and possible systemic or topical biomarkers, such as carnitines, for an early stage accurate diagnosis. Two studies compared the metabolic profiles of plasma taken from patients with POAG and from controls. In the first study conducted by Burgess et al., LC-MS was used for metabolic profiling analysis of plasma samples, and further statistical analysis of the data revealed several differentially expressed metabolites associated with POAG. Among the aforementioned metabolites palmitoylcarnitine (C16), a long-chain acylcarnitine, was found to be significantly elevated in the plasma of POAG patients in comparison with controls ( p = 3.69 −10 ) . It is well known that palmitoylcarnitine is part of the carnitine shuttle pathway, functioning for the transport of fatty acids for β-oxidation in the mitochondria. The aforementioned findings are in accordance with an earlier study regarding the deficiency of carnitine palmitoyl transferase II and its connection with normal tension glaucoma . However, since various factors could be related to the palmitoylcarnitine metabolism in the mitochondria, the elevated levels of palmitoylcarnitine in the plasma of POAG patients might be part of a more generalized defect of mitochondrial function and not specifically associated with POAG . Similarly, in the second study, Leruez et al. compared the metabolic profiles of plasma samples obtained from patients with POAG and controls with cataract. FIA-MS was used for the analysis of metabolites, and statistical analysis revealed 18 significantly altered metabolites between the two groups, six of which were identified to be carnitines. More specifically, univariate statistical analysis (with the use of the Wilcoxon test) showed an increase in propionylcarnitine (C3) (fold change = 1.30, p = 0.0036) in the POAG group in comparison to controls, while octadecenoylcarnitine (C18:1) was decreased (fold change = 0.76, p = 0.0017) in POAG patients. In addition, multivariate analysis (with the use of the least absolute shrinkage and selection (LASSO) operator) showed an elevation in the concentration of butyrylcarnitine (C4), decanoylcarnitine (C10:1), and dodecanoylcarnitine (C12:1) in the group of POAG patients compared with cataract controls (fold change and p value are not reported for these metabolites). Both univariate and multivariate analysis identified octadecadienylcarnitine (C18:2) to be statistically significantly increased (fold change = 0.82, p = 0.00068) in the POAG group compared with controls . The alterations in acylcarnitine concentrations delineate a perturbation in the fatty acid metabolism and are in accordance with the study of Burgess et al., where the long-chain palmitoylcarnitine was found to be increased. In addition, Rong et al. revealed five long-chain free fatty acids which were statistically significant in PACG patients but not acylcarnitines, and Mayordomo-Febrer et al. found elevated free fatty acid concentrations in the aqueous humor of their animal model (rats) of glaucoma . Taking into consideration the significant role of carnitines in the transport of fatty acids in the mitochondria and the inherited disorders of fatty acid oxidation, it could be presumed that when the oxidation of fatty acids is incomplete, this results in a reflux from mitochondria into the blood stream of smaller acylcarnitines, which may be the cause of increased acylcarnitine levels . Furthermore, the concentration of two long-chain acylcarnitines was found to be lower in POAG patients, which might be part of the ageing process, as it has been shown to decline with age in the blood of mice . A recent study conducted by Buisset et al. compared the metabolomic profiles obtained from the aqueous humor of POAG patients with that of controls undergoing cataract surgery. Again, FIA-MS was used to identify metabolites, and multivariate and univariate statistical analysis was performed to discriminate metabolites between the two groups. Among the metabolites which were found to be discriminant using multivariate analysis, three short-chain acylcarnitines, namely acetylcarnitine (C2), propionylcarnitine (C3), buryrylcarnitine (C4), and free carnitine (C0), were included. The concentrations of the aforementioned carnitines were statistically higher in the aqueous humor of POAG patients compared to controls. Additionally, univariate analysis after correction for the false discovery rate (FDR) demonstrated that C2 and C3 were significantly higher than other metabolites. Metabolites, including carnitines, were ranked based on importance using the median value of VIP, as well as the adjusted p-value (C3: VIP = 1.56, p = 0.013; C2: VIP = 1.52, p = 0.029; C0: VIP = 1.32, p = 0.067; C4: VIP = 1.11, p = 0.087) . The study by Buisset et al. was the first study to show elevated concentrations of short-chain carnitines (C2, C3, C4) and free carnitine in the aqueous humor of POAG patients. Furthermore, carnitine has shown neuroprotective properties, as well as antioxidant and antiapoptotic properties, in retinal cells in the eyes of mice with high intraocular pressure . Therefore, an increase in the concentration of carnitine could be involved in the protection of retinal cells in the case of disease-related stress and production of reactive oxygen species. Additionally, the lack of long-chain acylcarnitines in the metabolic profile of the aqueous humor of POAG patients is not in favor of mitochondrial oxidation impairment in the cells obtained by the aforementioned fluid. On the contrary, a defect in the amino acid metabolism could be presumed, since the concentration of several amino acids, along with that of short chain acylcarnitines, which are associated with their degradation, is increased . A previous study by Leruez et al. showed a higher concentration of C3 and C4 acylcarnitines in the plasma of POAG patients, demonstrating the important and systemic role of these short-chain acylcarnitines in the pathogenesis of POAG . Additionally, Lillo et al. investigated the metabolomic composition of the aqueous humor of patients with POAG, comparing them with healthy controls . This study confirmed the study of Buisset et al. regarding the changes in the levels of C0, C2, C3, and C4 that increased in glaucoma patients. In fact, C0, C2 and C3 were the most abundant carnitines, with concentration values of 9.8 μΜ, 1.7 μΜ, and 0.2 μΜ, respectively. However, the study of Lillo et al. showed an increase in the number of acylcarnitines whose concentration was above the detection limit, of which there were 13 in total; the levels of 10 acylcarnitines significantly increased in the group of glaucoma patients, whereas the concentrations of C4:1 and C14:2OH were similar between the two groups and the concentration of C10 decreased in the glaucoma samples. These alterations in the levels of acylcarnitines seem to be of mitochondrial origin, and the role of C10 carnitine, which was the one that decreased, should be evaluated further regarding its role in the functionality of the mitochondria. Indeed, in the past, the exogenous administration of this metabolite interestingly led to impairment in mitochondrial handling, as well as fatty acid oxidation and the inhibition of ketogenesis . A recent study conducted by Rossi et al. enrolled POAG patients and healthy controls, and determined their levels of metabolites. Among them, acylcarnitines were identified with the use of direct infusion mass spectrometry (DI-MS). Following a multivariate statistical approach, the levels of tear acetylcarnitine (C2) were found to be statistically significantly lower ( p = 0.008) in POAG patients compared with controls. Free carnitine (C0) showed a tendency towards lower levels ( p = 0.05) in the POAG group of patients in comparison to the healthy controls . Therefore, an easily accessible biofluid, such as tears, could be helpful in detecting molecules such as carnitines, and this correlation of POAG biomarkers and the progression of disease could lead to an early diagnosis and interesting aspect of investigation in future studies. 4.6. Rhegmatogenous Retinal Detachment (RRD) RRD is a condition that is caused when fluid passes from the vitreous cavity through a tear of the retina into the subretinal space, specifically between the neurosensory retina and the underlying retinal pigment epithelium (RPE), leading to the separation of the two tissues. RRD is a significant cause of visual impairment and it will result in total blindness if the retina is not repaired properly . Proliferative vitreoretinopathy (PVR) is a complication that might follow the RRD process and the most common reason for RRD repair failure. More particularly, PVR comprises the growth of cell membranes in the vitreous cavity and their contraction leads to retinal re-detachment and loss of vision, with poor visual outcomes even with repeated interventions . A study conducted by Li et al. performed a metabolomic analysis of human vitreous samples obtained from patients with RRD, patients with recurrent retinal detachment and PVR, and controls (donor eyes) in order to identify novel potential biomarkers that will help to clarify the mechanisms and pathogenesis of the formation of RRD and PVR. Relevant metabolites were revealed via a LC-Q-TOF-MS method and multivariate statistical analysis, which was used to distinguish the metabolite concentrations that were significantly altered between patient groups. Specifically, only eleven metabolites were statistically different between eyes with RRD and PVR. Among them, carnitine was found to be decreased in both groups. In fact, the extent of the L-carnitine decrease was significantly more prominent in RRD samples than in PVR samples ( p < 0.01 between the two groups) . In addition, a dysregulation of pathways linked to inflammation was detected . More particularly, in several reports, L-carnitine is associated with the control of inflammatory response, as well as the potential ability to suppress pro-inflammatory cytokines . Therefore, an elevated level of L-carnitine could potentially inhibit inflammation. The study by Li et al. showed that the lower levels of L-carnitine may cause an amplification of inflammation development in both patient groups, RRD and PVR. Since the decrease in the concentration of L-carnitine was significantly more evident in RRD samples compared to those with PVR, this led to the fact that the inflammation is more serious in eyes with RRD alone . Furthermore, this study showed that some of the identified metabolites were associated with proliferation of pathology-related cells, whereas others showed some anti-proliferation effects. It is stated that L-carnitine can suppress the proliferation and further differentiation of vascular smooth cells ; thus, it inhibits vessel growth and decreases the degree of visual loss. Interestingly, carnitine was elevated in PVR samples in comparison to those with RRD alone, which reveals a positive regulation made by the organism to inhibit the proliferation of pathological cells . 4.7. Dry Eye Syndrome (DES) DES is a multifactorial disease that affects the homeostasis of the tear film and, thus, the integrity of the ocular surface. In fact, tear film instability and hyperosmolarity can play an important etiological role in DES . Carnitines seem to be involved in the regulation of tear film osmolarity since they are thought to have osmoregulatory properties protecting the affected cornea surface . Pescosolido et al. conducted a case–control study using HPLC-MS, analyzing the presence of carnitine as well as its derivatives, i.e., L-acetylcarnitine and L-propionylcarnitine, in the tears of DES patients and healthy subjects. Comparing tear samples between the two groups, the concentrations of carnitine, L-acetylcarnitine, and L-propionylcarnitine were statistically significantly lower in the tears of DES patients ( p < 0.05). Therefore, it has been suggested that tear film osmolarity might be regulated by carnitines which could have a protective effect preventing damage of the ocular surface. An imbalance in the concentration of carnitine molecules in the tear film is suggested to be connected with DES, and further studies should be conducted to address their protective role as osmo-protection molecules and their possible use as biomarkers of the disease early diagnosis . 4.8. Animal Studies Metabolic perturbations and particularly alterations in the concentrations of several acylcarnitines have also been detected in animal studies related to ocular diseases. More specifically, in a study conducted by Yanshole et al., the metabolomic profiles of rat lenses were investigated by nuclear magnetic resonance (NMR) and HPLC-MS. The analysis regarding age-related changes in the metabolic composition of the lenses showed a decline in several metabolites, and among them, in carnitine. In fact, the most significant changes are observed between the ages of 1 and 3 months when the younger lenses of the Wistar rats are richer in metabolites compared to the older rats, since the period of enhanced metabolomic activity is terminated with the completion of the maturation process of the lens during the first month. A statistically significant difference was detected in the concentration of carnitine in the lens, which was decreased by approximately 100% between 1 and 3 months, and by 50% between 3 and 14 months. In addition, the concentrations of metabolites of Wistar rats were compared with those of senescence-accelerated OXYS rats. A statistically significant difference was found for carnitine in the OXYS lens, the concentration of which was on average 30% higher than in the Wistar lens. This could be explained by the fact that there is an excessive production of reactive oxygen species in tissues of the OXYS rats and, thus, the enhanced level of some metabolites, and among them of carnitine, which could be attributed to the compensatory response to the aforementioned oxidative stress . Another animal study conducted by Kurihara et al. investigated genetically triggered hypoxia in retinal pigment epithelium (RPE) cells that could lead to photoreceptor atrophy in mouse models, thus trying to gain an insight into the pathogenesis of AMD. In fact, the aforementioned hypoxia-induced metabolic stress triggered alterations in the lipid metabolism of hypoxic RPE cells and, more particularly, in a group of medium- and long-chain acylcarnitines. Therefore, the identification of a group of acylcarnitines that were dysregulated from 3 to 14 days post induction could be informative for the early diagnosis and a better understanding of diseases, such as AMD, in which defects in RPE and photoreceptors are common . Additionally, a study conducted by Rowan and coworkers investigated the contribution of dietary patterns that differ in the type of dietary carbohydrate on AMD in a mouse model. Given the fact that the consumption of a high-glycemic diet resulted in several AMD features, such as RPE hypopigmentation and atrophy and photoreceptor degeneration, whereas the consumption of low-glycemic dietary products did not, it is not unreasonable to think that switching from a high-glycemic diet to a low-glycemic diet could stop or reverse AMD features. The findings revealed an interaction between carbohydrate intake, AMD features, and the metabolome. In fact, among the metabolites which were statistically significantly different was C3-carnitine, which was found to be lower in the retina, plasma, or urine of affected mice, i.e., the ones that were given the high-glycemia diet. More specifically, C3-carnitine was a metabolite that performed nearly perfectly in ROC analysis regarding the ability of a metabolite to distinguish unaffected from affected retinas, and its plasma levels were statistically lower in affected individuals (AUC = 0.969, p = 0.00264). Therefore, C3-carnitine could act as one of several potential biomarkers for earlier diagnosis, prognosis, or evaluation of the efficacy of a new therapy, and provide a better understanding of the retinal maintenance and possible mechanisms of AMD features . Finally, in the aforementioned study of Paris et al., metabolomic analyses were performed on ocular samples from mice with oxygen-induced-retinopathy (OIR) which exhibits several comparable pathological retinal features also observed in PDR. The metabolomic profile of the OIR mouse model was analyzed at the 17th day of age, i.e., the time of maximal pre-retinal neovascularization. Among the metabolites that were altered in OIR mouse eyes compared with the normoxic ones were three acylcarnitines, namely octanoylcarnitine (C8), propionylcarnitine (C3), and acetylcarnitine (C2), that were increased in OIR models by 3.0-, 86.4-, and 2-fold compared to controls. Thus, octanoylcarnitine was the metabolite which was altered in both human and mouse eye samples, while decanoylcarnitine concentration was below the limit of detection in the OIR mouse model .
AMD is a disease that affects central vision progressively and is the major cause of visual impairment in people over 60 years of age. It constitutes 8.7% of worldwide total blindness, affecting approximately 150 million globally, and this number is projected to incline to 288 million by 2040 . AMD is classified mainly as early stage (small-medium drusen deposits), intermediate (larger drusen deposits and/or retinal pigmentary changes), and late AMD. The latter is subdivided into two categories: (i) geographic atrophy or dry AMD, characterized by a gradual degeneration of retinal cells, and (ii) neovascular or exudative or wet AMD, in which new choroidal blood vessels are formed and proliferate, leading to exudation, retinal edema, hemorrhages, and central vision loss . A combination of risk factors, including age, genetics, and environmental factors, such as tobacco use, a low dietary consumption of antioxidants, and hypercholesterolemia, leads to AMD . Although several factors associated with the development of AMD have been identified, the etiology and the pathogenesis of the disease have not been fully delineated. Moreover, the interactions among cells and the exchange of metabolites between the retina, the retina pigment epithelium, and the choroid complex highlight the significance of finding metabolic local and systemic biomarkers which are indicative of the disease and improve the outcome. Therefore, AMD progression is related with metabolic dysfunction, and alterations in the level of metabolites, such as carnitines, might act as a diagnostic biomarker for its development. Recently, a metabolomic study conducted by Mitchell et al. compared metabolite levels in plasma samples obtained from patients with intermediate AMD (IAMD), neovascular AMD (NVAMD), and non-AMD controls . The aforementioned levels of metabolites among all groups were compared. By using a liquid chromatography-mass spectrometry (LC-MS) platform to analyze samples, and partial least-squares discriminant analysis (PLS-DA) and linear regression (LR) to identify discriminatory metabolic features, six acylcarnitines were found to meet the significance criteria. Among them, two carnitines (specifically linoleyl and linolenyl carnitine) were nearly 1.5-fold (1.63 and 1.49, respectively) higher in AMD patients compared to disease-free controls. Four carnitines (heptadecanoyl carnitine, 11Z-octadecenylcarnitine, glutaconylcarnitine, and stearoylcarnitine) were between 1.7- and 2.10-fold (2.08, 1.74, 1.75, 1.83, respectively) higher in NVAMD patients compared to IAMD patients. It should be mentioned that five of the acylcarnitines belong to medium- or long-chain acylcarnitine species. Furthermore, seven additional discriminatory features were identified with a medium or high confidence match with acylcarnitines, which presented higher levels along with more advance disease. Regarding metabolic pathway analysis, the carnitine shuttle pathway was the one that was significantly altered in all comparisons (AMD vs. control, IAMD vs. control, NVAMD vs. control, and NVAMD vs. IAMD). This study reveals the fact that plasma levels of certain acylcarnitines were not only higher in AMD patients and NVAMD patients compared with controls, but also in NVAMD patients compared with IAMD patients . Furthermore, Mitchel et al. previously reported a metabolomic analysis of plasma samples obtained from NVAMD patients and healthy controls. Among the plethora of metabolic features analyzed, 39 of them were annotated with confidence, from which multiple were carnitine species. Using LC-MS/MS analysis, the identity of five acylcarnitine intermediates was confirmed. The plasma levels of the aforementioned five long-chain acylcarnitines (9-hexadecenoylcarnitine, heptadecanoylcarnitine, 11Z-octadecenylcarnitine, L-palmitoylcarnitine, and stearoylcarnitine) were significantly higher, approximately 2-fold (2.17, 2.16, 2.03, 1.95, and 1.65, respectively), in NVAMD patients compared to healthy controls. Furthermore, metabolite pathway analysis showed that the carnitine shuttle pathway was significantly changed in NVAMD patients . Two more case–control studies have addressed altered plasma acylcarnitines in AMD patients. The first study, conducted by Luo et al., was an untargeted metabolomic study. It compared metabolomic features in the morning plasma of Chinese patients with wet AMD and healthy controls. By using ultra-high-pressure liquid chromatography and quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF MS) to detect metabolic differences and multidimensional statistical methods to analyze samples, 10 metabolites were identified to differ significantly between the two groups of patients. Among them, L-palmitoylcarnitine was found to be lower in the group of patients with wet AMD (fold change 0.79) (variable importance for projection (VIP) >1, p < 0.05) . The second case–control study by Chao de la Barca et al., which was based on a targeted metabolomics approach, compared plasma samples from patients with exudative/wet AMD and healthy controls. Among the 188 metabolites that were analyzed, only six remained statistically significantly altered after Benjamini–Hochberg correction, and two of them were carnitines, specifically free L-carnitine (C0) and valerylcarnitine (C5). The concentration of both carnitines was statistically increased in the plasma of AMD patients compared to controls and, in fact, valerylcarnitine was approximately 1.4 more concentrated in the blood of AMD individuals . In another study conducted by Han et al., metabolomic changes in the aqueous humor of patients with wet AMD, in comparison to controls without AMD that underwent cataract surgery, were investigated. The untargeted metabolomics study, performed with the use of UHPLC-MS/MS and univariate analysis, showed 18 statistically significantly altered metabolites. Among them, two carnitines were identified, namely free L-carnitine (C0), which was decreased in the wet AMD group (approximately 0.72-fold change), and deoxycarnitine, which was increased in the patient AMD group (almost 1.87-fold change) compared to the control group. In addition, since the aforementioned molecule is the precursor substrate for carnitine biosynthesis, it should be mentioned that 6-N-trimethyl-L-lysine (TML) was significantly higher in the aqueous humor of AMD patients (1.84-fold change) compared with non-AMD controls. Therefore, alterations in free L-carnitine, deoxycarnitine, and TML suggest a disturbance in the carnitine pathway associated with mitochondrial dysfunction and fatty acid metabolism, possibly contributing to the pathogenesis of AMD .
DR, being a common complication of diabetes mellitus (DM), constitutes the main cause of blindness in the adult, working-age population . It affects approximately 103 million patients worldwide, and the number is estimated to rise to 160.5 million patients in 2045 . The disease leads to visual impairment, having a devastating impact on patients’ quality of life . DR is categorized mainly into two stages: non-proliferative DR (NPDR), characterized by microaneurysms, haemorrhages, cotton-wool spots, and intra-retinal microvascular anomalies (IRMAs); and proliferative DR (PDR), which is characterized by neovascularization and/or vitreous haemorrhage . The risk factors for DR include poor glycemic control and, thus, hyperglycemia, as well as hypertension and hyperlipidemia . Therapeutic strategies, such as anti-VEGF injections, are limited and the understanding of the DR pathogenesis is still incomplete. Therefore, further investigation should be undertaken and new metabolites, such as carnitines, could play an important role in the early detection of the disease and the optimization of patient care. In a recent study conducted by Sumarriva et al., metabolic profile differences of plasma samples between patients with type 2 DM, with DR and without DR (diabetic controls), and between patients with PDR and NPDR have been shown. LC-MS analysis was performed; features were selected using PLS-DA (VIP ≥ 1.5) and were significantly associated ( p < 0.05) in a Wilcoxon rank sum test. Several metabolic features were found to differ significantly between DR patients and diabetic controls, four of which constituted key contributors to the pathways analysis differences detected in the study; dehydroxycarnitine was among them and its concentration was found to be significantly increased in DR patients ( p = 0.0069). Regarding PDR, pathway analysis revealed alterations in the β-oxidation of saturated fatty acids ( p = 0.032), as well as fatty acid metabolism (0.038). Carnitine, being a key molecule in the metabolism and oxidation of fatty acids in mitochondria, was found to be statistically significantly increased in PDR patients in comparison with NPDR patients . Furthermore, Yun et al. performed a metabolomics analysis in serum samples obtained from patients with type 2 DM. Specifically, patients were divided into three groups, i.e., NDR, NPDR, and PDR. Metabolites were quantified with the use of LC-MS and FIA-MS, and statistical analysis was performed with R software. The concentrations of several carnitines were found to be significantly different among groups and they could act as potential disease markers. In fact, propionylcarnitine (C3) and butyrylcarnitine (C4) showed significantly higher concentrations in the DR patient group compared with the NDR group (a 1.16- and 1.31-fold change, respectively), while the concentrations of dodecanoylcarnitine (C12), tetradecenoylcarnitine (C14:1), tetradecadienylcarnitine (C14:2), hexadecanoylcarnitine (C16), octadecenoylcarnitine (C18:1), and octadecadienylcarnitine (C18:2) were lower in the DR group (0.89-, 0.87-, 0.87-, 0.86-, 0.87-, and 0.87-fold change, respectively). Additionally, comparing serum samples from NPDR and NDR patients, the concentrations of three acylcarnitines, namely L-carnitine (C0), tetradecenoylcarnitine (C14:1), and hexadecanoylcarnitine (C16), were found to be lower in the NPDR group (0.96-, 0.88-, and 0.87-fold change, respectively). Comparing the concentrations of metabolites between PDR and NDR patients, three short-chain acylcarnitines (propionylcarnitine (C3), butyrylcarnitine (C4), and valerycarnitine (C5)) showed statistically higher concentrations in PDR patients (1.25-, 1.63-, and 1.23-fold change, respectively), while five long-chain acylcarnitines, tetradecenoylcarnitine (C14:1), hexadecanoylcarnitine (C16), octadecanoylcarnitine (C18), octadecenoylcarnitine (C18:1), and octadecadienylcarnitine (C18:2), showed lower concentrations in the PDR group (0.84-, 0.82-, 0.83-, 0.81-, and 0.81-fold change, respectively). Moreover, among the metabolites that statistically differed between PDR and NDPR patients, the medium-chain pimelycarnitine (C7:DC) was found to be elevated in the PDR group, and its concentration was reported to be 1.10-fold higher. Finally, the concentrations of sixteen metabolites showed differences when comparing both PDR and NPDR patients to NDR patients. Two acylcarnitines, namely tetradecenoylcarnitine (C14:1) and hexadecanoylcarnitine (C16), were included among them, and their concentrations were lower in the PDR and NPDR groups than in the NDR group (0.87- and 0.86-fold, respectively) . Interestingly, in a prior study by Paris et al., the content of metabolites in vitreous humor samples from patients with PDR and non-diabetic controls were compared. Initial analysis was performed in a first set of patients, and afterwards, the findings were validated through the analysis of a second set of patient samples. The concentrations of two medium-chain carnitines, specifically decanoylcarnitine (C10) and octanoylcarnitine (C8), were increased in the group of PDR patients compared to non-diabetic controls in both sets of patients (C10 with 1.7- and 1.4-fold changes and C8 with 2.2- and 1.7-fold changes in the first and second set of patients, respectively) .
ROP is a disease characterized by immature retinal vascular development and the presence of retinal ischemia and neovascularization, which can lead to retinal detachment and vision loss. Both prematurity and low birth weight are factors strongly related to a high risk of the disease . It should be mentioned that ROP affects almost 100,000 children globally, according to an epidemiological study . Indeed, the incidence and the severity of ROP is still high, and the pathogenesis is not yet fully understood. Therefore, metabolites, such as carnitines, could act as potential biomarkers and contribute to the early diagnosis and better understanding of the underlying mechanisms of the disease. Recently, Yang et al. conducted a study based on a targeted metabolomics analysis of blood from premature infants, comprising two groups of cases: ROP patients and non-ROP controls . UPLC-MS targeted analysis was applied for the measurement of malonylcarnitine. Subsequently, standard multivariate and univariate analysis was performed for interpretation of the results. Malonyl carnitine (C3DC) was found and confirmed to be statistically significantly different ( p < 0.001) between ROP and non-ROP infants. C3DC constituted an independent strong risk factor for ROP, since the higher the value of the specific acylcarnitine, the higher the risk of ROP. Although a probable association is revealed between C3DC and the risk of ROP, this is not the case for the severity of the disease. In addition, the predictive ability of C3DC for ROP diagnosis was the best among the other discriminant metabolites (area under the curve (AUC) = 0.914, sensitivity = 97.5%, and specificity = 68.3%), showing that C3DC could be a potential biomarker for the diagnosis of ROP .
CRVO is a common disease of the retinal vasculature which may lead to visual loss, usually unilateral . It is estimated to affect approximately 0.1–0.5% of the population. Its pathogenesis is associated with atherosclerosis of the central retinal artery, which is situated adjacent to the vein within the same adventitia, compressing and inducing stasis and thrombosis in the vein lumen, and leading to CRVO . In this condition, there is an increase in intravenous hydrostatic pressure and a nonperfusion of the retinal capillaries, leading to a decrease in oxygen supply. The aforementioned alterations in the retinal blood flow result in ischemia and hypoxia-induced metabolic dysregulation . Therefore, changes in the concentrations of metabolic substances and dysfunction of several pathways, among which is the carnitine shuttle pathway, could reveal the pathological mechanisms and the progression of the disease. A study conducted by Wei et al. performed a metabolomic analysis of the aqueous humor obtained from fifteen patients with CRVO and twenty patients who underwent cataract surgery (controls). UHPLC-MS/MS was used to identify the involved metabolites . Butyrylcarnitine ( p = 0.003) and deoxycarnitine ( p value not reported) were found to be elevated and among the statistically significant metabolites in CRVO group. Furthermore, 6-N-trimethyllysine (TML), being the compound from which the biosynthesis of carnitines starts, was significantly altered in CRVO patients as well. A speculation that was suggested is that the dysregulated carnitine metabolism pathway might be involved in mitochondrial dysfunction in CRVO patients; however, regarding the pathogenesis of CRVO, any exact involvement of such metabolomic pathways is still unknown.
POAG represents the most common form of glaucoma and it is a group of chronic optic degenerative neuropathies characterized by progressive impairment of retinal ganglion cells and visual field loss without an identifiable cause . The risk of developing POAG is related to many factors, among which the most prominent one is elevated intraocular pressure (IOP) . POAG affects approximately 57.5 million people and is the leading cause of irreversible blindness globally; it is projected to reach 111.8 million cases in 2040 . Due to the slow and asymptomatic progression of the disease in the early stages, the diagnosis of POAG often does not occur until later when severe field damage and loss of central fixation are established . Therefore, the asymptomatic nature of POAG, combined with the worldwide prevalence of the disease and its estimated dramatic increase in the ageing population in the following years, necessitate the need of discovering new strategies and possible systemic or topical biomarkers, such as carnitines, for an early stage accurate diagnosis. Two studies compared the metabolic profiles of plasma taken from patients with POAG and from controls. In the first study conducted by Burgess et al., LC-MS was used for metabolic profiling analysis of plasma samples, and further statistical analysis of the data revealed several differentially expressed metabolites associated with POAG. Among the aforementioned metabolites palmitoylcarnitine (C16), a long-chain acylcarnitine, was found to be significantly elevated in the plasma of POAG patients in comparison with controls ( p = 3.69 −10 ) . It is well known that palmitoylcarnitine is part of the carnitine shuttle pathway, functioning for the transport of fatty acids for β-oxidation in the mitochondria. The aforementioned findings are in accordance with an earlier study regarding the deficiency of carnitine palmitoyl transferase II and its connection with normal tension glaucoma . However, since various factors could be related to the palmitoylcarnitine metabolism in the mitochondria, the elevated levels of palmitoylcarnitine in the plasma of POAG patients might be part of a more generalized defect of mitochondrial function and not specifically associated with POAG . Similarly, in the second study, Leruez et al. compared the metabolic profiles of plasma samples obtained from patients with POAG and controls with cataract. FIA-MS was used for the analysis of metabolites, and statistical analysis revealed 18 significantly altered metabolites between the two groups, six of which were identified to be carnitines. More specifically, univariate statistical analysis (with the use of the Wilcoxon test) showed an increase in propionylcarnitine (C3) (fold change = 1.30, p = 0.0036) in the POAG group in comparison to controls, while octadecenoylcarnitine (C18:1) was decreased (fold change = 0.76, p = 0.0017) in POAG patients. In addition, multivariate analysis (with the use of the least absolute shrinkage and selection (LASSO) operator) showed an elevation in the concentration of butyrylcarnitine (C4), decanoylcarnitine (C10:1), and dodecanoylcarnitine (C12:1) in the group of POAG patients compared with cataract controls (fold change and p value are not reported for these metabolites). Both univariate and multivariate analysis identified octadecadienylcarnitine (C18:2) to be statistically significantly increased (fold change = 0.82, p = 0.00068) in the POAG group compared with controls . The alterations in acylcarnitine concentrations delineate a perturbation in the fatty acid metabolism and are in accordance with the study of Burgess et al., where the long-chain palmitoylcarnitine was found to be increased. In addition, Rong et al. revealed five long-chain free fatty acids which were statistically significant in PACG patients but not acylcarnitines, and Mayordomo-Febrer et al. found elevated free fatty acid concentrations in the aqueous humor of their animal model (rats) of glaucoma . Taking into consideration the significant role of carnitines in the transport of fatty acids in the mitochondria and the inherited disorders of fatty acid oxidation, it could be presumed that when the oxidation of fatty acids is incomplete, this results in a reflux from mitochondria into the blood stream of smaller acylcarnitines, which may be the cause of increased acylcarnitine levels . Furthermore, the concentration of two long-chain acylcarnitines was found to be lower in POAG patients, which might be part of the ageing process, as it has been shown to decline with age in the blood of mice . A recent study conducted by Buisset et al. compared the metabolomic profiles obtained from the aqueous humor of POAG patients with that of controls undergoing cataract surgery. Again, FIA-MS was used to identify metabolites, and multivariate and univariate statistical analysis was performed to discriminate metabolites between the two groups. Among the metabolites which were found to be discriminant using multivariate analysis, three short-chain acylcarnitines, namely acetylcarnitine (C2), propionylcarnitine (C3), buryrylcarnitine (C4), and free carnitine (C0), were included. The concentrations of the aforementioned carnitines were statistically higher in the aqueous humor of POAG patients compared to controls. Additionally, univariate analysis after correction for the false discovery rate (FDR) demonstrated that C2 and C3 were significantly higher than other metabolites. Metabolites, including carnitines, were ranked based on importance using the median value of VIP, as well as the adjusted p-value (C3: VIP = 1.56, p = 0.013; C2: VIP = 1.52, p = 0.029; C0: VIP = 1.32, p = 0.067; C4: VIP = 1.11, p = 0.087) . The study by Buisset et al. was the first study to show elevated concentrations of short-chain carnitines (C2, C3, C4) and free carnitine in the aqueous humor of POAG patients. Furthermore, carnitine has shown neuroprotective properties, as well as antioxidant and antiapoptotic properties, in retinal cells in the eyes of mice with high intraocular pressure . Therefore, an increase in the concentration of carnitine could be involved in the protection of retinal cells in the case of disease-related stress and production of reactive oxygen species. Additionally, the lack of long-chain acylcarnitines in the metabolic profile of the aqueous humor of POAG patients is not in favor of mitochondrial oxidation impairment in the cells obtained by the aforementioned fluid. On the contrary, a defect in the amino acid metabolism could be presumed, since the concentration of several amino acids, along with that of short chain acylcarnitines, which are associated with their degradation, is increased . A previous study by Leruez et al. showed a higher concentration of C3 and C4 acylcarnitines in the plasma of POAG patients, demonstrating the important and systemic role of these short-chain acylcarnitines in the pathogenesis of POAG . Additionally, Lillo et al. investigated the metabolomic composition of the aqueous humor of patients with POAG, comparing them with healthy controls . This study confirmed the study of Buisset et al. regarding the changes in the levels of C0, C2, C3, and C4 that increased in glaucoma patients. In fact, C0, C2 and C3 were the most abundant carnitines, with concentration values of 9.8 μΜ, 1.7 μΜ, and 0.2 μΜ, respectively. However, the study of Lillo et al. showed an increase in the number of acylcarnitines whose concentration was above the detection limit, of which there were 13 in total; the levels of 10 acylcarnitines significantly increased in the group of glaucoma patients, whereas the concentrations of C4:1 and C14:2OH were similar between the two groups and the concentration of C10 decreased in the glaucoma samples. These alterations in the levels of acylcarnitines seem to be of mitochondrial origin, and the role of C10 carnitine, which was the one that decreased, should be evaluated further regarding its role in the functionality of the mitochondria. Indeed, in the past, the exogenous administration of this metabolite interestingly led to impairment in mitochondrial handling, as well as fatty acid oxidation and the inhibition of ketogenesis . A recent study conducted by Rossi et al. enrolled POAG patients and healthy controls, and determined their levels of metabolites. Among them, acylcarnitines were identified with the use of direct infusion mass spectrometry (DI-MS). Following a multivariate statistical approach, the levels of tear acetylcarnitine (C2) were found to be statistically significantly lower ( p = 0.008) in POAG patients compared with controls. Free carnitine (C0) showed a tendency towards lower levels ( p = 0.05) in the POAG group of patients in comparison to the healthy controls . Therefore, an easily accessible biofluid, such as tears, could be helpful in detecting molecules such as carnitines, and this correlation of POAG biomarkers and the progression of disease could lead to an early diagnosis and interesting aspect of investigation in future studies.
RRD is a condition that is caused when fluid passes from the vitreous cavity through a tear of the retina into the subretinal space, specifically between the neurosensory retina and the underlying retinal pigment epithelium (RPE), leading to the separation of the two tissues. RRD is a significant cause of visual impairment and it will result in total blindness if the retina is not repaired properly . Proliferative vitreoretinopathy (PVR) is a complication that might follow the RRD process and the most common reason for RRD repair failure. More particularly, PVR comprises the growth of cell membranes in the vitreous cavity and their contraction leads to retinal re-detachment and loss of vision, with poor visual outcomes even with repeated interventions . A study conducted by Li et al. performed a metabolomic analysis of human vitreous samples obtained from patients with RRD, patients with recurrent retinal detachment and PVR, and controls (donor eyes) in order to identify novel potential biomarkers that will help to clarify the mechanisms and pathogenesis of the formation of RRD and PVR. Relevant metabolites were revealed via a LC-Q-TOF-MS method and multivariate statistical analysis, which was used to distinguish the metabolite concentrations that were significantly altered between patient groups. Specifically, only eleven metabolites were statistically different between eyes with RRD and PVR. Among them, carnitine was found to be decreased in both groups. In fact, the extent of the L-carnitine decrease was significantly more prominent in RRD samples than in PVR samples ( p < 0.01 between the two groups) . In addition, a dysregulation of pathways linked to inflammation was detected . More particularly, in several reports, L-carnitine is associated with the control of inflammatory response, as well as the potential ability to suppress pro-inflammatory cytokines . Therefore, an elevated level of L-carnitine could potentially inhibit inflammation. The study by Li et al. showed that the lower levels of L-carnitine may cause an amplification of inflammation development in both patient groups, RRD and PVR. Since the decrease in the concentration of L-carnitine was significantly more evident in RRD samples compared to those with PVR, this led to the fact that the inflammation is more serious in eyes with RRD alone . Furthermore, this study showed that some of the identified metabolites were associated with proliferation of pathology-related cells, whereas others showed some anti-proliferation effects. It is stated that L-carnitine can suppress the proliferation and further differentiation of vascular smooth cells ; thus, it inhibits vessel growth and decreases the degree of visual loss. Interestingly, carnitine was elevated in PVR samples in comparison to those with RRD alone, which reveals a positive regulation made by the organism to inhibit the proliferation of pathological cells .
DES is a multifactorial disease that affects the homeostasis of the tear film and, thus, the integrity of the ocular surface. In fact, tear film instability and hyperosmolarity can play an important etiological role in DES . Carnitines seem to be involved in the regulation of tear film osmolarity since they are thought to have osmoregulatory properties protecting the affected cornea surface . Pescosolido et al. conducted a case–control study using HPLC-MS, analyzing the presence of carnitine as well as its derivatives, i.e., L-acetylcarnitine and L-propionylcarnitine, in the tears of DES patients and healthy subjects. Comparing tear samples between the two groups, the concentrations of carnitine, L-acetylcarnitine, and L-propionylcarnitine were statistically significantly lower in the tears of DES patients ( p < 0.05). Therefore, it has been suggested that tear film osmolarity might be regulated by carnitines which could have a protective effect preventing damage of the ocular surface. An imbalance in the concentration of carnitine molecules in the tear film is suggested to be connected with DES, and further studies should be conducted to address their protective role as osmo-protection molecules and their possible use as biomarkers of the disease early diagnosis .
Metabolic perturbations and particularly alterations in the concentrations of several acylcarnitines have also been detected in animal studies related to ocular diseases. More specifically, in a study conducted by Yanshole et al., the metabolomic profiles of rat lenses were investigated by nuclear magnetic resonance (NMR) and HPLC-MS. The analysis regarding age-related changes in the metabolic composition of the lenses showed a decline in several metabolites, and among them, in carnitine. In fact, the most significant changes are observed between the ages of 1 and 3 months when the younger lenses of the Wistar rats are richer in metabolites compared to the older rats, since the period of enhanced metabolomic activity is terminated with the completion of the maturation process of the lens during the first month. A statistically significant difference was detected in the concentration of carnitine in the lens, which was decreased by approximately 100% between 1 and 3 months, and by 50% between 3 and 14 months. In addition, the concentrations of metabolites of Wistar rats were compared with those of senescence-accelerated OXYS rats. A statistically significant difference was found for carnitine in the OXYS lens, the concentration of which was on average 30% higher than in the Wistar lens. This could be explained by the fact that there is an excessive production of reactive oxygen species in tissues of the OXYS rats and, thus, the enhanced level of some metabolites, and among them of carnitine, which could be attributed to the compensatory response to the aforementioned oxidative stress . Another animal study conducted by Kurihara et al. investigated genetically triggered hypoxia in retinal pigment epithelium (RPE) cells that could lead to photoreceptor atrophy in mouse models, thus trying to gain an insight into the pathogenesis of AMD. In fact, the aforementioned hypoxia-induced metabolic stress triggered alterations in the lipid metabolism of hypoxic RPE cells and, more particularly, in a group of medium- and long-chain acylcarnitines. Therefore, the identification of a group of acylcarnitines that were dysregulated from 3 to 14 days post induction could be informative for the early diagnosis and a better understanding of diseases, such as AMD, in which defects in RPE and photoreceptors are common . Additionally, a study conducted by Rowan and coworkers investigated the contribution of dietary patterns that differ in the type of dietary carbohydrate on AMD in a mouse model. Given the fact that the consumption of a high-glycemic diet resulted in several AMD features, such as RPE hypopigmentation and atrophy and photoreceptor degeneration, whereas the consumption of low-glycemic dietary products did not, it is not unreasonable to think that switching from a high-glycemic diet to a low-glycemic diet could stop or reverse AMD features. The findings revealed an interaction between carbohydrate intake, AMD features, and the metabolome. In fact, among the metabolites which were statistically significantly different was C3-carnitine, which was found to be lower in the retina, plasma, or urine of affected mice, i.e., the ones that were given the high-glycemia diet. More specifically, C3-carnitine was a metabolite that performed nearly perfectly in ROC analysis regarding the ability of a metabolite to distinguish unaffected from affected retinas, and its plasma levels were statistically lower in affected individuals (AUC = 0.969, p = 0.00264). Therefore, C3-carnitine could act as one of several potential biomarkers for earlier diagnosis, prognosis, or evaluation of the efficacy of a new therapy, and provide a better understanding of the retinal maintenance and possible mechanisms of AMD features . Finally, in the aforementioned study of Paris et al., metabolomic analyses were performed on ocular samples from mice with oxygen-induced-retinopathy (OIR) which exhibits several comparable pathological retinal features also observed in PDR. The metabolomic profile of the OIR mouse model was analyzed at the 17th day of age, i.e., the time of maximal pre-retinal neovascularization. Among the metabolites that were altered in OIR mouse eyes compared with the normoxic ones were three acylcarnitines, namely octanoylcarnitine (C8), propionylcarnitine (C3), and acetylcarnitine (C2), that were increased in OIR models by 3.0-, 86.4-, and 2-fold compared to controls. Thus, octanoylcarnitine was the metabolite which was altered in both human and mouse eye samples, while decanoylcarnitine concentration was below the limit of detection in the OIR mouse model .
Metabolomics is an emerging and potentially powerful tool in ophthalmology research. Metabolic profiling studies that have been performed in the past ten years have demonstrated that among the various metabolites that were found to differ between patients with common ocular diseases and controls , there were also L-carnitine and various short-, medium- and long-chain acylcarnitines. Acylcarnitines have already been identified as important indicators in metabolic studies of many diseases, including metabolic disorders, cardiovascular diseases, diabetes, depression, neurologic disorders, and certain cancers, and, interestingly, there is growing evidence on their role in ocular dysfunctions. According to the findings that have been presented above, the alterations in the levels of either free L-carnitine or certain acylcarnitines suggest a possible important role of the carnitine shuttle pathway in several ocular diseases. The aforementioned pathway is responsible for the transport of fatty acids, especially medium- and long-chain ones, into the mitochondria for the subsequent catabolism via β-oxidation, which is a process that requires acyl-CoA and leads to the esterification of L-carnitine to form acylcarnitine derivatives . It is possible that changes in the levels of acylcarnitines and the impairment of the carnitine shuttle pathway indicate a subsequent dysfunction in the mitochondrial fatty acid metabolism . Furthermore, there are links between fatty acid oxidation and the regulation of angiogenesis. It has been demonstrated that fatty acid oxidation is essential for maintaining redox homeostasis and preventing dysfunction in endothelial cells . Moreover, the inhibition of fatty acid oxidation via carnitine palmitoyl transferase 1 (CPT1), a rate-limiting enzyme, repressed proliferation and neovascularization in endothelial cells and inhibited pathological ocular angiogenesis in mice . NVAMD such as PDR are characterized by new vascular growth. Evidence showed that in both diseases, acylcarnitines are altered and these changes in fatty acid metabolism are associated with ischemia or neovascularization . For example, it has been reported that long-chain acylcarnitine levels were increased in patients with neovascular AMD , whereas L-carnitine has the potential to protect the retina from ischemia-reperfusion injury . Furthermore, it has been suggested that the perturbation of the carnitine shuttle pathway and the compromised mitochondrial function could decrease cellular capacity to handle reactive oxygen species, resulting in increased cellular dysfunction and cell death . The eyes are organs that highly demand energy and mitochondrial fatty acid metabolism is expected to have an impact on their function. This impact has been investigated in retinal cells . According to a retinal model of mitochondrial impairment, disorders in carnitine metabolism and in fatty acid β-oxidation may lead to retinopathy . It is tempting to speculate that a possible decrease in free L-carnitine and an increase in various chain acylcarnitines may lead to compromised mitochondrial function. This could be explained due to the fact that the possible decreased level of L-carnitine found in studies may indicate the increase in esterification to generate acylcarnitine derivatives, while an incomplete oxidation of fatty acids results in a reflux from mitochondria toward the blood of various acyl-carnitines, especially smaller ones, as was the case in POAG patients , which may be the origin of increased acyl-carnitine levels. In fact, this phenomenon has been reported in inherited disorders of fatty acid oxidation . On the other hand, long-chain acylcarnitines were also found downregulated in some studies in POAG or PDR patients, which is possibly in accordance with a senescence-like phenotype since the concentration of long-chain acyl-carnitines has been shown to decrease with age in the blood of mice . Furthermore, studies have explored the role that L-carnitine presents as an anti-inflammatory, antioxidant and antiapoptotic agent. Specifically, scattered reports have shown that L-carnitine may suppress pro-inflammatory cytokines and control the inflammatory and immune response . For example, the anti-inflammatory response of L-carnitine seems to be connected with its downregulated expression in RRD where inflammation development is present . Moreover, experimental studies showed that L-carnitine could improve carbohydrate metabolism, reduce oxidative stress and free radical levels, and prevent subsequent cell death during ischemia , while other experimental models have indicated that L-carnitine, as well as its derivative, acetylcarnitine, have been shown to have neuroprotective, antioxidant, and antiapoptotic reactivity in the retinal cells of mouse models with high intraocular pressure . Moreover, L-carnitine in the AH has been reported to play a role in maintaining AH homeostasis and osmosis . An important issue in the studies that have been presented is the type of biospecimen, since the levels of free/acylcarnitines should be related to it. For example, in AMD patients, the higher level of free carnitine present in the AH leads to lower levels of its esterified form, such as acylcarnitine, than in the choroid-retina and plasma . Furthermore, L-carnitine seems to play an important role in the osmoregulation of the tear fluid. Although tear fluid is known to be hyperosmolar in the DES, the levels of L-carnitine were found to be reduced. Decreased amounts of L-carnitine in the tear film may be related to increased concentrations of other tear solutes, to active transport across the cell membrane of ocular tissues and to lessen the retention of water in the aqueous layer, resulting in hyperevaporation, hyperosmolarity, reduced control of oxidative stress in the tear film, and increased apoptosis in the corneal surface. Therefore, L-carnitine might have a possible protective effect by preventing the damaging impact of a hypertonic tear film, while the reduced L-carnitine concentration in the tears of these patients could be consequential of the DES and may lend itself to the development of a future diagnostic test for the disease .
In conclusion, the literature data on the application of carnitine metabolites in ophthalmology seem promising, and carnitines may be proven useful tools in the treatment of ocular diseases. However, there are challenges to be faced in the field. Although the alterations of specific acylcarnitines and L-carnitine are evident, there seems to be no clear pattern yet. This could be easily explained since this is an evolving field with a small number of different studies regarding each ocular disease and biospecimen, and only few human samples. Studies with large numbers of patients should be designed and several factors, for example, the sex, the age or the medication, and other systemic diseases, should be taken into consideration. Specifically, systematic diseases, such as cardiovascular disease and the metabolic syndrome, could influence the levels of acylcarnitines in blood samples. Indeed, identifying acylcarnitines as systemic biomarkers remains a significant challenge because of the potential overlap or interaction between increased or decreased plasma levels of acylcarnitines in cases of the coexistence of the aforementioned systematic diseases and the ocular diseases. Moreover, the published studies were mainly with humans and, according to our knowledge, only few animal studies have been reported in the literature . The usual biospecimen was plasma/serum, but there are also a few studies where aqueous or vitreous humor or tears have been used. Specifically, aqueous humor was taken in three studies regarding AMD, POAG, and CRVO , vitreous humor was examined in two studies regarding DR and RRD , and tears were the specimen obtained in two studies regarding POAG and DES . Although aqueous or vitreous humor are more difficult to be obtained than blood or tears, it would also be interesting if ocular tissues or fluid could be used in a greater extent in future studies. Due to its unique metabolome, eye specimen metabolic profiling shows promise as an application area . Carnitines are a class of metabolites that could act as potential biomarkers and contribute to the early diagnosis and better understanding of the underlying mechanisms of the disease, and for this, future investigation is needed.
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A novel Y-shaper and chopper for small pupil management in cataract surgery: The initial experience | 526acfec-2f6b-4200-9e16-c1086581ea64 | 10229943 | Ophthalmology[mh] | Description of the instrument The instrument is made of stainless steel; at one end is a straight rod, and the other end has a curved chopper with a tip dimension of 0.8 mm which allows for easy entry through the side ports. In scenarios wherein we have moderately dilated pupil (5 mm) with any amount of iris billowing or miosis, it allows for simultaneous stretching of the floppy iris, thus enabling the phaco probe to hold a nuclear fragment and emulsifying the nucleus, whereas the curved chopper allows for easy nucleus chopping and allowing us to manage the small pupil and nucleus emulsification without the use of any pupil-expanding devices. Clinical applications Intra-operative miosis because of FIS: Intra-operative FIS is classified upon the presence of floppy iris stroma, which billows and ripples responding to phaco fluidics with progressive intra-operative miosis, independent of the use of mydriatic agents, and the iris stroma’s tendency to prolapse through the incisions. The Y-shaped chopper allows stretching the floppy iris to the periphery and preventing iris plugging in the phaco probe during aspiration and concomitantly performing nuclear fragmentation without risking anterior capsular tears or PCR and thus prevents the prolapse of the iris through the side ports or main wound . Small Pupil management: The Y-shaped chopper design allows for both capsular and iris stretching to enable the phacoemulsification in eyes in small pupils (<5 mm), allowing easy sculpting, nuclear fragmentation, and chopping of the nucleus fragments into smaller pieces. The rotation of the nucleus and epinuclear plate can also be achieved with ease, thus allowing the surgeon to sail through the cataract surgery without the use of pupil-expanding devices which can cause sphincter damage and post-operative photophobia to the patient . The instrument is made of stainless steel; at one end is a straight rod, and the other end has a curved chopper with a tip dimension of 0.8 mm which allows for easy entry through the side ports. In scenarios wherein we have moderately dilated pupil (5 mm) with any amount of iris billowing or miosis, it allows for simultaneous stretching of the floppy iris, thus enabling the phaco probe to hold a nuclear fragment and emulsifying the nucleus, whereas the curved chopper allows for easy nucleus chopping and allowing us to manage the small pupil and nucleus emulsification without the use of any pupil-expanding devices. Intra-operative miosis because of FIS: Intra-operative FIS is classified upon the presence of floppy iris stroma, which billows and ripples responding to phaco fluidics with progressive intra-operative miosis, independent of the use of mydriatic agents, and the iris stroma’s tendency to prolapse through the incisions. The Y-shaped chopper allows stretching the floppy iris to the periphery and preventing iris plugging in the phaco probe during aspiration and concomitantly performing nuclear fragmentation without risking anterior capsular tears or PCR and thus prevents the prolapse of the iris through the side ports or main wound . Small Pupil management: The Y-shaped chopper design allows for both capsular and iris stretching to enable the phacoemulsification in eyes in small pupils (<5 mm), allowing easy sculpting, nuclear fragmentation, and chopping of the nucleus fragments into smaller pieces. The rotation of the nucleus and epinuclear plate can also be achieved with ease, thus allowing the surgeon to sail through the cataract surgery without the use of pupil-expanding devices which can cause sphincter damage and post-operative photophobia to the patient . Successful surgical outcomes have been achieved with both mechanical iris dilation and iris retention devices, and these devices add to overall surgical cost and generally require more time in the operating theater than a mechanical pupillary stretch. One of the most important inventions in the history of mechanical pupil expansion was introduction of the iris hooks. Since the very first reports, the technique gained wide popularity all over the world. The advantages of this technique include ease of manipulations and wide availability of the hooks manufactured in different sizes, materials, and designs. However, there are chances of iris sphincter tears and risk of bleeding. It is generally recommended not to extend the pupil over 5.0 mm in size to decrease the chances of iris tissue over-stretching and in turn producing irregular and atonic pupils post-operatively, which can lead to post-operative photophobia and Haloes . Pupil stretching till date has been performed with the help of two instruments (spatulas, Kuglen hook, or similar) introduced through paracentesis incisions located contralateral to each other. However, pupillary stretching maneuvers are more traumatic to the iris and also possibly to the corneal endothelium, but this does not appear to detract from the surgical outcomes. The major drawback is that intra-operatively, the iris can get pulled into the phaco probe and in the corneal wounds, which makes it difficult to perform nuclear fragmentation and simultaneously salvage the iris. This problem gets alleviated with our Y-shaper chopper which allows us to manage the FIS and perform nuclear emulsification. In our initial experience, we have not observed any loss of iris tone or permanent damage or any patient complaining of post-operative photophobia. Significant variations in the ocular and systemic co-morbidities require the whole spectrum of pharmacological and surgical strategies to be in the armamentarium of the modern cataract surgeon. The easiness of manipulations and the final results vary significantly with different devices. Iris hooks and Malyugin Ring are the current standard of care for intra-operative mechanical pupil expansion in patients not responding to the pharmacological protocols. Some of these methods are associated with bleeding, loss of iris sphincter function, and an abnormal pupil shape post-operatively. The author’s initial experience with the Y-shaper and chopper minimizes the risk of intra-operative complications, yet enabling surgeons to aim at similar surgical outcomes. Further studies are needed to compare the various available techniques and assess the learning curve associated with this instrument. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. Nil. There are no conflicts of interest. www.ijo.in |
Acceptability of the Cytosponge procedure for detecting Barrett's oesophagus: a qualitative study | 4cabd662-8c1f-487d-86ed-7cf567e23273 | 5353314 | Pathology[mh] | In the UK, 8784 individuals were diagnosed with oesophageal cancer in 2013. The majority of oesophageal cancers occur as either squamous cell carcinomas or adenocarcinomas (EACs), two cancers with different aetiologies. EAC mainly originates from Barrett's mucosa. Barrett's oesophagus (BE), a precursor of EAC, is a complication of chronic gastro-oesophageal reflux disease (GERD), where the stomach contents rise into the oesophagus often due to a malfunctioning lower oesophageal sphincter muscle. The reflux of acid and bile damages the squamous cells lining the oesophagus, which are then repaired through metaplastic columnar epithelium instead of the regeneration of more squamous cells. The risk of malignant progression is low, with an estimated annual incidence of EAC of 0.1–0.5% in individuals with BE. Risk factors for BE include male gender, Caucasian ethnicity, over 50 years of age, and having a family history of BE. Lifestyle factors include being overweight or obese, high abdominal fat, smoking and drinking alcohol. US and UK data suggest approximately one in five adults experience GERD in their lifetime, and 5–20% of adults with GERD are affected by BE. BE is an asymptomatic condition that is usually only discovered during endoscopy performed for evaluation of GERD symptoms. The majority of EACs, however, present de novo without a prior diagnosis of BE, and symptomatically detected EACs are normally advanced. In the UK, the overall EAC 5-year survival rate is as low as 19%. Identifying patients with undiagnosed BE may reduce mortality; however, routinely investigating all patients with dyspepsia and GERD using endoscopy would be time and resource intensive, and not feasible in the National Health Service (NHS). The Cytosponge is a non-endoscopic, ingestible oesophageal sampling device, which is both minimally-invasive and potentially cost-effective. It can be administered in various settings including primary care. Patients with a history of GERD symptoms are asked to swallow a gelatine capsule on a string, which dissolves in the stomach. As the capsule dissolves a small sponge expands and is pulled out of the mouth, collecting cells from the oesophagus in the process. The Cytosponge takes 5 min to administer. Immunohistochemistry is performed on the sponge to detect Trefoil Factor 3 (TFF3), a biomarker for BE. The acceptability and accuracy of the Cytosponge-TFF3 test for screening for BE has been tested in several studies, including the Barrett's Oesophagus Trial 1 (BEST1), a cohort study in patients with previous prescriptions for acid suppressants in a UK primary care setting, and BEST2, a case–control study in patients with GERD with or without BE in a UK hospital setting. It was shown to have high sensitivity (73.3% and 79.9%, respectively) and specificity (93.8% and 92.4%, respectively) for detecting BE. Moreover, over 93% of patients successfully swallowed the Cytosponge and 82% reported low levels of anxiety before the test, suggesting high acceptability. We are currently in the process of setting up a randomised control trial, BEST3, to evaluate if the Cytosponge-TFF3 test leads to an increase in the number of patients diagnosed with BE in primary care, and to gain an in-depth understanding of the associated health economics. The acceptability of the procedure and preferences regarding the presentation of information on the test are an important part of this process, and has not been investigated in any detail until now. To ensure information on the procedure will be presented to BEST3 participants in the most accessible manner, and to encourage rapid implementation in clinical practice at a later stage, public perceptions of the procedure need to be explored in greater depth. The aim of this study was to obtain a detailed understanding of the acceptability of the Cytosponge-TFF3 test in a sample of individuals with GERD. This population was chosen because they reflect the population to which the test will be offered as part of the BEST3 trial and if it is implemented in routine primary care.
Participants The study was advertised to adults aged 50–69 years by a recruitment company email, and respondents were screened on the telephone for eligibility. This age group was selected as this population will be eligible for the Cytosponge test within the BEST3 trial. GERD severity was assessed using the GERD impact scale. Individuals were eligible if they experienced one of five GERD symptoms (eg, heartburn), or used GERD medications, ‘sometimes’, ‘often’ or ‘daily’. Individuals with a medical occupation or previous cancer diagnosis were excluded. The recruitment company were provided with quotas to ensure at least half the sample were men and half did not have an academic degree. Respondents with an academic degree were classified as having a high level of education. The number of people who were approached or refused is not known. The first 10 eligible respondents were invited to participate in a one-to-one interview with a researcher (MF). The interviews were used to inform the focus group presentation. After 10 interviews were completed four focus groups were arranged. The focus groups were stratified by gender (male and female) and education (high vs basic). Participants were recruited until data saturation was reached and no new themes emerged from the focus groups or interviews. Procedure Semi-structured interviews are an effective method for obtaining detailed opinions of an isolated individual, whereas focus groups generate discussion from multiple perspectives. A combination of both face-to-face interviews and focus group discussions were used for this study to explore how individuals perceived the Cytosponge test when they were alone and in a group setting. Interviews and focus group discussions took place in meeting rooms at the Wolfson Institute for Preventive Medicine, Queen Mary, University of London. Written consent was obtained at the beginning of the interview or focus group by a female research assistant trained in qualitative research (MF, MSc Health Psychology). The interviewer had not met any participant prior to the study, and participants were given little information beyond her research interests. MF had no personal interest in oesophageal cancer research that would influence her behaviour towards the participants. No other person was present within the interview or focus group. A demographic and lifestyle questionnaire was completed prior to starting. This assessed GERD severity and management with the GERD impact scale, as well as experience of endoscopy, age, ethnicity, education and smoking status. All interviews and focus group discussions were audio recorded. Participants were compensated £40 for their time and travel. The consolidated criterion for reporting qualitative research was used. Interviews The interview schedule was designed by the research team and tested with three adults aged over 50 years to ensure participants could follow all questions in the different sections. In response to these test interviews background sections were expanded or simplified where necessary and a small number of questions about the Cytosponge were added. These interviews, however, were not recorded or included in the analysis. The interview schedule had six sections, also see online : participants' understanding of GERD and its association with BE and EAC, their first impressions of the Cytosponge test, their perceptions of the BEST3 trial, their attitudes towards endoscopy, the potential impact of cancer prevention on their decision-making . At prespecified points of the interview, photographs of the Cytosponge were shown , as well as physical examples of the device and a video of a patient undergoing the Cytosponge test. 10.1136/bmjopen-2016-013901.supp1 supplementary document Focus groups A presentation was created by the research team. This followed a similar structure to the interview schedule, although questions relating to the topic of ‘Cancer Prevention’ were removed, as participants had difficulty answering these in the interviews. The presentation contained more visual cues than the interview and participants could read the text, which was in bullet-point format, and projected onto a screen (see online ). As with the interviews, photographs , physical examples and a video of the Cytosponge test were shown at prespecified points. Each focus group was moderated by MF who was supported by a second member of the research team (SS or AM). Notes were taken by the second researcher. 10.1136/bmjopen-2016-013901.supp2 supplementary document Analysis Interviews and focus group recordings were transcribed verbatim by MF. Thematic analysis, a technique for identifying patterns (themes) within qualitative data was used for the analysis. Transcripts were read by MF, SS and JO to familiarise themselves with the data. The transcripts were then coded by MF to identify initial themes in the data. Using these themes, MF and SS created an analytical model on which to base the remaining analysis. Themes within the model were revised by MF, SS and JO iteratively throughout the analysis. Representative quotes were chosen to illustrate the themes identified within the data set. Consideration was given to ensure all sociodemographic groups were adequately represented in the quotations. It was agreed by the three researchers involved in the analysis that data saturation had been reached, and no further interviews or focus group discussions were necessary. Participants were not given an opportunity to review the transcripts. Microsoft Excel was used to support the analysis.
The study was advertised to adults aged 50–69 years by a recruitment company email, and respondents were screened on the telephone for eligibility. This age group was selected as this population will be eligible for the Cytosponge test within the BEST3 trial. GERD severity was assessed using the GERD impact scale. Individuals were eligible if they experienced one of five GERD symptoms (eg, heartburn), or used GERD medications, ‘sometimes’, ‘often’ or ‘daily’. Individuals with a medical occupation or previous cancer diagnosis were excluded. The recruitment company were provided with quotas to ensure at least half the sample were men and half did not have an academic degree. Respondents with an academic degree were classified as having a high level of education. The number of people who were approached or refused is not known. The first 10 eligible respondents were invited to participate in a one-to-one interview with a researcher (MF). The interviews were used to inform the focus group presentation. After 10 interviews were completed four focus groups were arranged. The focus groups were stratified by gender (male and female) and education (high vs basic). Participants were recruited until data saturation was reached and no new themes emerged from the focus groups or interviews.
Semi-structured interviews are an effective method for obtaining detailed opinions of an isolated individual, whereas focus groups generate discussion from multiple perspectives. A combination of both face-to-face interviews and focus group discussions were used for this study to explore how individuals perceived the Cytosponge test when they were alone and in a group setting. Interviews and focus group discussions took place in meeting rooms at the Wolfson Institute for Preventive Medicine, Queen Mary, University of London. Written consent was obtained at the beginning of the interview or focus group by a female research assistant trained in qualitative research (MF, MSc Health Psychology). The interviewer had not met any participant prior to the study, and participants were given little information beyond her research interests. MF had no personal interest in oesophageal cancer research that would influence her behaviour towards the participants. No other person was present within the interview or focus group. A demographic and lifestyle questionnaire was completed prior to starting. This assessed GERD severity and management with the GERD impact scale, as well as experience of endoscopy, age, ethnicity, education and smoking status. All interviews and focus group discussions were audio recorded. Participants were compensated £40 for their time and travel. The consolidated criterion for reporting qualitative research was used.
The interview schedule was designed by the research team and tested with three adults aged over 50 years to ensure participants could follow all questions in the different sections. In response to these test interviews background sections were expanded or simplified where necessary and a small number of questions about the Cytosponge were added. These interviews, however, were not recorded or included in the analysis. The interview schedule had six sections, also see online : participants' understanding of GERD and its association with BE and EAC, their first impressions of the Cytosponge test, their perceptions of the BEST3 trial, their attitudes towards endoscopy, the potential impact of cancer prevention on their decision-making . At prespecified points of the interview, photographs of the Cytosponge were shown , as well as physical examples of the device and a video of a patient undergoing the Cytosponge test. 10.1136/bmjopen-2016-013901.supp1 supplementary document
A presentation was created by the research team. This followed a similar structure to the interview schedule, although questions relating to the topic of ‘Cancer Prevention’ were removed, as participants had difficulty answering these in the interviews. The presentation contained more visual cues than the interview and participants could read the text, which was in bullet-point format, and projected onto a screen (see online ). As with the interviews, photographs , physical examples and a video of the Cytosponge test were shown at prespecified points. Each focus group was moderated by MF who was supported by a second member of the research team (SS or AM). Notes were taken by the second researcher. 10.1136/bmjopen-2016-013901.supp2 supplementary document
Interviews and focus group recordings were transcribed verbatim by MF. Thematic analysis, a technique for identifying patterns (themes) within qualitative data was used for the analysis. Transcripts were read by MF, SS and JO to familiarise themselves with the data. The transcripts were then coded by MF to identify initial themes in the data. Using these themes, MF and SS created an analytical model on which to base the remaining analysis. Themes within the model were revised by MF, SS and JO iteratively throughout the analysis. Representative quotes were chosen to illustrate the themes identified within the data set. Consideration was given to ensure all sociodemographic groups were adequately represented in the quotations. It was agreed by the three researchers involved in the analysis that data saturation had been reached, and no further interviews or focus group discussions were necessary. Participants were not given an opportunity to review the transcripts. Microsoft Excel was used to support the analysis.
Sample characteristics Thirty-three individuals participated in the study, 16 women and 17 men . Ages ranged from 50 to 69 years, with a mean age of 57 years. The majority of participants were white British (73%), approximately half of them were educated to degree level (52%), and 45% of the sample had experience of endoscopy. Sampling of roughly equal numbers of participants with previous experience with endoscopy occurred by chance rather than through purposive sampling. Ten individuals were interviewed and 23 took part in focus groups. Interviews ranged in length between 26 and 57 min, and focus group discussions between 64 and 86 min, with participants spending the longest discussing their personal heartburn experience and their impression of the Cytosponge. Participants expressed enthusiasm about the Cytosponge, with most indicating they would take the test if invited. The three major themes identified were the anticipated physical experience, information preferences and comparisons with endoscopy. Subthemes are presented below, along with representative quotes. Anticipated physical experience Individuals discussed their anticipated physical experience of the Cytosponge test. This included swallowing the Cytosponge and extracting it. The anticipated physical experience appeared to be shaped by both handling examples of the capsule and sponge, and watching the video of the test being carried out. Swallowing the sponge Based on initial images and verbal descriptions most individuals imagined the capsule to be bigger than its actual size. People expressed surprise and felt reassured after handling a Cytosponge. It was generally felt that the act of swallowing the Cytosponge capsule would not be problematic, because it was of a similar size and shape to other tablets they take regularly. It's smaller than I imagined … The capsule is just like a normal vitamin capsule, so that's perfectly fine (Participant 4, Focus Group 3, female, age 56, high education) A few people were concerned about swallowing the string attached to the capsule. It's just the string following it down I think I might have a problem with. I’ve never actually swallowed a ball of string (Participant 4, Focus Group 2, male, age 51, basic education) Extracting the sponge After feeling an expanded Cytosponge in the demonstration, participants said it was rougher than they expected, with many comparing it to a ‘Brillo pad’. As a result, there was concern that an expanded Cytosponge would damage their oesophagus when it was extracted. You’re going to feel it, it's going to be as if somebody's got their fingers and scratching the inside of your windpipe (Participant 6, Focus Group 2, male, age 56, basic education) Some people were worried they would gag or even vomit when the Cytosponge was withdrawn from the stomach. I’d be frightened that I’d throw up. I’ll be honest (Participant 5, Focus Group 4, female, age 55, basic education) A consistent concern was the possibility of the string breaking and the Cytosponge getting stuck in the oesophagus or stomach. What if it got stuck? Because you know sometimes when a sweet goes down the wrong way…and it gets stuck? That is scary (Participant 2, Focus Group 3, female, age 52, high education) Video reassurance After watching the video, individuals were more positive about the Cytosponge test compared with their initial reactions. The main reason for this attitude shift was because the extraction of the sponge was considerably quicker than expected. In my mind I was thinking of you know [removing the sponge would be like] catching a fish (Participant 5, Focus Group 2, male, age 50, basic education) Additionally, the ease with which the patient swallowed the Cytosponge was commented on. The fact the patient did not gag during its extraction was considered comforting. He didn’t show any gagging or anything. He was absolutely fine (LK, Interview, female, age 59, basic education) Finally, people commented that seeing the patient in the video looking relaxed put them at ease. He looked very relaxed, surprisingly… I think I'm sort of happier now, after watching the video (Participant 6, Focus Group 1, male, age 60, high education) Information preferences Individuals differed in their preferences for what information they would receive if they were asked to do the Cyosponge test. This included the amount of information they were given on the link between GERD, BE and EAC, on the Cytosponge test itself, whether they were shown an expanded Cytosponge before taking the test, and the optimal timing for informing patients about endoscopy referral. People also discussed their preferred format for communicating information. Information on cancer link Most participants had no or low awareness of the relationship between GERD, BE and EAC. I’ve never taken it [GERD] to the next step in my mind (Participant 2, Focus Group 3, female, age 52, high education) Some people said they would want to be told GERD can lead to EAC before they took the Cytosponge test, while others said they would not want to know about the association. Some perceived that discussing a link with cancer may frighten patients and reduce uptake. If you’re reading something with ‘cancer’, you’re frightening them anyway… You say cancer, people won't take the pill (Participant 1, Focus Group 2, male, age 58, basic education) Information on Cytosponge test Most participants felt the amount of information provided about the Cytosponge test during the interview or focus group was sufficient for them to decide whether they would have the Cytosponge test. I'd want to know everything you told us here [referring to earlier verbal descriptions, sample demonstrations and the video]. … What it does, how it does it, why you're doing it? (Participant 4, Focus Group 2, male, age 51, basic education) In addition, some people wanted to be informed about other details for example whether the test had to be performed on an empty stomach. There was also demand for information on side effects, even though they felt that those would probably be unlikely or mild. If there was any side effect, whether it has to be done on an empty stomach, like, you know, should it be done in the morning? (Participant 5, Focus Group 4, female, age 55, basic education) Other information people felt should be included regarded timings, specifically how long the test takes in total, how long the sponge stays in the stomach and the time it takes to receive results. Yeah, so what happens? Once it goes to the lab, how long will it take before you find out whether you've got it? (Participant 6, Focus Group 3, female, age 54, high education) Furthermore, individuals felt that information on the accuracy of the test should be included. I'd want to know how, like I say, I'd want to know how accurate it is as well. There's no point doing it if it's not accurate (Participant 4, Focus Group 2, male, age 51, basic education) Demonstration of Cytosponge test During the demonstration, some participants expressed surprise at the appearance of the Cytosponge after the capsule had dissolved and it had expanded. They felt that prospective patients would be unnerved by its change in appearance if they had not been shown how the Cytosponge looks after it has been extracted before the test. If they don’t tell you, you’re going to be in for a fright when you pull it out of your throat (Participant 4, Focus Group 2, male, age 51, basic education) One person conceded this may not be necessary, and that a similar approach would not be taken with other medical procedures. They wouldn’t do that with any other test, would they? They wouldn’t let you feel the needle before they took your blood (Participant 2, Focus Group 3, female, age 52, high education) Another approach would be to offer each patient a choice. I think it would be nice to be offered the choice. Maybe be asked ‘would you like to see how it looks? (Participant 5, Focus Group 2, male, age 50, basic education) Test outcomes There was debate over whether or not one should be told before taking the Cytosponge test that a positive result will necessitate further investigation. Some people insisted they would expect to be fully informed about the whole process at the outset, and compared it with the existing NHS screening programmes. I imagine it's just like NHS screening actually, when you have a mammogram you’re actually told what the programme is (Participant 5, Focus Group 3, female, age 60, high education) Others felt they would prefer to be told this information in a step by step approach, only being informed about the need for endoscopy after they have received a positive result. This preference was perceived to prevent unnecessary anxiety. I think that just having one test is scary enough for someone, thinking they might have cancer. So just stick to that test, and when the results come back, then they can be told what the next step is (LK, Interview, female, age 59, basic education) Information format Peoples' preferences for the modality in which information about the Cytosponge test was delivered varied. Suggestions included pictures, a leaflet, a website and a video. The preferred modality was an information leaflet with a website link to the video of the test being carried out. A link within the leaflet that you can actually go and see that video of the guy. I think that's really important (Participant 2, Focus Group 1, male, age 53, high education) A video was perceived by some individuals as being easier to understand than written information. Sometimes if it's late at night and my brain has, sort of, had enough for the day, I’d much rather just watch a little clip, you know I can get to see, and I can listen, I don’t have to read… reading is something you really have to really concentrate. So, the clip is almost doing it all for you (RH, Interview, female, age 54, basic education) One participant mentioned that she would also like to see a follow-up video of the patient so that she would know what to expect after the test had been performed. I would like to see him saying how he feels the next day… just another follow-up video…you know, ‘I’ve got a really bad sore throat, and, I feel a bit raw down there (AL, Interview, female, age 51, high education) Comparisons with endoscopy While the Cytosponge test is not intended to replace endoscopy, it was felt that the new device was preferable physically, practically and economically. Discomfort Fifteen participants had previously undergone endoscopies for their heartburn, and almost all reported an unpleasant experience. By comparison, the Cytosponge test was perceived to be a more comfortable procedure. The last [endoscopy] the nurse had to practically sit on me to stop me moving, because it was so unpleasant. And I was just regurgitating all this sort of yellow stuff, it was like something from a horror film (GC, Interview, male, age 51, high education) [The Cytosponge] is such an innocuous test. You swallow a pill, you pull it out, two weeks later, done. I can’t see anybody saying no to that (IF, Interview, male, age 55, high education) Practical factors Individuals were enthusiastic that the Cytosponge test would be a quicker procedure than endoscopy, could be carried out by their general practitioner, and would not require an anaesthetic. It's quicker, my doctor can do it and there's no messing around, no hospital appointments (AL, Interview, female, age 51, high education) The fact that people would be able to resume their everyday activities immediately after the procedure was also seen as a benefit. You can walk out of the doctor's surgery… you can get on with your everyday life (Participant 5, Focus Group 4, female, age 55, basic education) Economic factors A minority of people considered the superior cost-effectiveness of the Cytosponge test, and the benefits this would have for the healthcare system. That's going to be an awful lot cheaper to do than an endoscopy at an hour a go with a gastroenterologist (KP, Interview, female, age 58, basic education)
Thirty-three individuals participated in the study, 16 women and 17 men . Ages ranged from 50 to 69 years, with a mean age of 57 years. The majority of participants were white British (73%), approximately half of them were educated to degree level (52%), and 45% of the sample had experience of endoscopy. Sampling of roughly equal numbers of participants with previous experience with endoscopy occurred by chance rather than through purposive sampling. Ten individuals were interviewed and 23 took part in focus groups. Interviews ranged in length between 26 and 57 min, and focus group discussions between 64 and 86 min, with participants spending the longest discussing their personal heartburn experience and their impression of the Cytosponge. Participants expressed enthusiasm about the Cytosponge, with most indicating they would take the test if invited. The three major themes identified were the anticipated physical experience, information preferences and comparisons with endoscopy. Subthemes are presented below, along with representative quotes.
Individuals discussed their anticipated physical experience of the Cytosponge test. This included swallowing the Cytosponge and extracting it. The anticipated physical experience appeared to be shaped by both handling examples of the capsule and sponge, and watching the video of the test being carried out. Swallowing the sponge Based on initial images and verbal descriptions most individuals imagined the capsule to be bigger than its actual size. People expressed surprise and felt reassured after handling a Cytosponge. It was generally felt that the act of swallowing the Cytosponge capsule would not be problematic, because it was of a similar size and shape to other tablets they take regularly. It's smaller than I imagined … The capsule is just like a normal vitamin capsule, so that's perfectly fine (Participant 4, Focus Group 3, female, age 56, high education) A few people were concerned about swallowing the string attached to the capsule. It's just the string following it down I think I might have a problem with. I’ve never actually swallowed a ball of string (Participant 4, Focus Group 2, male, age 51, basic education) Extracting the sponge After feeling an expanded Cytosponge in the demonstration, participants said it was rougher than they expected, with many comparing it to a ‘Brillo pad’. As a result, there was concern that an expanded Cytosponge would damage their oesophagus when it was extracted. You’re going to feel it, it's going to be as if somebody's got their fingers and scratching the inside of your windpipe (Participant 6, Focus Group 2, male, age 56, basic education) Some people were worried they would gag or even vomit when the Cytosponge was withdrawn from the stomach. I’d be frightened that I’d throw up. I’ll be honest (Participant 5, Focus Group 4, female, age 55, basic education) A consistent concern was the possibility of the string breaking and the Cytosponge getting stuck in the oesophagus or stomach. What if it got stuck? Because you know sometimes when a sweet goes down the wrong way…and it gets stuck? That is scary (Participant 2, Focus Group 3, female, age 52, high education) Video reassurance After watching the video, individuals were more positive about the Cytosponge test compared with their initial reactions. The main reason for this attitude shift was because the extraction of the sponge was considerably quicker than expected. In my mind I was thinking of you know [removing the sponge would be like] catching a fish (Participant 5, Focus Group 2, male, age 50, basic education) Additionally, the ease with which the patient swallowed the Cytosponge was commented on. The fact the patient did not gag during its extraction was considered comforting. He didn’t show any gagging or anything. He was absolutely fine (LK, Interview, female, age 59, basic education) Finally, people commented that seeing the patient in the video looking relaxed put them at ease. He looked very relaxed, surprisingly… I think I'm sort of happier now, after watching the video (Participant 6, Focus Group 1, male, age 60, high education)
Based on initial images and verbal descriptions most individuals imagined the capsule to be bigger than its actual size. People expressed surprise and felt reassured after handling a Cytosponge. It was generally felt that the act of swallowing the Cytosponge capsule would not be problematic, because it was of a similar size and shape to other tablets they take regularly. It's smaller than I imagined … The capsule is just like a normal vitamin capsule, so that's perfectly fine (Participant 4, Focus Group 3, female, age 56, high education) A few people were concerned about swallowing the string attached to the capsule. It's just the string following it down I think I might have a problem with. I’ve never actually swallowed a ball of string (Participant 4, Focus Group 2, male, age 51, basic education)
After feeling an expanded Cytosponge in the demonstration, participants said it was rougher than they expected, with many comparing it to a ‘Brillo pad’. As a result, there was concern that an expanded Cytosponge would damage their oesophagus when it was extracted. You’re going to feel it, it's going to be as if somebody's got their fingers and scratching the inside of your windpipe (Participant 6, Focus Group 2, male, age 56, basic education) Some people were worried they would gag or even vomit when the Cytosponge was withdrawn from the stomach. I’d be frightened that I’d throw up. I’ll be honest (Participant 5, Focus Group 4, female, age 55, basic education) A consistent concern was the possibility of the string breaking and the Cytosponge getting stuck in the oesophagus or stomach. What if it got stuck? Because you know sometimes when a sweet goes down the wrong way…and it gets stuck? That is scary (Participant 2, Focus Group 3, female, age 52, high education)
After watching the video, individuals were more positive about the Cytosponge test compared with their initial reactions. The main reason for this attitude shift was because the extraction of the sponge was considerably quicker than expected. In my mind I was thinking of you know [removing the sponge would be like] catching a fish (Participant 5, Focus Group 2, male, age 50, basic education) Additionally, the ease with which the patient swallowed the Cytosponge was commented on. The fact the patient did not gag during its extraction was considered comforting. He didn’t show any gagging or anything. He was absolutely fine (LK, Interview, female, age 59, basic education) Finally, people commented that seeing the patient in the video looking relaxed put them at ease. He looked very relaxed, surprisingly… I think I'm sort of happier now, after watching the video (Participant 6, Focus Group 1, male, age 60, high education)
Individuals differed in their preferences for what information they would receive if they were asked to do the Cyosponge test. This included the amount of information they were given on the link between GERD, BE and EAC, on the Cytosponge test itself, whether they were shown an expanded Cytosponge before taking the test, and the optimal timing for informing patients about endoscopy referral. People also discussed their preferred format for communicating information. Information on cancer link Most participants had no or low awareness of the relationship between GERD, BE and EAC. I’ve never taken it [GERD] to the next step in my mind (Participant 2, Focus Group 3, female, age 52, high education) Some people said they would want to be told GERD can lead to EAC before they took the Cytosponge test, while others said they would not want to know about the association. Some perceived that discussing a link with cancer may frighten patients and reduce uptake. If you’re reading something with ‘cancer’, you’re frightening them anyway… You say cancer, people won't take the pill (Participant 1, Focus Group 2, male, age 58, basic education) Information on Cytosponge test Most participants felt the amount of information provided about the Cytosponge test during the interview or focus group was sufficient for them to decide whether they would have the Cytosponge test. I'd want to know everything you told us here [referring to earlier verbal descriptions, sample demonstrations and the video]. … What it does, how it does it, why you're doing it? (Participant 4, Focus Group 2, male, age 51, basic education) In addition, some people wanted to be informed about other details for example whether the test had to be performed on an empty stomach. There was also demand for information on side effects, even though they felt that those would probably be unlikely or mild. If there was any side effect, whether it has to be done on an empty stomach, like, you know, should it be done in the morning? (Participant 5, Focus Group 4, female, age 55, basic education) Other information people felt should be included regarded timings, specifically how long the test takes in total, how long the sponge stays in the stomach and the time it takes to receive results. Yeah, so what happens? Once it goes to the lab, how long will it take before you find out whether you've got it? (Participant 6, Focus Group 3, female, age 54, high education) Furthermore, individuals felt that information on the accuracy of the test should be included. I'd want to know how, like I say, I'd want to know how accurate it is as well. There's no point doing it if it's not accurate (Participant 4, Focus Group 2, male, age 51, basic education) Demonstration of Cytosponge test During the demonstration, some participants expressed surprise at the appearance of the Cytosponge after the capsule had dissolved and it had expanded. They felt that prospective patients would be unnerved by its change in appearance if they had not been shown how the Cytosponge looks after it has been extracted before the test. If they don’t tell you, you’re going to be in for a fright when you pull it out of your throat (Participant 4, Focus Group 2, male, age 51, basic education) One person conceded this may not be necessary, and that a similar approach would not be taken with other medical procedures. They wouldn’t do that with any other test, would they? They wouldn’t let you feel the needle before they took your blood (Participant 2, Focus Group 3, female, age 52, high education) Another approach would be to offer each patient a choice. I think it would be nice to be offered the choice. Maybe be asked ‘would you like to see how it looks? (Participant 5, Focus Group 2, male, age 50, basic education) Test outcomes There was debate over whether or not one should be told before taking the Cytosponge test that a positive result will necessitate further investigation. Some people insisted they would expect to be fully informed about the whole process at the outset, and compared it with the existing NHS screening programmes. I imagine it's just like NHS screening actually, when you have a mammogram you’re actually told what the programme is (Participant 5, Focus Group 3, female, age 60, high education) Others felt they would prefer to be told this information in a step by step approach, only being informed about the need for endoscopy after they have received a positive result. This preference was perceived to prevent unnecessary anxiety. I think that just having one test is scary enough for someone, thinking they might have cancer. So just stick to that test, and when the results come back, then they can be told what the next step is (LK, Interview, female, age 59, basic education) Information format Peoples' preferences for the modality in which information about the Cytosponge test was delivered varied. Suggestions included pictures, a leaflet, a website and a video. The preferred modality was an information leaflet with a website link to the video of the test being carried out. A link within the leaflet that you can actually go and see that video of the guy. I think that's really important (Participant 2, Focus Group 1, male, age 53, high education) A video was perceived by some individuals as being easier to understand than written information. Sometimes if it's late at night and my brain has, sort of, had enough for the day, I’d much rather just watch a little clip, you know I can get to see, and I can listen, I don’t have to read… reading is something you really have to really concentrate. So, the clip is almost doing it all for you (RH, Interview, female, age 54, basic education) One participant mentioned that she would also like to see a follow-up video of the patient so that she would know what to expect after the test had been performed. I would like to see him saying how he feels the next day… just another follow-up video…you know, ‘I’ve got a really bad sore throat, and, I feel a bit raw down there (AL, Interview, female, age 51, high education)
Most participants had no or low awareness of the relationship between GERD, BE and EAC. I’ve never taken it [GERD] to the next step in my mind (Participant 2, Focus Group 3, female, age 52, high education) Some people said they would want to be told GERD can lead to EAC before they took the Cytosponge test, while others said they would not want to know about the association. Some perceived that discussing a link with cancer may frighten patients and reduce uptake. If you’re reading something with ‘cancer’, you’re frightening them anyway… You say cancer, people won't take the pill (Participant 1, Focus Group 2, male, age 58, basic education)
Most participants felt the amount of information provided about the Cytosponge test during the interview or focus group was sufficient for them to decide whether they would have the Cytosponge test. I'd want to know everything you told us here [referring to earlier verbal descriptions, sample demonstrations and the video]. … What it does, how it does it, why you're doing it? (Participant 4, Focus Group 2, male, age 51, basic education) In addition, some people wanted to be informed about other details for example whether the test had to be performed on an empty stomach. There was also demand for information on side effects, even though they felt that those would probably be unlikely or mild. If there was any side effect, whether it has to be done on an empty stomach, like, you know, should it be done in the morning? (Participant 5, Focus Group 4, female, age 55, basic education) Other information people felt should be included regarded timings, specifically how long the test takes in total, how long the sponge stays in the stomach and the time it takes to receive results. Yeah, so what happens? Once it goes to the lab, how long will it take before you find out whether you've got it? (Participant 6, Focus Group 3, female, age 54, high education) Furthermore, individuals felt that information on the accuracy of the test should be included. I'd want to know how, like I say, I'd want to know how accurate it is as well. There's no point doing it if it's not accurate (Participant 4, Focus Group 2, male, age 51, basic education)
During the demonstration, some participants expressed surprise at the appearance of the Cytosponge after the capsule had dissolved and it had expanded. They felt that prospective patients would be unnerved by its change in appearance if they had not been shown how the Cytosponge looks after it has been extracted before the test. If they don’t tell you, you’re going to be in for a fright when you pull it out of your throat (Participant 4, Focus Group 2, male, age 51, basic education) One person conceded this may not be necessary, and that a similar approach would not be taken with other medical procedures. They wouldn’t do that with any other test, would they? They wouldn’t let you feel the needle before they took your blood (Participant 2, Focus Group 3, female, age 52, high education) Another approach would be to offer each patient a choice. I think it would be nice to be offered the choice. Maybe be asked ‘would you like to see how it looks? (Participant 5, Focus Group 2, male, age 50, basic education)
There was debate over whether or not one should be told before taking the Cytosponge test that a positive result will necessitate further investigation. Some people insisted they would expect to be fully informed about the whole process at the outset, and compared it with the existing NHS screening programmes. I imagine it's just like NHS screening actually, when you have a mammogram you’re actually told what the programme is (Participant 5, Focus Group 3, female, age 60, high education) Others felt they would prefer to be told this information in a step by step approach, only being informed about the need for endoscopy after they have received a positive result. This preference was perceived to prevent unnecessary anxiety. I think that just having one test is scary enough for someone, thinking they might have cancer. So just stick to that test, and when the results come back, then they can be told what the next step is (LK, Interview, female, age 59, basic education)
Peoples' preferences for the modality in which information about the Cytosponge test was delivered varied. Suggestions included pictures, a leaflet, a website and a video. The preferred modality was an information leaflet with a website link to the video of the test being carried out. A link within the leaflet that you can actually go and see that video of the guy. I think that's really important (Participant 2, Focus Group 1, male, age 53, high education) A video was perceived by some individuals as being easier to understand than written information. Sometimes if it's late at night and my brain has, sort of, had enough for the day, I’d much rather just watch a little clip, you know I can get to see, and I can listen, I don’t have to read… reading is something you really have to really concentrate. So, the clip is almost doing it all for you (RH, Interview, female, age 54, basic education) One participant mentioned that she would also like to see a follow-up video of the patient so that she would know what to expect after the test had been performed. I would like to see him saying how he feels the next day… just another follow-up video…you know, ‘I’ve got a really bad sore throat, and, I feel a bit raw down there (AL, Interview, female, age 51, high education)
While the Cytosponge test is not intended to replace endoscopy, it was felt that the new device was preferable physically, practically and economically. Discomfort Fifteen participants had previously undergone endoscopies for their heartburn, and almost all reported an unpleasant experience. By comparison, the Cytosponge test was perceived to be a more comfortable procedure. The last [endoscopy] the nurse had to practically sit on me to stop me moving, because it was so unpleasant. And I was just regurgitating all this sort of yellow stuff, it was like something from a horror film (GC, Interview, male, age 51, high education) [The Cytosponge] is such an innocuous test. You swallow a pill, you pull it out, two weeks later, done. I can’t see anybody saying no to that (IF, Interview, male, age 55, high education) Practical factors Individuals were enthusiastic that the Cytosponge test would be a quicker procedure than endoscopy, could be carried out by their general practitioner, and would not require an anaesthetic. It's quicker, my doctor can do it and there's no messing around, no hospital appointments (AL, Interview, female, age 51, high education) The fact that people would be able to resume their everyday activities immediately after the procedure was also seen as a benefit. You can walk out of the doctor's surgery… you can get on with your everyday life (Participant 5, Focus Group 4, female, age 55, basic education) Economic factors A minority of people considered the superior cost-effectiveness of the Cytosponge test, and the benefits this would have for the healthcare system. That's going to be an awful lot cheaper to do than an endoscopy at an hour a go with a gastroenterologist (KP, Interview, female, age 58, basic education)
Fifteen participants had previously undergone endoscopies for their heartburn, and almost all reported an unpleasant experience. By comparison, the Cytosponge test was perceived to be a more comfortable procedure. The last [endoscopy] the nurse had to practically sit on me to stop me moving, because it was so unpleasant. And I was just regurgitating all this sort of yellow stuff, it was like something from a horror film (GC, Interview, male, age 51, high education) [The Cytosponge] is such an innocuous test. You swallow a pill, you pull it out, two weeks later, done. I can’t see anybody saying no to that (IF, Interview, male, age 55, high education)
Individuals were enthusiastic that the Cytosponge test would be a quicker procedure than endoscopy, could be carried out by their general practitioner, and would not require an anaesthetic. It's quicker, my doctor can do it and there's no messing around, no hospital appointments (AL, Interview, female, age 51, high education) The fact that people would be able to resume their everyday activities immediately after the procedure was also seen as a benefit. You can walk out of the doctor's surgery… you can get on with your everyday life (Participant 5, Focus Group 4, female, age 55, basic education)
A minority of people considered the superior cost-effectiveness of the Cytosponge test, and the benefits this would have for the healthcare system. That's going to be an awful lot cheaper to do than an endoscopy at an hour a go with a gastroenterologist (KP, Interview, female, age 58, basic education)
In this sample of UK adults with GERD, the Cytosponge test was considered to be an acceptable and simple test to detect BE. Three key themes emerged from the data, including the anticipated physical experience of the test, preferences for the format and content of information, and comparisons with the current gold-standard test. These data can be used to inform the development of information materials and assist clinicians communicating with the public about the test. Initial reactions to the Cytosponge test focused on the anticipated physical experience. Common concerns included worries about swallowing the capsule and string, the texture of the sponge once the capsule had dissolved, and the potential for the device to break and become stuck. Despite these worries, nearly all participants were reassured by seeing examples of the device and a video of a previous patient undergoing the procedure. The observation that participants felt more inclined to take the Cytosponge test after watching the video demonstration reflects previous research into the use of multimedia to promote health behaviours, including screening attendance. Video demonstration of the test could be an important communicative approach to reduce test anxiety and promote informed uptake. Participants' information preferences varied. Opinions differed on how much information should be presented on the link between GERD, BE and EAC, test outcomes, and whether an example of an expanded Cytosponge should be shown. The content of health information materials is generally driven by the ethical and legal responsibility to promote informed decision-making. The preference of some patients to receive limited information therefore creates a tension between respecting public opinion, while also acting ethically and responsibly. The volume of questions asked by participants in the study and the variety of preferences suggests care should be taken during the design and evaluation of information resources. Ensuring the perspective of patient stakeholders is considered during this process may help to accommodate patient preferences. Evidence-based techniques and strategies for promoting comprehension should also be used to develop such resources. Overall, participants felt that the Cytosponge would be preferable for potential patients compared with the current gold-standard test, endoscopy. The device was considered superior in terms of the physical experience, the practical simplicity and the potential for economic savings to the healthcare service. Initial concerns about the device notwithstanding, public enthusiasm for the Cytosponge test is encouraging. On the basis of this initial acceptability study, recruitment of patients to the BEST3 trial and subsequently implementing the device within a primary care setting is unlikely to face insurmountable barriers from the public. Our study is the first to explore in-depth attitudes towards the Cytosponge test in a sample of adults with GERD. The use of interviews and focus groups provided a rich data set consisting of opinions expressed individually as well as in a group environment. The sample was diverse in terms of educational background and gender, allowing multiple perspectives to be considered. The study did have limitations. The sample was recruited using a market research company, and respondents may have been particularly motivated or interested in the topic. We did not collect quantitative data on acceptability or attitudes towards the test. Finally, the participants were not attending a medical appointment and therefore their attitudes towards the Cytosponge test were hypothetical, and may not reflect the opinions of patients in a clinical setting. This study has several implications for clinical practice. Both for BEST3 and if the Cytosponge test is implemented in routine care it will be important for information materials and health professionals to reassure patients about the procedure. In particular, concerns about the size of the device, the string breaking and the physical sensation on the throat when the device is withdrawn should be addressed. The speed with which the procedure can be completed should be emphasised to prevent exaggerated assumptions about a lengthy extraction. The study showed individuals hold strong preferences regarding the amount and format of information they are given. Consequently, health professionals should consider eliciting information preferences during the consultation. Finally, we demonstrated a need to raise awareness of the link between GERD, BE and EAC. Clinicians should not assume that patients are aware of the associations, and should carefully explain the context when discussing why recurrent GERD symptoms should be investigated.
This qualitative study revealed adults with GERD considered the Cytosponge test to be acceptable physically, practically and economically, as well as being preferred to endoscopy. If the device is implemented in routine clinical practice, it will be necessary to reassure patients about the Cytosponge and provide materials that meet the information needs of the patient group.
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Development and evaluation of a text analytics algorithm for automated application of national COVID-19 shielding criteria in rheumatology patients | 99e309eb-db55-4212-8234-f9990166ee1f | 11287580 | Internal Medicine[mh] | In April 2020, several Western countries used varying population-level strategies to identify patients who were clinically extremely vulnerable (CEV) to help mitigate the most severe adverse outcomes associated with COVID-19. For rheumatology patients in the UK, identification of these subgroups was based on a national risk-based score incorporating age, comorbidities and being on immunosuppressive therapies including biologics. It required manual review of semistructured clinical letters by rheumatology teams at a time, which was resource-intensive and inefficient. Natural language processing, a branch of artificial intelligence, offers opportunities to extract meaningful information from unstructured text data; however, there are few large-scale implemented examples in rheumatology. A text analytics-based algorithm for automated application of the COVID-19 CEV criteria was developed and applied to rheumatology clinical letters. Compared with manual scores performed by clinicians during the pandemic, it performed well with a sensitivity of 80% and specificity of 92%, with deployment of the algorithm requiring considerably less time. An automated algorithm for risk stratification could help reduce clinician time for manual review to allow more time for direct care and rapid deployment of a complex set of rules on all patient records, improving efficiency and transparently communicating decisions based on individual patient risk. It could also be adapted for future clinical use requiring rapid risk stratification of patients for pandemic planning or in other public health initiatives. In April 2020, at the start of the COVID-19 pandemic, the UK’s Scientific Committee issued extreme social distancing measures termed ‘shielding’. These were aimed at a subset of the UK population who were deemed clinically extremely vulnerable (CEV) to infection. Approximately 4.1 million patients over the course of the pandemic who were identified as CEV and perceived at increased risk of becoming seriously ill from COVID-19 were therefore asked to shield. It was advised that these individuals should not leave their homes and avoid face to-face contact based on individual risk factors. To guide efficient and systematic identification of these patients, national specialty societies produced risk stratification tools based on a patient’s age, immunosuppressive therapies and diagnoses directed at clinicians to quickly identify such patients . In outpatient-based specialties, such as rheumatology, where communication is commonly based on unstructured outpatient clinic letters, it was not possible to run a rapid search for patients with diagnostic and medication codes. Diagnoses for outpatient visits, outpatient prescribed medications (ie, majority of high-cost drugs such as biologics) and measures such as disease severity are only recorded in semistructured letters and are thus not machine readable, searchable or analysable across the population. Hospital clinicians needed to manually review sequential clinic letters of patients to manually score their risk as per national guidance and identify which patients should receive communications about shielding measures. This took whole rheumatology teams many dedicated sessions amounting to scores of hours to read through recent letters for all their patient cohort. Given the considerable clinician time required, manual evaluation of clinical records can be inefficient and inconsistent. This approach may not allow evaluation of every single patient attending the department due to the large patient volume. Reviewing only a subset of patients based on pre-existing likelihood of being high risk is error-prone and would likely miss many patients, and manually reviewing every patient record is cost and time prohibitive. A recent retrospective analysis based on National Health Service (NHS) digital data shows substantial regional variation in the proportions of people who were asked to shield across the country. While these differences may be in part due to variation in the prevalence of long-term conditions, the heterogeneity in the approaches between clinical teams due to workload pressures to identify CEV patients is likely to contribute to these discrepancies. In hospitals where manual evaluation was not possible due to clinical workload pressures, strategies included sending a generic letter to all patients about shielding and asking patients to self-identify if they met the set of complex criteria. However, personal interpretation of the complex guidance may lead to added anxiety during a period that was fraught with uncertainty. This approach also put the onus on the patient informing clinical teams about their CEV status to access additional support. For example, timely shielding classification was especially important to CEV patients for practical reasons such as obtaining a supermarket priority delivery slot and to provide evidence for the need to work from home. The process of identifying individuals to go onto the shielded list of patients was retrospectively critiqued for being reliant on inaccessible data and a ‘lack of joined up systems’. In this retrospective study, we explored the feasibility and potential benefits of using an automated approach to assist in efficient identification of patients who needed shielding, with a future benefit of adaptability as the guidance evolved. We based our approach on automated text analytics, which we applied to rheumatology department outpatient letters in a large foundation trust hospital providing secondary and tertiary-level care. Our aim was to implement and evaluate an algorithm that used patient demographics, automatically text-mined diagnoses and medications from outpatient letters to apply shielding rules in rheumatology. If successful, this could reduce the need for manual review of such tasks in the future. Specific objectives were to: Automatically extract and code comorbidities and medication exposures from outpatient letters. Implement and assess the performance of an algorithm to apply the British Society for Rheumatology (BSR) shielding guidelines, comparing identification of shielding requirements in a subset of patients with an automated approach with the manual record review by clinicians. Evaluate the time required to deploy the automated algorithm on electronic health records (EHRs) of all patients who attended the rheumatology clinic with at least two clinical letters prior to April 2020, when the shielding guidance was issued. Data source We retrieved outpatient letters for this study from the rheumatology department of Salford Royal Hospital, part of the Northern Care Alliance, a large foundation trust in the UK. Outpatient letters in the UK are the main method of communication between hospitals and primary care physicians. They are written as per the Professional Records Standards Body (PRSB) guidance and comprise of semistructured data that include relevant diagnoses and medications with additional free-text narrative. Since publication of the PRSB guidelines, it is expected that all outpatient clinical letters would follow this standard format in the UK. 10.1136/ard-2024-225544.supp1 Supplementary data Extraction of diagnoses and medications We retrieved all letters between 10 June 2013, when semistructured letters were consistently implemented in the hospital according to the national PRSB published guidelines, and 13 November 2020. Prior to June 2013, there was considerable heterogeneity in how clinicians structured their own outpatient letters. To obtain an appropriate level of clinical data required to address the objectives, we retrieved the two most recent letters prior to 1 April 2020 for each patient, with information about diagnoses and medications taken from the semistructured part of the letter only. We extracted outpatient letter subheading titles from the semistructured part of letters initially (n=3465) and reviewed by the research team. Headings used to denote diagnoses (eg, ‘rheumatological diagnoses’, ‘other diagnoses’) and medications (eg, ‘current medications’, ‘previous DMARDs’ (disease-modifying antirheumatic drugs)) were then selected. The hospital data science team used these headings to extract the relevant lists of diagnosis and medication text from the semistructured part of the letters to run the subsequent algorithms. No personal identifiable information was available to the research team. The lists of diagnoses were processed with Intelligent Medical Objects (IMO) software (Concept Tagger), which used IMO’s core interface terminology to map each free-text description of a condition to Systematized Nomenclature of Medicine Clinical Terms (SNOMED-CT) codes. IMO was chosen as the software as it specialises in developing, managing and licensing medical vocabularies, and it had an existing partnership in place with the research team to evaluate its performance compared with manual coding performed by experienced clinicians. In our previous work, we had demonstrated that the performance of this software is comparable with human coding of diagnoses. SNOMED-CT codes were then mapped to lists of codes for each specific comorbidity that was used in the risk scoring according to the BSR shielding guidance . For the purposes of this work, we assumed all diagnoses in the letter be current diagnoses. Based on previous work, we developed the Medication Concept Recognition tool (Named Entity Recognition) to retrieve medications (including their type, for example, immunosuppressant), dose, duration and the status (active/past) at the time of the letter from their textual description . We used a combination of existing text-mining tools, including MedEx and Stanza, to process the data and extract the necessary information . The medication status: past, current/active, planned medications or unknown, was established mainly based on the heading where they appeared. For instance, a medication appearing under ‘previous DMARDs’ meant that the medication’s status was past. The extraction algorithm also considered any additional information that might have been reported in the free-text medication list. This included any information that indicated that a medication was stopped or not started, even though it appeared under ‘current medication’. RxNorm relationships were used to map identified medications to the drug classes required for shielding decisions (eg, ‘Humira’ to ‘biological drugs’). Only those medications which were ‘active’ in April 2020 contributed towards scoring by the algorithm. We consolidated data for each patient by combining information from the latest two letters prior to April 2020, which were assumed to contain the most up-to-date patient information on diagnoses and active medications. For the information that appeared in both letters such as data on a patient has a particular comorbidity or on a particular medication, we kept only the information from the most recent letter, along with the date of the clinical encounter. If the diagnosis and medication information from the last two letters was different, the latest letter’s information provided the input for the algorithm, as would be commonly observed with medication/dose changes. Shielding algorithm The BSR published a rule-based score that specified numerical thresholds for three categories: shielding (score of 3 or above), self-isolation/social distancing according to patient discretion (score of 2) or social distancing alone as per the current government guidance (score of 1 or 0). illustrates the weight provided to different diagnoses and medications. The rule as specified by the BSR scoring grid was heavily weighted on specific medications such as biological drugs, immunosuppressive medications, glucocorticoids and cyclophosphamide. Highest scores were allocated to higher doses and recent exposure to glucocorticoids or being on cyclophosphamide within the last 6 months, requiring text-mining of dose and timing of these medications . Being over 70 years old or having one of several comorbidities added an additional 1 point. The exception was patients with pulmonary hypertension, who were recommended to be automatically placed in the shielding category. For instance, a patient aged 71 years on 1 April 2020 with type 2 diabetes (1 point total), on adalimumab and methotrexate (2 points) would score a total of 3 points, therefore requiring them to shield . Following dissemination of the BSR scoring grid, a similar risk stratification guide (without scores) was also published including measures such as disease activity alongside medication use . For the purposes of developing the algorithm, we focused on the BSR scoring grid as the default input, as it was easier to interpret clinically and was the one most implemented by the hospital clinical team. The medication, diagnosis and demographic variables were combined to output a score computed by an algorithm with R (V.4.2.2). Comparison with gold standard . At the time of the pandemic, manual review by the rheumatology consultant team was performed in a subset of rheumatology patients deemed likely to fall into the CEV category. Patient records for manual review were identified from pharmacy records indicating prescription of high-cost drugs (eg, biologics and Janus kinase inhibitors), cyclophosphamide and specific scoring conventional synthetic DMARDs (eg, methotrexate), as groups most likely to require shielding based on the BSR criteria. In the absence of being able to easily search for rheumatological diagnoses or medications as this information was not machine readable, locally held databases with details for patients with myositis and systemic sclerosis were also reviewed, as the hospital is a tertiary referral site for these conditions. For the purposes of this study, these decisions were deemed as the ‘gold standard’ against which the performance of the algorithm was calculated using standard performance metrics including sensitivity, specificity and F1 score. F1 score is a measure of predictive performance with values ≥0.7 indicating a good score. Statistical Disclosure Control was applied in line with the ethics approvals for the project. Evaluation Following algorithm development, all algorithm mismatches were manually reviewed by a clinical researcher on the binary measure of shielding or not. We first calculated the number of mismatched patients who had been identified as requiring shielding as per the algorithm but not through manual review (false positives), and vice versa (false negatives). These cases were then reviewed by evaluating the semistructured pseudonymised data (age, diagnoses, medications) extracted from letters without personal identifiable information, rather than the source letters themselves in line with the study approvals. Application of the algorithm to all patients and processing time. Following development of the shielding algorithm and iterative improvements on the data where a gold standard was available, we deployed the automated algorithm on a separate set of EHRs. This included all patients who attended the rheumatology department with at least two clinical letters prior to April 2020, excluding those from the training dataset described above. The time required to deploy the algorithm, once developed, was recorded. Patient and public involvement This work was informed and refined following discussions between a COVID-19 working group that included three to four patient representatives with long-term musculoskeletal conditions, one of whom had experienced a hospital admission with COVID-19. One patient partner who was interested in this work collaborated with the research and NHS teams on this project (LL) and contributed to the protocol and manuscript. We retrieved outpatient letters for this study from the rheumatology department of Salford Royal Hospital, part of the Northern Care Alliance, a large foundation trust in the UK. Outpatient letters in the UK are the main method of communication between hospitals and primary care physicians. They are written as per the Professional Records Standards Body (PRSB) guidance and comprise of semistructured data that include relevant diagnoses and medications with additional free-text narrative. Since publication of the PRSB guidelines, it is expected that all outpatient clinical letters would follow this standard format in the UK. 10.1136/ard-2024-225544.supp1 Supplementary data We retrieved all letters between 10 June 2013, when semistructured letters were consistently implemented in the hospital according to the national PRSB published guidelines, and 13 November 2020. Prior to June 2013, there was considerable heterogeneity in how clinicians structured their own outpatient letters. To obtain an appropriate level of clinical data required to address the objectives, we retrieved the two most recent letters prior to 1 April 2020 for each patient, with information about diagnoses and medications taken from the semistructured part of the letter only. We extracted outpatient letter subheading titles from the semistructured part of letters initially (n=3465) and reviewed by the research team. Headings used to denote diagnoses (eg, ‘rheumatological diagnoses’, ‘other diagnoses’) and medications (eg, ‘current medications’, ‘previous DMARDs’ (disease-modifying antirheumatic drugs)) were then selected. The hospital data science team used these headings to extract the relevant lists of diagnosis and medication text from the semistructured part of the letters to run the subsequent algorithms. No personal identifiable information was available to the research team. The lists of diagnoses were processed with Intelligent Medical Objects (IMO) software (Concept Tagger), which used IMO’s core interface terminology to map each free-text description of a condition to Systematized Nomenclature of Medicine Clinical Terms (SNOMED-CT) codes. IMO was chosen as the software as it specialises in developing, managing and licensing medical vocabularies, and it had an existing partnership in place with the research team to evaluate its performance compared with manual coding performed by experienced clinicians. In our previous work, we had demonstrated that the performance of this software is comparable with human coding of diagnoses. SNOMED-CT codes were then mapped to lists of codes for each specific comorbidity that was used in the risk scoring according to the BSR shielding guidance . For the purposes of this work, we assumed all diagnoses in the letter be current diagnoses. Based on previous work, we developed the Medication Concept Recognition tool (Named Entity Recognition) to retrieve medications (including their type, for example, immunosuppressant), dose, duration and the status (active/past) at the time of the letter from their textual description . We used a combination of existing text-mining tools, including MedEx and Stanza, to process the data and extract the necessary information . The medication status: past, current/active, planned medications or unknown, was established mainly based on the heading where they appeared. For instance, a medication appearing under ‘previous DMARDs’ meant that the medication’s status was past. The extraction algorithm also considered any additional information that might have been reported in the free-text medication list. This included any information that indicated that a medication was stopped or not started, even though it appeared under ‘current medication’. RxNorm relationships were used to map identified medications to the drug classes required for shielding decisions (eg, ‘Humira’ to ‘biological drugs’). Only those medications which were ‘active’ in April 2020 contributed towards scoring by the algorithm. We consolidated data for each patient by combining information from the latest two letters prior to April 2020, which were assumed to contain the most up-to-date patient information on diagnoses and active medications. For the information that appeared in both letters such as data on a patient has a particular comorbidity or on a particular medication, we kept only the information from the most recent letter, along with the date of the clinical encounter. If the diagnosis and medication information from the last two letters was different, the latest letter’s information provided the input for the algorithm, as would be commonly observed with medication/dose changes. The BSR published a rule-based score that specified numerical thresholds for three categories: shielding (score of 3 or above), self-isolation/social distancing according to patient discretion (score of 2) or social distancing alone as per the current government guidance (score of 1 or 0). illustrates the weight provided to different diagnoses and medications. The rule as specified by the BSR scoring grid was heavily weighted on specific medications such as biological drugs, immunosuppressive medications, glucocorticoids and cyclophosphamide. Highest scores were allocated to higher doses and recent exposure to glucocorticoids or being on cyclophosphamide within the last 6 months, requiring text-mining of dose and timing of these medications . Being over 70 years old or having one of several comorbidities added an additional 1 point. The exception was patients with pulmonary hypertension, who were recommended to be automatically placed in the shielding category. For instance, a patient aged 71 years on 1 April 2020 with type 2 diabetes (1 point total), on adalimumab and methotrexate (2 points) would score a total of 3 points, therefore requiring them to shield . Following dissemination of the BSR scoring grid, a similar risk stratification guide (without scores) was also published including measures such as disease activity alongside medication use . For the purposes of developing the algorithm, we focused on the BSR scoring grid as the default input, as it was easier to interpret clinically and was the one most implemented by the hospital clinical team. The medication, diagnosis and demographic variables were combined to output a score computed by an algorithm with R (V.4.2.2). Comparison with gold standard . At the time of the pandemic, manual review by the rheumatology consultant team was performed in a subset of rheumatology patients deemed likely to fall into the CEV category. Patient records for manual review were identified from pharmacy records indicating prescription of high-cost drugs (eg, biologics and Janus kinase inhibitors), cyclophosphamide and specific scoring conventional synthetic DMARDs (eg, methotrexate), as groups most likely to require shielding based on the BSR criteria. In the absence of being able to easily search for rheumatological diagnoses or medications as this information was not machine readable, locally held databases with details for patients with myositis and systemic sclerosis were also reviewed, as the hospital is a tertiary referral site for these conditions. For the purposes of this study, these decisions were deemed as the ‘gold standard’ against which the performance of the algorithm was calculated using standard performance metrics including sensitivity, specificity and F1 score. F1 score is a measure of predictive performance with values ≥0.7 indicating a good score. Statistical Disclosure Control was applied in line with the ethics approvals for the project. Following algorithm development, all algorithm mismatches were manually reviewed by a clinical researcher on the binary measure of shielding or not. We first calculated the number of mismatched patients who had been identified as requiring shielding as per the algorithm but not through manual review (false positives), and vice versa (false negatives). These cases were then reviewed by evaluating the semistructured pseudonymised data (age, diagnoses, medications) extracted from letters without personal identifiable information, rather than the source letters themselves in line with the study approvals. Application of the algorithm to all patients and processing time. Following development of the shielding algorithm and iterative improvements on the data where a gold standard was available, we deployed the automated algorithm on a separate set of EHRs. This included all patients who attended the rheumatology department with at least two clinical letters prior to April 2020, excluding those from the training dataset described above. The time required to deploy the algorithm, once developed, was recorded. This work was informed and refined following discussions between a COVID-19 working group that included three to four patient representatives with long-term musculoskeletal conditions, one of whom had experienced a hospital admission with COVID-19. One patient partner who was interested in this work collaborated with the research and NHS teams on this project (LL) and contributed to the protocol and manuscript. To train the algorithm and compare with manual decisions, we focused on 895 patients who were reviewed clinically at the start of the pandemic. Of 895 patients, 64 patients (7.1%) had not consented for their data to be used for research as part of the national opt-out scheme, hence have been excluded from subsequent analysis. Following removal of duplicate patients (n=3) and missing records (n=24), 803 patient records were available for analysis. Cohort derivation to obtain the final training data is described in . In this cohort, the baseline characteristics of the patients identified as those requiring shielding (29.8%, n=239) compared with those who did not (70.2%, n=564) following algorithm deployment, are presented in . Patients in the shielding category were older (mean age 67 years, SD 13) compared with those who were identified as not requiring shielding (55 years; SD 14). Patients identified as requiring shielding had a higher proportion of specific comorbidities such as pre-existing lung disease (n=123; 51%), diabetes (n=49; 21%) and rheumatoid arthritis (n=164; 69%), as well as being on glucocorticoids (n=84; 35%), in line with the BSR guidance . A total of 5942 free-text diagnoses were extracted and mapped to SNOMED-CT (average 7 per patient), along with a list of 13 665 free-text medication descriptions (average 17 per patient). This included 11 339 medications (83%) as those identified as being on current/active in the most recent letter prior to April 2020; 2130 medications (16%) as past medications; 164 (1%) as planned medications to be commenced in the future and 32 (0.2%) that were of unknown status. The automated algorithm for numerical risk scoring performed well and demonstrated a sensitivity of 80% (95% CI: 75%, 85%) and specificity of 92% (95% CI: 90%, 94%). Positive likelihood ratio was 10 (95% CI: 8, 14) and negative likelihood ratio was 0.21 (95% CI: 0.16, 0.28). False positive rate was 8%, while false negative rate was 20% . The performance of the algorithm when implementing the rules of the BSR stratification guide that incorporated disease activity was very similar, with a sensitivity of 80% and a specificity of 92% . Evaluation of mismatches Of the 43 patients who were identified as positive when the BSR scoring grid rules were deployed by the algorithm but where the clinical team had not allocated to shielding, evaluation revealed the following three themes: (1) genuine false positives due to how medications or comorbidities were text-mined by the algorithm (n=3, 7%); (2) no text-mining reason (n=5, 11%), which may indicate clinical judgement over-riding the national guidance; (3) clinical interpretation of national guidance (n=35, 81%). Further details and examples where possible are provided below. Of the three genuine false positives, two patients had issues regarding the temporality of the medication extraction. For instance, one letter containing the immunosuppressant medication in its ‘current’ section was followed by the reason for stopping in the future, which was still extracted as an active medication contributing to the shielding score. In one patient’s records, the mention of ‘pneumonia’ in the diagnosis section (that may be considered inactive/or an acute episode by the clinician) was mapped to pneumonitis as per the SNOMED dictionary, pneumonitis being a ‘parent code’, with pneumonia being one of the children of this term. According to the BSR guidance, any pre-existing lung disease (such as ‘pneumonitis’) would score a point. The majority of false positives were due to the interpretation of the sentence in both sets of BSR guidance: ‘Patients who have rheumatoid arthritis (RA) or connective tissue disease-related interstitial lung disease (ILD) are at additional risk and may need to be placed in the shielding category.’ This sentence may have been interpreted by the clinical team as patients with RA alone or those with RA-ILD. The developed algorithm scored an additional 1 point for having RA, thereby tipping them into the shielding category. When RA was removed as scoring a point in the algorithm, the algorithm performance dropped considerably. The 47 false negatives were also evaluated (ie, those patients who were deemed as requiring shielding but not detected as such by the algorithm). The primary reason for false negatives was medication metadata, specifically prednisolone dose not being correctly captured and incorporated towards the shielding score (n=28, 60%). This was often due to more complex instructions to the primary care team such as ‘Prednisolone 30 mg to be reduced in 5 mg increments every x days until reduced to a dose of 10 mg ’ . The algorithm captured the medication name correctly but was unable to calculate the current dose at the time of the pandemic, hence affecting the total score. No text-mining reason accounted for 25% (n=12) of false negatives. This included instances of patients being on a combination of two biological drugs for very active psoriatic arthritis and psoriasis, which would score a maximum of 2 points according to the guidance (3 required for shielding). Other examples included patients who had been receiving multiple intramuscular steroids, despite being on immunosuppressants for active/uncontrolled inflammatory arthritis, who may also be deemed clinically to be at much higher risk. In four individual patients (8.5%), there were extraction issues such as typographical errors contributing to misclassification. Examples include the prednisolone dose not extracted from the letters; hence, the patient scored 1 point rather than 2 as per the algorithm. Finally, clinical interpretation of the guideline accounted for 6% (n=3) false negatives. This primarily included patients with RA and ILD, where the decision for shielding as per the guidance was left up to the clinician (as above). Deployment of the algorithm and processing time There were an additional 15 865 patients’ outpatient letters that had two letters available prior to April 2020 (including those that may no longer be active patients in the department). shows the results of deploying the algorithm on these additional patients, which identified between 729 and 848 patients who met criteria for shielding using the two published BSR criteria. In terms of processing time, extracting diagnoses and medications (natural language processing (NLP) step) from free-text data took 18 hours for the 15 865 patients. However, once medications were extracted, running the shielding algorithm took an hour. Of the 43 patients who were identified as positive when the BSR scoring grid rules were deployed by the algorithm but where the clinical team had not allocated to shielding, evaluation revealed the following three themes: (1) genuine false positives due to how medications or comorbidities were text-mined by the algorithm (n=3, 7%); (2) no text-mining reason (n=5, 11%), which may indicate clinical judgement over-riding the national guidance; (3) clinical interpretation of national guidance (n=35, 81%). Further details and examples where possible are provided below. Of the three genuine false positives, two patients had issues regarding the temporality of the medication extraction. For instance, one letter containing the immunosuppressant medication in its ‘current’ section was followed by the reason for stopping in the future, which was still extracted as an active medication contributing to the shielding score. In one patient’s records, the mention of ‘pneumonia’ in the diagnosis section (that may be considered inactive/or an acute episode by the clinician) was mapped to pneumonitis as per the SNOMED dictionary, pneumonitis being a ‘parent code’, with pneumonia being one of the children of this term. According to the BSR guidance, any pre-existing lung disease (such as ‘pneumonitis’) would score a point. The majority of false positives were due to the interpretation of the sentence in both sets of BSR guidance: ‘Patients who have rheumatoid arthritis (RA) or connective tissue disease-related interstitial lung disease (ILD) are at additional risk and may need to be placed in the shielding category.’ This sentence may have been interpreted by the clinical team as patients with RA alone or those with RA-ILD. The developed algorithm scored an additional 1 point for having RA, thereby tipping them into the shielding category. When RA was removed as scoring a point in the algorithm, the algorithm performance dropped considerably. The 47 false negatives were also evaluated (ie, those patients who were deemed as requiring shielding but not detected as such by the algorithm). The primary reason for false negatives was medication metadata, specifically prednisolone dose not being correctly captured and incorporated towards the shielding score (n=28, 60%). This was often due to more complex instructions to the primary care team such as ‘Prednisolone 30 mg to be reduced in 5 mg increments every x days until reduced to a dose of 10 mg ’ . The algorithm captured the medication name correctly but was unable to calculate the current dose at the time of the pandemic, hence affecting the total score. No text-mining reason accounted for 25% (n=12) of false negatives. This included instances of patients being on a combination of two biological drugs for very active psoriatic arthritis and psoriasis, which would score a maximum of 2 points according to the guidance (3 required for shielding). Other examples included patients who had been receiving multiple intramuscular steroids, despite being on immunosuppressants for active/uncontrolled inflammatory arthritis, who may also be deemed clinically to be at much higher risk. In four individual patients (8.5%), there were extraction issues such as typographical errors contributing to misclassification. Examples include the prednisolone dose not extracted from the letters; hence, the patient scored 1 point rather than 2 as per the algorithm. Finally, clinical interpretation of the guideline accounted for 6% (n=3) false negatives. This primarily included patients with RA and ILD, where the decision for shielding as per the guidance was left up to the clinician (as above). There were an additional 15 865 patients’ outpatient letters that had two letters available prior to April 2020 (including those that may no longer be active patients in the department). shows the results of deploying the algorithm on these additional patients, which identified between 729 and 848 patients who met criteria for shielding using the two published BSR criteria. In terms of processing time, extracting diagnoses and medications (natural language processing (NLP) step) from free-text data took 18 hours for the 15 865 patients. However, once medications were extracted, running the shielding algorithm took an hour. We successfully developed an automated text-mining algorithm to identify patients who required shielding as per national guidance based on patient demographics, medications and diagnoses. The automated algorithm demonstrated good specificity and reasonable sensitivity when compared with manual clinical decisions. Evaluation revealed the algorithm predominantly performed correctly against the rules. An automated algorithm for risk stratification has several advantages including reducing clinician time for manual review to allow more time for direct care, rapid deployment of a complex set of rules on all patient records across the whole department (rather than a subset) and improving efficiency and transparently communicating decisions based on individual risk. With further development, it has the potential to be adapted in other specialties that used similar risk stratification and for future public health initiatives that require prompt automated review of hospital outpatient letters. When run across 15 865 patients, the data extraction and the developed algorithm deployment took 19 hours. If it is assumed that manual review for each patient, which requires review of multiple letters, takes on average between 7 and 10 min per patient, clinicians would be required to spend a total of between 104 and 149 hours reviewing a high-risk subset of 895 patients. Reviewing all 15 865 patients would have taken between 1850 and 2644 hours (77–110 days), which was simply not feasible given the need for prompt communication with patients in this setting. A recent survey in 2022, providing insights from nearly 1000 healthcare professionals, revealed that the average time spent generating clinical documentation was 13.5 hours a week. Consultant doctors spent on average 4.7 hours outside of normal working hours on such tasks. Additionally, the value of time for a consultant creating/adding to clinical documentation and searching for missing information was nearly £57 000 per doctor per annum. Automation of shielding guidance or future public health guidance requiring outpatient EHRs has several additional benefits, including cost implications of person-time. Importantly, it also allows for improving transparency of decisions, not only for patients deemed CEV but also those who were deemed to be of intermediate or low risk. While we used clinical manual scores as the gold standard for assessing algorithm performance as it was the best available, physicians used their clinical discretion in some cases that over-rode the scoring system. An example may include an off-license but clinically appropriate use of two biologics in the same patient, which would score 2 according to the national rules, but a clinician may decide that this patient would be at high risk of infection therefore appropriately allocate them to the shielding category. Another key reason for lower sensitivity of 80% and false negatives from the evaluation was due to limitations in accurately assessing medication use during the risk window. This is a challenging area that needs to incorporate a range of complex tasks, including identifying the following: (1) the heading under which a given medication was listed; (2) any additional medication start/stop information that can over-ride the heading (eg, a medication listed under ‘current medications’ but with a note that the patient stopped taking it due to an adverse event but plans to restart at a later date); (3) the dose of drug in April 2020, particularly if there are plans to taper, pause or escalate, as was frequently the case with glucocorticoids; (4) duration of medication required to score specific drugs as per the national rules may not be always stated in the letter. For instance, the rule ‘intravenous cyclophosphamide within the last 6 months ’ requires a date of the last cyclophosphamide infusion within the letter that is clearly dated to calculate if it was administered within 6 months of 1 April 2020. To address these challenges, we combined a set of available tools to extract relevant information. However, the evaluation highlights the need for further refinement to improve identification and profiling of medications and diagnoses as reported in outpatient letters. One way of addressing this would be to allow the algorithm to have a third result, apart from the advice to shield or not to shield. This could be using a rule-based approach to identify and single out expressions that contain complex medication instructions, such as multiple dosage mentions and temporal elements associated with a single medication name, to output a third category ‘for manual review’. Those records could be assessed individually by the medical team. Another solution would be to decide between the clinical team a set of a priori additional ‘rules’ that would allow the algorithm to increase the score when certain clinical exceptions were encountered (e.g., being on two biological drugs). While the algorithm performed correctly against the set of rules, in the case of 11% of false positives and 25% of false negatives were not due to failures in the automated extraction and algorithm. Evaluation of these instances implied that clinical judgement correctly over-rode the national set of rules, as discussed above. The later published BSR risk stratification guide also included assessment of disease activity with rules such as ‘well-controlled patients with minimal disease activity ’ . However, disease activity measures are not always captured in the semi-structured parts of letters and may be recorded in the free-text part of the letter. As opposed to clinicians, the algorithm did not have access to the narrative part of clinical outpatient letters, which might have further information on the current medication status, as well as information on disease severity used in the decision-making. Therefore, this rule was out of scope for the algorithm, however, could be introduced following future development. From a patient perspective, qualitative research has described the increased emotional work of feeling forgotten about and navigating the conflicting advice given when notifications came from multiple different sources. Patients value timely, clear communication about their shielding status. Efficient deployment of the developed algorithm may have improved this aspect of the shielding process. From a clinician perspective, during the pandemic, rheumatology clinicians were also redeployed to help with the front line, which in some cases led to clinicians feeling there was less time to directly support rheumatology patients despite wanting to do so. Automating clinical processes that are administrative and time-consuming would have allowed more time for these interactions with rheumatology patients. The results of the study need to be interpreted in the context of certain limitations. The first is in line with the information governance and approvals of the study; we did not access the patient records of the 64 patients who opted out as part of the national data opt-out scheme. Therefore, should such algorithms be implemented in real time in the future for clinical purposes, consideration needs to be made on how to handle decisions and communicate those clinical decisions. Second, we did not have access to the free-text information within letters due to the higher risk of patient re-identification. Therefore, information regarding disease activity, medication dose adjustments or other social factors that may have influenced the clinical decision to shield were not available. We have however used this work to leverage further funding to build on the existing tools. Within this programme, we plan to use a suite of deidentification software that will be applied to the free-text narrative of each letter to remove explicit mentions of key personal identifiable information. This would reduce the risk of accidental reidentification to researchers and enable further NLP development to extract other measures of interest such as disease activity and quality of life. Third, the process to develop the algorithm was an iterative one, which was not included in the above deployment time as not accurately captured across different team members. However, once developed, it would be able to be easily adapted for future public health risk stratification if needed. Since development of the algorithm that relied on rule-based NLP, large language models (LLMs) such as GPT4 have surged in popularity and are being leveraged in several settings. However, LLMs are associated with their own limitations including black box models, LLM hallucinations and introducing different types of bias. How they could be implemented in a healthcare setting on outpatient letters that contain sensitive patient information to replace text analytics has not been well established. Beyond COVID-19, the algorithm has the future potential to be adapted for several purposes that include addressing questions regarding service provision, clinically important research and related to population health. For instance, it is currently not possible to easily determine the prevalence of a particular group of conditions such as inflammatory arthritis within a hospital, due to a lack of structured coded diagnoses in an outpatient setting. The developed algorithm could be adapted to address what proportion of rheumatology patients with a certain diagnosis are on a specific biological drug or medication. This type of information would be extremely useful for cohort selection to aid clinical trial recruitment, for quality improvement purposes and to estimate financial costs for local authorities with a specific condition/medications with consequent policy implications. Quickly identifying patients on specific medications within clinic letters could help provide a safety net for DMARD monitoring. Capturing dose and frequency of the medications across letters can enable insights into specific clinical/research questions such quantifying dose tapering across a department and subsequent cost-savings. The COVID-19 pandemic has illustrated the pertinent public health need to efficiently use data collected at the point of care to rapidly evaluate comorbidities and medications timely and efficiently. Establishing partnerships between academia, secondary care business intelligence teams, patient and clinical representatives can allow development of new algorithms using text analytics that could ultimately improve patient care and save clinician time, in an already overstretched health service. |
J-ACCESS investigation and nuclear cardiology in Japan: implications for heart failure | ea50abf4-0a37-4dc0-8b8d-719c23aad896 | 10195738 | Internal Medicine[mh] | Coronary artery disease (CAD) remains the leading cause of mortality and morbidity in developed countries, including Japan. The global prevalence of HF is increasing despite advances in pharmacological and non-pharmacological therapies, and early recognition and appropriate treatments are recognized as major health-related issues. The number of patients aged > 20 years with HF is 6.5 million in the USA, and > 1.2 million in Japan with HF are outpatients . The Japanese Registry of All Cardiac and Vascular Diseases (JROAD) statistics generated by the Japan Circulation Society ( https://www.j-circ.or.jp/jittai_chosa/about/report/https://www.j-circ.or.jp/jittai_chosa/about/report/ , accessed on December 2022) indicate that ~ 0.3 million patients were diagnosed and hospitalized with HF in Japan during 2020 (Fig. ). The prevalence of HF among persons aged ≥ 20 years in the USA was 2.1% (5.1 million) during 2010 , when the estimated prevalence in Japan was 1%. However, the population of people aged > 75 years is increasing and is predicted to reach 20% by 2025. In fact, statistics from the Japanese Registry of Acute Decompensated Heart Failure (JROADHF) show that the mean age of 13,238 patients at 128 hospitals was 78.0 ± 12.5 years, with those aged > 75 years accounting for 68.9%. During a median follow-up of 4.3 years, the respective rates of cardiovascular death and the re-hospitalization for HF were 7.1 and 21.1 per 100 person-years . The relatively low incidence (26.6%) of ischemic etiology in this multicenter registry is characteristic of Japanese patients with HF, but the proportion of HF caused by ischemic heart disease has recently increased. In contrast, the proportion of ischemic etiology in the European Society of Cardiology HF long-term (ESC-HF-LT) registry is 56.5% . Thus, we anticipate a rapid increase in the numbers of patients with HF, which will also increase the burden on the Japanese healthcare system. Moreover, the social frailty of super-aged elderly and the increasing proportions of individuals with dementia are important targets in Japan . The rate of pump-failure death is high in aged populations with HF. Moreover, HF with preserved ejection fraction (HFpEF) might have contributed to 16–50% of HF according to recent databases , and hypertension and ischemic heart disease are major underlying diseases in Japan. Strategies for avoiding sudden cardiac death could also be an important target in younger patients. The Chronic Heart Failure Analysis and Registry in the Tohoku District (CHART) studies 1 and 2 have identified westernized ischemic etiology and clinical characteristics in Japan as increased comorbidity and HF admissions . An overview of imaging modality statistics in JROAD showed that the rate of coronary CT angiography (CCTA) evaluations has rapidly increased, whereas that of myocardial perfusion imaging (MPI) has gradually decreased over the past decade (Fig. ). Coronary angiography (CAG) and elective percutaneous coronary interventions (PCI) have slightly decreased since 2018, which was partly due to changes in reimbursement policies that are discussed in the section entitled Guidelines for stable CAD updated by the Japanese Circulation Society (JCS) below. With respect to nuclear cardiology practice, few multi-center registries in Japan include patients with CAD and HF, and the prospective prognostic study of Japanese Assessment of Cardiac Events and Survival Study by Quantitative Gated SPECT (J-ACCESS) 1‒4 are important databases for understanding the role of MPI . Although J-ACCESS includes patients with definite or suspected CAD and investigations of HF outcomes were not intended, the most prevalent outcome was severe HF requiring hospitalization. Therefore, the incidence and the reasons for HF events in patients who underwent nuclear studies require clarification. This article reviews J-ACCESS investigations conducted since 2001 to the present, as well as recent Japanese trends in nuclear cardiology, and discusses possible roles of nuclear imaging with a focus on HF.
Epidemiological and race differences between Japan and the USA as well as European countries should be considered when evaluating prognoses associated with MPI. However, such a database was not available in Japan during the year 2000. We therefore created the first nationwide database that specifically focused on evaluations by quantitative myocardial perfusion single-photon emission computed tomography (SPECT), namely the Japanese Assessment of Cardiac Events and Survival Study by Quantitative Gated SPECT (J-ACCESS; QGS; Mount Sinai Medical Center, Los Angeles, CA, USA) . Four series of J-ACCESS investigations over the next two decades focused on different types of targeted patients (Table , Fig. ). The J-ACCESS registered 4629 consecutive patients with confirmed or suspected CAD from 117 institutions in 2001. All patients underwent 99m Tc-tetrofosmin stress-rest studies and quantitative perfusion defect scoring with summed stress/rest/difference scores (SSS/SRS/SDS) and quantitative left ventricular functional analysis . The major events were cardiac death, non-fatal myocardial infarction (MI), and severe HF requiring hospitalization. These patients were followed up for 3 years during the J-ACCESS investigations 1‒3, when 175 (4.3%) patients developed major cardiac events, of which half were severe HF requiring hospitalization (Fig. A,B,C). Although the rate of major events was lower in Japan than in the USA between 1998 and 2003 , the major predictors of events were similar among several multicenter studies. For example, normal myocardial perfusion SPECT with normal ventricular function indicated a good prognosis . Myocardial perfusion defects at stress and a lower left ventricular ejection fraction (LVEF) were major predictors of cardiac events. Having comorbid diabetes mellitus or chronic kidney disease (CKD) increased major event risk ~ twofold, which was less fatal but similar to findings in the USA . Perfusion abnormalities revealed by coronary angiography (CAG) had significant additive value over coronary stenosis for predicting cardiac events . A propensity-score-matched analysis of early revascularization and medications revealed that such abnormalities were effective for patients with > 10% ischemia (Fig. E) . While the study included a high proportion of serious HF events (49‒77%; J-ACCESS-1, 3 and 4), CKD, greater SSS, and higher end-systolic volume (ESV) or low LVEF were independent and additive predictors of refractory HF risk in patients with confirmed or suspected CAD . The J-ACCESS 2 study investigated the prognosis of patients with type-2 diabetes and asymptomatic CAD . Abnormal perfusion findings indicated myocardial ischemia and/or scar in 32% of 485 patients. Five (1.0%) of 485 patients developed fatal cardiovascular events, and severe HF and other events comprised mostly non-fatal acute coronary syndrome, new onset of stable angina pectoris, and cerebrovascular accidents in 9 (1.9%), 10 (2.1%), and 15 (3.1%) of these patients, respectively, during a 3-year follow-up. Multivariate analysis revealed that SSS ≥ 9, low eGFR, and currently smoking were significant variables, indicating that this group of patients needed active treatment strategies. The J-ACCESS 3 prospective cohort study of CKD investigated predictions of cardiac events among patients with eGFR < 50 mL/min/1.73 m 2 without definitive CAD . The event-free survival rate was lower among patients with renal dysfunction and a higher SSS (Fig. D). A C-reactive protein value of ≥ 0.3 mg/dL independently predicted cardiac events, suggesting that this additional inflammatory parameter is important as a pathophysiological basis for developing cardiovascular events. The highest fraction of the events consisted of severe HF (77%), which confirmed the importance of therapeutic intervention for patients with CKD (Fig. F). The J-ACCESS 4 study investigated patients with coronary revascularization . Although cardiology guidelines describe myocardial ischemia as an indication for coronary revascularization in patients with chronic stable CAD, whether myocardial ischemia could be a target of coronary revascularization to reduce cardiac events was not verified in a Japanese population. When patients were classified into two groups with a threshold of 5% ischemic reduction, the prognosis of those with ≥ 5% improvement after revascularization was significantly better, and essentially agreed with the findings of a large cohort in the COURAGE study . The J-ACCESS series revealed a lower prevalence of CAD and a better prognosis for Japanese patients than those in other developed countries. The hard cardiac event rate of Japanese patients was < 3% over 3 years, which was less than half that of patients in the USA .
Appropriate risk stratification is a major target of MPI studies because total risk to patients can be determined by comorbidities in addition to perfusion and function. We therefore proposed a risk chart or software based on statistical multivariable models derived from the J-ACCESS results. The risk model included the variables of age, SSS, LVEF, and diabetes in the initial model, then was revised by adding eGFR as a variable . The risk chart and Heart Risk View software (Nihon Medi Physics, Tokyo, Japan) have been incorporated into current clinical practice. The risk model calculates major event rates as ratios per 3 years. This model was intended for application to patients with suspected CAD and a background similar to that in the J-ACCESS study . We validated the model by comparing predicted and actual outcomes between a new patient cohort in the APPROACH study and patients in J-ACCESS 3 . The results showed that the risk model effectively stratified risk, whereas actual event rates were equally high across all risk groups among patients with eGFR < 15 mL/min/1.73 m 2 . The model was not intended simply to numerically report event rates. More importantly, risk stratification incorporating MPI and clinical variables allowed the classification of patients at low, intermediate, and high risk for cardiovascular events. This provided useful information upon which to base decisions regarding medications and other types of therapies such as coronary revascularization (Fig. ). Although risk can be evaluated by stress MPI, how prognostic indices can be applied in clinical practice is more important . Even when degrees of ischemia are similar, event rates will be higher in patients with, than without comorbid diabetes, CKD and a reduced LVEF. This might have a practical value when considering invasive therapeutic strategies. Table shows multivariable logistic analyses recalculated to predict major cardiac events using the continuous variable of age, and the categorical variables of LVEF (< 35%, 35‒50%, > 50%), eGFR stage (G1, ≥ 90; G2, 60‒89; G3, 30‒59; G4, 15‒29; G5, < 15 mL/min/1.73 m 2 ), diabetes (0, 1), and SSS grades (0, 1, 2, and 3: scores < 4; 4‒8; 9‒12 and > 12, respectively). This table was recalculated based on previous information using the same categorical variables except for age. The model structure revealed how each variable affects major event outcomes. Combining moderate-to-high SSS, low eGFR, low LVEF, and diabetes synergistically doubled the calculated risk (Fig. ). Figure shows levels of risk for major cardiac events estimated by the model to understand the contribution of each factor. Since the model included severe HF as a major event, we created another risk model, namely cardiac death and non-fatal MI for estimating hard events. Diagnostic use of multivariable risk model incorporating SSS, left ventricular volume, diabetes, hypertension, and number of risk factors has also been proposed for the diagnosis of multivessel CAD and indications for stress-only imaging . A combination of diabetes and CKD offers important predictors when considering the background of Japanese patients with HF, because the JROAD HF statistics revealed that 30.1% and 38.9% of patients, respectively, had comorbid diabetes and CKD . The applicability of such a risk model requires careful consideration, as it is not universally applicable to diverse populations. The backgrounds of patients upon which to create the model, contemporary medications and interventions, comorbidities, healthcare systems, and many other factors affect predictive models. Our model was applicable to Japan, and an Italian prognostic study found its direct use limited . However, the principal ideas including major variables, such as age, myocardial perfusion or extent of ischemia, LV function, and comorbid diabetes, and CKD fit Japanese patients might be acceptable choices of predictors in general.
The ISCHEMIA trial highlighted the effectiveness of a contemporary optimal medical therapy (OMT) strategy including risk factor modification to prevent serious cardiac events . The COURAGE study also found that adding coronary revascularization to OMT did not necessarily result in a prognostic benefit, whereas patients with ≥ 5% reduction in myocardial ischemia had lower risk for death and myocardial infarction irrespective of treatment strategies . These new perspectives also affected Japanese clinical practice, and the JCS suggested strategic algorithm , whereas the prevalence of CCTA evaluations has rapidly increased. The updated guidelines incorporated strategies starting from pre-test probability and a clinical likelihood of CAD to guide downstream non-invasive tests for stable CAD. Patients with intermediate-to-high pre-test probability can undergo further non-invasive imaging modalities, including CCTA and stress imaging. When both CCTA and stress imaging are available, CCTA is preferred to rule out CAD, whereas functional stress imaging is preferred as an initial screen of patients with a high pre-test probability or known history of CAD for risk assessment. The approaches of CCTA and stress imaging are typically summarized as rule-out and rule-in strategies, respectively. After post-test risk assessment, OMT is generally applied in the next step, and a decision to proceed with invasive coronary angiography will be reached for patients with uncontrolled angina despite OMT, high-risk CAD such as left main CAD or its equivalent, inconclusive non-invasive imaging tests, and HF with suspected CAD. Measurements of fractional flow reserve (FFR) are also important to determine indications of PCI aimed at reducing cardiovascular events. The prospective, multicenter Japanese CVIT-DEFER registry that included 3,228 of 3,804 consecutive patients with angiographically moderate lesions of the coronary artery found frequent mismatches between angiographic stenosis and FFR, suggesting the clinical importance of physiological assessments to guide PCI . Japanese reimbursement for elective PCI in patients with CAD was updated in 2018 to include a need for functionally proven ischemia, which slightly decreased the prevalence of CAG and elective PCI (Fig. ). Considering the recent rapid increase in the numbers of patients undergoing CCTA in Japan, a CCTA-first strategy might prevail. Nonetheless, the increase in aged patients with multiple comorbidities and the recommendations for physiology-based PCI support the application of nuclear cardiology approaches. Not simply taking a dogmatic CT-first approach is an important viewpoint. The most appropriate test should be selected based on the findings of careful clinical evaluation to ensure the “right test for the right patient” . The patient-first approach is the key to clinical practice in an era when multimodal imaging has become part of a widespread clinical workup. Types of coronary revascularization, namely PCI and CABG, and medications have also become important topics. Ten-year data from the STICH studies have associated lower all-cause mortality with surgical revascularization compared with medical therapy . The JROAD statistics (2021) revealed ~ 76,000 and ~ 172,000 emergency and elective PCIs, respectively, as well as ~ 9100 (on pump) and 7300 (off pump) coronary artery bypass surgeries (CABGs). The ratio of CABG to PCI was 6.6%. In contrast, trends in coronary revascularization in the USA revealed a CABG-to-PCI ratio of 39%, while the numbers of both PCIs and CABGs decreased between 2003 and 2016 . A comparison of FFR-guided PCI and CABG in the FAME-3 investigation of three-vessel disease showed that PCI was not non-inferior to CABG with respect to the incidence of a composite of death, MI, stroke, or repeat revascularization at one year . The CREDO-Kyoto PCI/CABG registry Cohort-3 (14,927 patients; follow-up, 5.7 years) assessed all-cause and cardiovascular death among patients with three-vessel disease . That study associated PCI with a significantly higher risk for all-cause death than CABG, whereas risk for cardiovascular death among those treated by PCI did not exceed CABG in the real-world clinical era of drug-eluting stents in Japan. While stent technology and materials have improved, coronary artery patency might be prolonged with a less invasive approach such as CABG. The indications for CABG are therefore moving to more complex coronary lesions, reduced left ventricular function, and complications of diabetes and/or CKD.
While risk for major cardiac event rates were analyzed, further investigation focused more specifically on HF in a subset of the J-ACCESS database . Among 3835 patients with confirmed or suspected CAD, 71 required aggressive medical treatment due to new-onset congestive HF symptoms for 3 years. Multivariable Cox hazard models revealed that CKD, the ESV index, and moderate to high SSS predicted refractory HF (Table ). This combination of factors had the greatest prognostic value compared with single or combined variables. The reasons for the high event rate in CKD might be attributed to fluid retention, myocardial stiffness and endothelial dysfunction in arterioles, none of which is estimated by anatomical studies using invasive and coronary CT angiography. One of the major factors for predicting HF was SSS. The J-ACCESS studies did not select SDS as a major variable to predict events, probably due to relatively small ischemic lesions and a relatively small proportion of acute coronary syndrome after entry. In addition, patients with early coronary revascularization within the first 60 days were excluded from the analysis of prognosis because the choice of revascularization might have been influenced by SPECT diagnoses at the time. The ESV was also a reasonable variable showing that dilated left ventricular volume and a low LVEF could predict HF events. One-third to half of patients with HF has a background of HFpEF. However, HFpEF was not investigated using variables such as diastolic dysfunction determined by gated SPECT and/or echocardiography in the J-ACCESS study. The mean LVEF was higher and the prevalence of HF with reduced EF was lower in the JROADHF, than in other registries, while higher mortality rates in that registry suggested a relatively higher prevalence of HFpEF partly due to the more advanced age of the patients . A recent meta-analysis has associated diabetes with new-onset HF especially in young populations and recurrent HF particularly in women or individuals with HFpEF . The contributions of risk factors, such as sex, aging, hypertension, and diabetes to systolic and diastolic functions, and actual prognostic impact require further evaluation of Japanese patients with HF.
123 I-labeled radiopharmaceuticals in CAD and HF The situation in Japan regarding the use of 123 I-labeled radiopharmaceuticals, such as 123 I-beta-methyliodophenylpentadecanoic acid (BMIPP) and 123 I-metaiodobenzylguanidine (MIBG), for patients with CAD and HF is unique. These 123 I-labeled radiopharmaceutical tracers are included in 20% of nuclear cardiology studies, and they have been available since the 1990s . Current JCS clinical guidelines for Diagnosis of Chronic Coronary Heart Diseases were issued by the JCS in Japanese and English during 2018 and 2021, respectively . They recommend BMIPP imaging for myocardial ischemia and risk/prognosis (Classes I‒IIa). Myocardial sympathetic imaging is also recognized in the prognostic evaluation of HF and ventricular tachyarrhythmias. A Japanese pooled 123 I-MIBG database has verified powerful prognostic values for patients with chronic HF . Defect scores for 123 I-BMIPP indicate possible risk stratification in patients with acute and chronic HF . Since these 123 I radiopharmaceuticals have not been approved outside Japan, their clinical role might have been universally underestimated. However, HF has been investigated in patients by innervation imaging using 123 I-MIBG for research purposes in the USA and Europe . The possibility of optimizing patient selection for evaluation using expensive cardiac devices to determine lethal cardiac arrhythmias has also been investigated. The effectiveness of MIBG indices for predicting arrhythmic and overall mortality risk and the possible integration of the clinical variables into the prediction model require further investigation and refinement for clinical practice . Simultaneous assessment of myocardial perfusion and innervation in patients with HF using contemporary semiconductor cadmium–zinc–telluride (CZT) detectors might also become an option with reduced radiation exposure . Large-scale studies are mandatory to support the clinical application of 123 I tracers as a useful adjunct to acquiring perfusion images of patients with CAD and HF.
The J-ACCESS studies conducted between 2001 and 2014 have several limitations. The most recent advances in technology and quantitation, such as integrated attenuation and scatter correction, and CZT detectors that allow reduced administered doses were not included. Treatment strategies for CAD and HF have been updated during the past two decades although appropriate contemporary strategies were applied during all J-ACCESS studies. The results cannot be applied to patients with valvular heart disease, and dilated, hypertrophic, and secondary cardiomyopathies, as they were excluded from the multicenter registry. Patients with severe HF were also not included at the timing of registration, although we discussed new-onset of HF retrospectively in this article. Moreover, recent advances in imaging technology and analytics, such as radiomics and artificial intelligence, or machine learning might offer novel strategies in cardiology, neurology, and oncology . Although a new patient cohort has not been included in the J-ACCESS registry, the application of artificial intelligence to quantify perfusion defects and/or ischemia is under investigation using current image databases.
The J-ACCESS investigations (series 1‒4) established a nationwide prognostic database of Japanese patients from 2001 to the present. An overview of current trends in HF indicated that a high proportion of HF among major cardiac events in J-ACCESS might be associated with increasing numbers of elderly persons, a high fraction of HFpEF, and a higher frequency of non-ischemic etiology in Japanese patients with HF. The predictors of major adverse events including hospitalization for severe HF are age, baseline left ventricular function, myocardial perfusion defects, and comorbid diabetes and CKD. Thus, comprehensive risk-based approaches using appropriate multicenter databases are required for groups of patients with CAD and HF. While changes in strategic algorithms now include non-invasive imaging cardiology guidelines and clinical practice in Japan, approaches to risk assessment require updating, and medical care should be appropriately personalized for patients.
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Population Proteomics: A Tool to Gain Insights Into the Inflamed Periodontium | c1544e9d-b46d-4140-bc56-6b0c9ca50b8f | 11735663 | Biochemistry[mh] | Introduction Periodontitis refers to a microbial‐caused pathology, which is characterized by inflammation and subsequent destruction of the tooth‐supporting tissues (i.e., gingiva, cementum, periodontal ligament, and the alveolar bone). Although other oral diseases are declining sharply, periodontitis remains at an alarmingly high prevalence. It affects around 60% of dentate adults worldwide, with severe cases accounting for approximately 24% . In addition to the local consequences that ultimately lead to tooth loss if untreated, periodontitis is considered a risk factor for a number of systemic diseases, including diabetes, dementia, and adverse pregnancy outcomes . The onset of periodontitis, attributed to an excessive inflammatory response to the poorly controlled attachment of bacterial plaque to the tooth, is influenced by various factors such as the oral microbiome, predominant risk factors, and genetic influences . This interplay of host‐specific characteristics and infections was described at the beginning of the millennium as “nightmarishly complex and impossible to study (…).” . However, even then, there was hope of deciphering this system with the help of proteomics . Proteins play a pivotal role in regulating metabolic pathways as signaling and effector molecules. Their presence in body fluids, through active secretion or cellular leakage, provides insights into the current state of human health. Moreover, they serve as therapeutic targets for numerous pharmacological agents . Collecting large‐scale proteomics data provides opportunities to reveal hidden associations, capture markers of genetic, lifestyle, and environmental exposure, stratify individuals according to their state of health, and monitor the progression of diseases . Such insights can be leveraged to analyze, and potentially therapeutically influence periodontal inflammation. In this review, we focus on the possibilities (and drawbacks) of profiling the proteome in large population studies, with a special focus on its linkage to genomics, to gain insights into inflamed periodontium.
Periodontitis and the Proteome—Clinical Challenges Periodontal diseases are divided into two main categories: Gingivitis, a reversible inflammation of the gingiva and periodontitis, an additional immune‐inflammatory infiltration of deeper areas of the periodontium resulting in an irreversible loss of clinical attachment . Particular focus in periodontitis pathology lies on the transition between the two forms, which is linked to a shift from a primarily protective defense mechanism against dental plaque to an exaggerated inflammatory response, leading to adverse effects on local tissue and systemic health. In susceptible individuals, prolonged exposure to plaque and inflammatory stimuli appears to disrupt gingival tissue homeostasis . As a result, the gingival sulcus deepens and forms the periodontal pocket—a gateway through which biofilm and accompanying inflammation can penetrate deeper into the periodontium. Interestingly, the stages of active advancement and remission of the inflammation appear to alternate . Well‐established risk factors known to promote this transition include smoking and diabetes . Furthermore, a mechanistic understanding of the systemic effects of periodontitis has emerged. Broadly speaking, bacteria, inflammatory cytokines, and lymphocytes are thought to translocate via the circulation and lymphatic system, resulting in chronic low‐grade inflammation . Nevertheless, the molecular mechanisms occurring in and around the periodontium remain largely elusive. This poses unmet clinical challenges concerning early detection, identification of active inflammatory phases, classification of progression, and securing an inflammation‐free state of the disease. Former research has shown that relying on a single marker alone is insufficient to shed light on the complex dynamics within the inflamed periodontium . Consequently, research endeavors are increasingly turning their attention toward the proteome, which encompasses the totality of proteins within an organism, cell, or tissue. By identifying and quantifying all proteins of a functional unit, the structure, mechanisms of action, and interactions at the cellular level can be assessed . However, due to the complexity of the oral cavity and the large number of players involved (host, microbiome, exogen factors), there is no stable, let alone standardized, proteome. Previous studies in dentistry have therefore tended to focus on defining a “proteotype” associated with disease. A “proteotype” describes the dynamic state of a proteome at a certain point in time in a certain protein source . The oral cavity harbors a plethora of potential “proteomic source constituents,” with proteins being prevalent in both hard and soft tissues of the periodontium, spanning from enamel to periodontal ligament to gingiva . Moreover, the teeth are in direct contact with gingival crevicular fluid (GCF) and saliva. Remarkably, proteomics can even be applied to historical dental calculus and provide insights into the evolution of the oral microbiome . The selection of the appropriate medium therefore depends on specific research objectives. For inquiries into local periodontal differences, emphasis is currently placed on intraoral tissues and fluids. Conversely, serum or plasma analysis is often used to investigate systemic effects of periodontitis and to facilitate personalized risk assessment . Nonetheless, establishing associations between protein signatures and corresponding disease occurrence or outcome requires large sample sizes with long‐term follow‐up of participants. The acquisition of such datasets mandates the utilization of large‐scale proteomic technologies within population‐based studies.
Assessing the Periodontal Proteome in Population‐Based Studies With High Throughput Conducting population‐based studies to elucidate the relationship between the proteome and periodontal inflammation poses distinct challenges for researchers. One crucial aspect involves determining the optimal medium in which proteomic methods are applied. Due to constraints in time and financial resources, source material that can be collected quickly, cost‐effectively, and in a less invasive manner is preferable. Furthermore, the selected medium should enable the exploration of diverse conditions . In large‐scale studies, data collection typically occurs within an interdisciplinary context, addressing multiple research questions simultaneously. It is further important to recall that to perceive distinctions in protein patterns, we first need to identify what counts as “normal composition,” and therefore already available information is beneficial, especially for non‐explorative queries. Body fluids suitable for investigating periodontal inflammation include blood products, saliva, and GCF . Plasma and serum are by far the most established choices for clinical testing in medicine. Consequently, routine diagnostic tests and the majority of population‐based studies focus on collecting and analyzing blood samples . This emphasis has led to a comprehensive understanding of the human proteome, particularly within the blood compartment. The blood proteome combines proteins from all kinds of tissues. Some are secreted, while others leak from damaged cells. With regard to the oral cavity, about 27% of salivary proteins are also detectable in blood . However, one of the greatest challenges in analyzing blood samples is the wide range of concentrations in which proteins occur and the abundance of some proteins may vary widely in response to disease or other physiological causes. Although around 5000 circulating proteins have been detected using mass spectrometry (MS) or immunoassays—approximately 25% of the whole human proteome —only 20 makeup around 90% of the total protein mass in plasma . In addition to the technical difficulties in measuring rare proteins, overlapping effects must be assumed, especially when the effect of periodontitis on the whole organism is being investigated. Saliva analysis is increasingly used in dentistry and medicine. Saliva offers several valuable advantages, including noninvasive and painless collection (an attractive alternative to blood samples in children and the elderly, where blood sampling often impairs compliance), constant availability, and the low need for specialized equipment; both for sample collection and storage . Therefore, it can be easily obtained in resource‐limited settings and by untrained examiners . Saliva can be collected as individual secretions from the major and minor salivary glands, as well as whole saliva in unstimulated (resting) or stimulated conditions. Detailed procedures were defined for this purpose in order to ensure comparability . Due to its direct proximity to the periodontium, saliva is suited for the examination of less frequently occurring (inflammatory) proteins. A major challenge, however, is given by the complexity of the oral cavity. The salivary proteome is subject to strong exogenous influences such as oral hygiene or food intake . This complexity, combined with a limited utilization in routine practice, resulted in the salivary proteome being less well‐researched than its blood counterpart. However, numerous initiatives have been launched in recent years to solve this problem, for example https://www.salivaryproteome.org/ . The term GCF refers to the fluid that emerges between the surface of the tooth and the periodontal structures and is therefore in direct contact with the local inflammatory process in periodontitis. It represents an ultrafiltration of the blood of the surrounding capillaries. As such, it contains classic blood serum and plasma components, immunological markers and cells, as well as various products of tissue breakdown. At healthy sites, the protein concentration is similar to that of the interstitial fluid, while at inflamed areas the protein concentration of the GCF is similar to that of the serum . Due to its site specificity, it is an excellent source for investigating pathologies around a single tooth. Sample collection is non‐invasive, yet significantly more technique‐sensitive compared to saliva and blood. In fully dentate jaws (third molars excluded) there are theoretically 6*28 sites for potential sampling. The sampling volume per pocket itself is very limited, especially in healthy areas. Pooling of samples is possible, but results in losing pocket specificity. Furthermore, the low relative concentration of proteins (substantially smaller compared to saliva), such as cytokines, further complicates their detection and analysis using proteomic techniques like MS ). Although the use of GCF diagnostics in large‐scale investigations is theoretically viable, due to these constraints, they have been used mainly in studies particularly concentrating on periodontal issues thus far. Table provides an overview of the key characteristics of the described body fluids. Another particularly fascinating aspect of saliva and GCF from a periodontal perspective is the presence of proteins of microbial origin. The oral microbiome displays a complexity comparable to the gut microbiome, and there have even been correlations found between the two . Omics analyses, which distinguish human proteins from bacterial proteins, show that more than 2000 microbial proteins from different bacterial genera contribute to the salivary proteome, forming a dynamic and complex unit . The exploration of the metaproteome of oral microbial communities and its relevance for dental diseases is gaining momentum. However, the identification and quantification of this metaproteome is challenging and exceeds the complexity of conventional proteomic analyses of individual organisms. At the same time, bacterial proteins, though numerous, represent only a minor component in terms of abundance. For example, Grassl et al. found that of the 1000 most abundant proteins in saliva, only 5% were of bacterial origin . As a result, metaproteomic analyses of the oral microbiome are still conducted outside the context of large population‐based studies. Nevertheless, we would like to emphasize seminal works of Xiao et al. and Manoil et al., which outline the current development on this topic . Irrespective of sample type, the proteomic method must enable high‐throughput analysis of the respective material. A combination of high sensitivity, high specificity, large sample throughput, and low costs is ideal . In large population studies, MS‐ and affinity‐based methods have been established for this purpose. MS is the primary technology in proteomics, mainly utilizing enzymatic digestion to produce peptides for protein identification and quantification. At its core, MS measures the mass‐to‐charge ratio of ionized molecules. Although certain MS techniques allow the identification of several thousand proteins in a single sample, the number of detectable proteins is substantially smaller for large‐scale applications analyzing many samples concurrently . Affinity‐based proteomics relies on antibodies or other binding agents as protein‐specific detection probes. Sandwich immunoassays (e.g., Olink or Luminex)—requiring two antibodies binding to different sites of the same measured protein—are frequently used in clinical settings for analyte quantification and are thought to be the most sensitive techniques for detecting low‐abundance proteins . Aptamer‐based immunoassays (such as SomaScan) employ DNA or RNA scaffolds as binding partners . These aptamer‐based proteomics platforms capture proteins in solution, followed by labeling and purification stages. Olink and SomaLogic have built libraries including upwards of 3000 and 4000 protein‐specific binding partners, respectively. These methods also allow to analyze several thousand samples at once. For large‐scale proteomic analyses, there is no one‐fits‐all method. MS‐based approaches benefit from a large community of users and their capability to identify proteoforms, that is capturing multiple post‐translational modifications. However, sample throughput and analytical sensitivity are relatively low. Affinity‐based methods provide higher sample throughput and analytical sensitivity, making them the preferred approach for analyzing data sets with over 5000 participants, each with around 1000 proteins to be determined. Nonetheless, some concerns have been raised regarding the vulnerability to off‐target binding and a lack of assay reproducibility . 3.1 Determining the Disease Phenotype In scientific context the determination of periodontal burden primarily occurs through clinical parameters such as pocket probing depth, bleeding on probing, and clinical attachment loss. Various protocols are available for varying extent of assessment, the most common being the Full Mouth Protocol, Half Mouth Protocol, or measurement of specific index teeth . The definition of a periodontitis case and its respective severity has changed multiple times in last decades. The current consensus was published in 2018 and incorporates a staging and grading approach. However, this definition is primarily intended for clinical use and therefore includes parameters such as radiographic measurements or progression markers like previous tooth loss due to periodontitis. Lastly, it includes some room for “sound clinical judgment” enabling a clinician to arrive at a diagnosis that best describes the individual considering other factors . However, implementing this in epidemiological evaluations is challenging since, for example, radiological examinations without cause are ethically unjustifiable or information on disease progression is lacking, especially in cross‐sectional studies. Holtfreter et al. provide more precise instructions for applying the definition in epidemiological investigations . Alternative indices can also be found, such as the American Academy of Periodontology and the American Dental Association's community periodontal index of treatment needs (CPITN) for periodontal screening, or the 2018 consensus definition of an intact periodontium . Given the alternating active and passive phases of periodontitis, that might influence the inflammatory load at the time of measurement, there have been further attempts to directly quantify inflammation . However, since periodontitis is a slowly progressing disease, observation periods over years to decades are critical. To allow for possible conversion to more current definitions, a minimum of basic parameters should be collected and reported. Another increasingly used approach in population‐based studies lacking direct oral measurements is further to include the analysis of claims data. Digital health records, prescriptions, and other registry data offer a broad spectrum of information to derive disease phenotypes. However, due to considerable variations in data quality, it is crucial to rigorously check the validity of generated phenotypes to ensure reliable results . Nevertheless, a certain degree of misclassification bias, that is, the difference between a measured quantity and its true value, must be interpreted on a case‐by‐case basis with respect to the applied definition . Table provides examples of current population‐based studies with proteomic data and differing approaches to accessing oral health data.
Determining the Disease Phenotype In scientific context the determination of periodontal burden primarily occurs through clinical parameters such as pocket probing depth, bleeding on probing, and clinical attachment loss. Various protocols are available for varying extent of assessment, the most common being the Full Mouth Protocol, Half Mouth Protocol, or measurement of specific index teeth . The definition of a periodontitis case and its respective severity has changed multiple times in last decades. The current consensus was published in 2018 and incorporates a staging and grading approach. However, this definition is primarily intended for clinical use and therefore includes parameters such as radiographic measurements or progression markers like previous tooth loss due to periodontitis. Lastly, it includes some room for “sound clinical judgment” enabling a clinician to arrive at a diagnosis that best describes the individual considering other factors . However, implementing this in epidemiological evaluations is challenging since, for example, radiological examinations without cause are ethically unjustifiable or information on disease progression is lacking, especially in cross‐sectional studies. Holtfreter et al. provide more precise instructions for applying the definition in epidemiological investigations . Alternative indices can also be found, such as the American Academy of Periodontology and the American Dental Association's community periodontal index of treatment needs (CPITN) for periodontal screening, or the 2018 consensus definition of an intact periodontium . Given the alternating active and passive phases of periodontitis, that might influence the inflammatory load at the time of measurement, there have been further attempts to directly quantify inflammation . However, since periodontitis is a slowly progressing disease, observation periods over years to decades are critical. To allow for possible conversion to more current definitions, a minimum of basic parameters should be collected and reported. Another increasingly used approach in population‐based studies lacking direct oral measurements is further to include the analysis of claims data. Digital health records, prescriptions, and other registry data offer a broad spectrum of information to derive disease phenotypes. However, due to considerable variations in data quality, it is crucial to rigorously check the validity of generated phenotypes to ensure reliable results . Nevertheless, a certain degree of misclassification bias, that is, the difference between a measured quantity and its true value, must be interpreted on a case‐by‐case basis with respect to the applied definition . Table provides examples of current population‐based studies with proteomic data and differing approaches to accessing oral health data.
Proteomic Patterns as (Predictive) Biomarkers A classic application of proteomics data is the search for biomarkers to predict disease onset, prevalence, or progression. Here, the term “biomarker” is not limited to a single measurement but encompasses a range of characteristics and their combinations that can indicate specific physiological or pathological processes . In addition to the ongoing search for a single marker for periodontitis, the emergence of large‐scale proteomics has brought protein patterns into focus. Patterns of protein concentrations can be used to derive risk profiles for various diseases and pathological mechanisms, including the transition from gingival health to periodontal diseases . An example illustrating development and validation of a disease risk score is the 9‐protein risk score for cardiovascular outcomes, designed for patients with stable coronary heart disease. In brief, researchers measured 1130 plasma proteins across a total of 2496 samples from two independent, prospective, population‐based cohorts. After quality control and Bonferroni correction, 200 significant associations were identified between individual proteins and cardiovascular outcomes. Further proteins were excluded by statistical variable selection techniques, resulting in a final model incorporating nine proteins. This model was then tested in a validation cohort and demonstrated superior performance compared to a traditional risk prediction model . Currently, a similarly validated score does not exist in dentistry. However, there is evidence that proteomics data can enhance a model with classical predictors of periodontitis . Furthermore, Beydoun et al. investigated proteomic biomarkers for the link between poor oral health problems (POHP) and neurodegenerative diseases. Comparable to the development phase of the 9‐protein risk score, they used affinity‐based proteomics to analyze 1472 proteins in 5089 individuals from the UK Biobank study and further examined associations between each individual protein and POHP. Applying Bonferroni correction and an effect size criterion, they ultimately identified 15 plasma proteins significantly associated with POHP. Further analyses showed that Growth Differentiation Factor 15 (GDF15) and WAP Four‐Disulfide Core Domain Protein 2 (WFDC2) were the most significant plasma proteome mediators between POHP and neurodegenerative outcomes . Nonetheless, the increasing number of identifiable proteins and the resulting diverse concentration ratios create a vast array of potential combinations, which rapidly exceed sample sizes, even in population‐based cohorts. This leads to both conceptual and technical challenges. To avoid false‐positive protein(‐combinations) to periodontitis associations, the multiple comparison problem must be adequately addressed based on the research question. However, there is a fine line between being too lenient and overly restrictive. Current methods for adjusting multiple testing, such as the Bonferroni correction as used in the examples above or the False Discovery Rate proposed by Benjamini and Hochberg, are widely used . However, these methods have the disadvantage of neglecting potential hierarchical structures in the tested hypotheses. For instance, in proteomics, proteins can often be organized into pathways or functional groups. Utilizing a hierarchical structure among tested hypotheses is currently being investigated, as it may enable new testing strategies that control error rates for reported discoveries while retaining statistical power . Table provides examples of functional or hierarchical protein subgroups in current periodontal research. Since there is no definitive solution to the multiple testing burden yet, validation of results is crucial. However, validation approaches are currently limited by the suboptimal correlation between assay technologies. For example, a recent head‐to‐head study showed significant discrepancies between SomaScan and Olink measurements . Currently, no gold standard exists for comparing different large‐scale proteomic platforms. Due to technical and practical constraints, respective assays often use relative quantification scales. Although relative values are sufficient for determining protein‐disease associations, absolute values have the advantage of offering interpretable effect sizes and comparable reference values. However, due to these limitations, replication of results in independent datasets employing different assays is advised . Furthermore, it is crucial to remember that a credible biomarker may not necessarily have a causal link with the disease, and thus an intervention on such a biomarker does not always result in an improvement of the condition. However, causal proteins are particularly valuable for therapeutic applications. In the absence of randomization and in light of our incomplete understanding of numerous protein pathways, the risk of confounding must always be considered. A technique of leveraging proteomic data that is increasingly being utilized to address this challenge is the combination with other omics approaches, like genomics .
Genome Meets Proteome—Identifying Potential Targets for Resolving Periodontal Inflammation One of the primary objectives of medical research is to pinpoint potential targets for intervention to ameliorate a disease or at least impede its progression. The majority of successful pharmaceutical drugs exert their effects by interacting with and altering the activity of proteins. Thus, the human proteome remains the most important source of therapeutic targets . However, despite the substantial growth in our knowledge of the human proteome in recent years, the rate of discovering new drugs has slowed down. Given that proteins are encoded by genes, integrating genetics into drug development turned out to be an effective way to enhance this process, as genetically supported drugs are significantly more likely to succeed in clinical trials . For a protein to qualify as a potential drug target, it must fulfill two fundamental criteria. Firstly, it must possess the ability to bind drug‐like molecules (druggability). At the outset of this millennium, Hopkins and Groom introduced the concept of the “Druggable Genome” to encompass the entirety of genes encoding such proteins, estimated to be around 20,000. Secondly, the protein must be linked to causal disease pathways . To establish these pathways, researchers are increasingly turning to genetic Mendelian randomization (MR) analysis. Named after Gregor Mendel's observations on the transmission of genetic traits, this methodology utilizes genetic variants as instrumental variables (IVs), that is, variables reliably associated with an exposure but not influenced by confounding factors or reverse causality . Leveraging Mendel's laws of segregation and independent assortment—stating that genetic variants are randomly distributed regardless of environmental or genetic factors—MR estimates the causal effect of exposure on outcome by analyzing the association estimates for the genetic variants with these same phenotypes . Data for this method comes from genome‐wide association studies (GWAS), in which associations between genetic variations (typically single nucleotide polymorphisms [SNPs]) and certain conditions or traits are investigated, ranging from complex diseases such as periodontitis to protein concentrations (Table ). With advancements in technology, proteomics has increasingly been integrated into large‐scale GWAS. For instance, recent GWAS of plasma proteins have revealed significant SNP effect estimates for thousands of different proteins . It is worth noting that genetic variations affecting protein concentrations are not limited to single SNPs. Protein quantitative trait loci (pQTLs) refer to genetic variations (including SNPs, insertions, deletions, and other [structural] variants) that are associated with protein concentrations on a genome‐wide scale. Significant genetic variants are particularly biologically “plausible” if they are located in the immediate vicinity of the gene coding for the protein. Such variants are referred to as “cis” and are supposed to immediately affect protein synthesis and function. Those that do not fall within a defined gene window are called “trans” (Figure ) . A curated list of pQTL studies can be found at: http://www.metabolomix.com/a‐table‐of‐all‐published‐gwas‐with‐proteomics/ . This newly accessible proteomics data enables in‐depth research in periodontal inflammation, such as the investigation of known or suspected inflammatory mechanisms . For example, recent findings indicated that excessive activation of the complement system is involved in periodontal breakdown. Complement component 3 (C3) plays a key role within the complement cascade. Laboratory, animal, and first clinical trials in humans suggest that its inhibition could lead to a reduction in periodontal inflammation . A cis‐MR study using large proteomics GWAS was able to replicate this potential effect . A cis‐MR is a specialized form of MR. In simplified terms, it operates on the principle that having a genetically predicted lower level of C3 is akin to administering a drug that reduces the proteins’ activity. Logically, if SNPs, which serve as valid proxies for an individuals’ C3 concentration are associated with periodontitis, this information allows researchers to assess the potential of altering the protein concentration on the disease . Like genome‐wide MR, cis‐MR relies on key assumptions: First, SNPs chosen as IVs should be associated with the exposure. Second, there are no confounders between the IVs and the outcome. Third, IVs should influence the outcome solely through the exposure. In practice, it is not possible to fully test if all conditions are met. However, selecting biologically plausible cis‐SNPs may help reduce some sources of error . Comparable studies investigated the involvement of various other proteins, including interleukin (IL) 17 and IL‐6 . Currently, MR analyses are limited by the size of GWAS datasets. Especially when dental phenotypes are considered as exposures, lack of statistical power constitutes a barrier. To date, the gene‐lifestyle interactions in dental endpoints (GLIDE) consortium remains the most comprehensive data source for dental association estimates . Fortunately, a much larger GWAS has been announced by the consortium . Further details on the MR design and its specific use in periodontitis research can be found in the work of Haworth et al. . Another restriction is that protein data in GWAS are frequently limited to blood measurements. It is reasonable to assume that there are several local, tissue‐specific mechanisms of action that could serve as potential drug targets (local host modulation). In principle, the aforementioned methods are also applicable to such situations, but currently the data foundation for such cases is very limited. Nevertheless, efforts are underway to characterize the genome of various tissues, such as the Human Protein Atlas ( https://www.proteinatlas.org/ ) . The merit of such oral proteomics is exemplified by Offenbacher et al. discovering two missense mutations in the anti‐inflammatory IL‐37 gene locus associated with higher IL‐1β levels in GCF: rs3811046 and rs2708943 . The first variant is associated with increased expression of IL‐1β in periodontal tissues, while the second is associated with lower IL‐37 mRNA expression. IL‐37 is known to inhibit innate immunity. Consistent with this, the authors demonstrated an association between rs2811046 and severe chronic periodontitis, 10‐year tooth loss, and aggressive periodontitis in an independent population .
Concluding Remarks Proteomics has undergone a remarkable evolution in the last decades including technical improvements as well as public data collaborations and methods for data interrogation . In dentistry, this progression has enabled a shift from previously limited inquiries with small sample sizes to large‐scale interdisciplinary research endeavors . Great effort has been put into mapping the human protein expression across several organs and tissues including the oral cavity, providing the promise of valuable insights into periodontal inflammation in the near future . The combination of various OMICS technologies is particularly promising for any mechanistic insights into molecular pathways. The integration of dental examinations and the collection of oral samples such as saliva or GCF in interdisciplinary studies can further shed light on the link between the periodontium and systemic health . Looking ahead, recent advances in periodontal research bring us closer to combatting periodontitis beyond traditional mechanical or surgical interventions. Host modulation approaches by acting on protein targets may offer new prevention and treatment strategies . The integration of large‐scale proteomics in extensive population studies could be the gateway for addressing periodontal health in the years to come.
S.L.R. and M.N. participated in conceptualizing the manuscript. S.L.R. drafted the initial version of the manuscript. M.N., S.‐E.B., D.H., and Z.A. contributed to various sections of the manuscript. All authors were involved in writing, reviewing, and/or revising the manuscript.
All authors declare no conflicts of interest.
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Population structure and pathogen interaction of | 5de6a440-aebc-4277-90be-365873e4d85e | 11297283 | Microbiology[mh] | Escherichia coli is used by many water managers as a reliable indicator of faecal contamination in water quality assessments (Till et al., ). This bacterium, which normally inhabits the intestines of warm‐blooded animals (Tenaillon et al., ), serves as a sentinel faecal indicator bacterium (FIB), providing valuable insights into the potential presence of harmful pathogens in water sources (Cookson et al., ; Hörman et al., ; Nowicki et al., ; Weller et al., ). The use of E. coli as an indicator is rooted in its prevalence and high concentration in the gastrointestinal tracts of humans and other animals, making it a practical and efficient marker for assessing water safety (Touchon et al., ). Water quality is a critical concern for public health, as contaminated water can harbour a variety of pathogens such as Shiga toxin‐producing Escherichia coli (STEC), Campylobacter , Salmonella and Cryptosporidium and Giardia that pose serious risks to human well‐being (Hörman et al., ; Weller et al., ). Faecal contamination is a key focus in water quality assessments due to its association with waterborne diseases; while there are numerous bacteria, viruses, and protozoa that may indicate faecal contamination, E. coli is favoured as it is relatively easy and cost‐effective to culture and detect. Standardized methods such as membrane filtration and Colilert (IDEXX) allow for efficient and reliable identification and quantification of E. coli to assess public health risk (Till et al., ). Moreover, E. coli has a short incubation period, meaning that its presence in water samples can be detected relatively quickly (12–24 h). This characteristic is crucial for timely responses to potential waterborne outbreaks, allowing water managers to take prompt action to protect public health. The presence of E. coli in water is indicative of recent faecal contamination, suggesting a higher likelihood of the presence of other, potentially harmful, waterborne pathogens which often are slower growing and require more complex culture detection methods (Devane et al., ). An association between E. coli and the risk of gastroenteric illness (Wiedenmann et al., ) makes it a valuable tool for assessing the overall safety of water sources (Till et al., ). Recent studies utilizing high resolution genomics have demonstrated that several benign ‘cryptic’ Escherichia clades, indistinguishable from generic E. coli phenotypically, may also survive and persist in the environment (Cookson et al., ; Luo et al., ; Mire et al., ; Walk et al., ). Formerly identified as E. coli strains, Escherichia whittamii (Clade II) (Gilroy et al., ), Escherichia ruysiae (Clades III and IV) (van der Putten et al., ) and Escherichia marmotae (Clade V) (Liu et al., ) have been described as novel species, however, current culture‐based methods, used to enumerate E. coli as a proxy for faecal contamination and pathogenic microorganisms, cannot distinguish between environmental Escherichia clades and faecal strains (Walk, ). This lack of discrimination could lead to a potential overestimation of health risk using standard culture‐based methods of E. coli determination. In addition to the naturalized Escherichia spp., whole genome sequencing of faecal E. coli has provided important phylogenetic insights into intra‐species variation with the identification of at least eight distinct phylotypes (Clermont et al., ; Clermont et al., ; Clermont et al., ). Paradoxically, some E. coli , particularly phylotypes B1 (Berthe et al., ; Nowicki et al., ; Power et al., ; Ratajczak et al., ; Touchon et al., ; Walk et al., ) and B2 (Cookson et al., ; Petit et al., ), have also been associated with prolonged survival and growth in environmental samples (e.g., water and sediment), where no obvious faecal contamination event has been noted. These observations have been described as relating to non‐recent or aged contamination events and typically refer to incidents involving faecal contamination into a particular water source occurring some time previously and not being of recent origin. Thus, there appear to be two groups of naturalized Escherichia ; from environmental, and those from enteric sources, but the extent to which either or both confound microbial water quality assessments is unknown (Devane et al., ). Measuring FIB offers insights into contamination levels but standard culture‐based methods are unable to identify faecal sources (Harwood et al., ; Tran et al., ). The implementation of molecular methods to identify host‐associated microbial source tracking (MST) markers can provide valuable insights into the faecal sources of pathogens impacting human health in water samples (Devane et al., ; Devane et al., ; Devane et al., ; Korajkic et al., ). Links with host‐associated faecal markers from humans, ruminants and avian sources can assist water managers with targeted mitigation strategies to reduce pathogen levels and for more accurate public health risk assessments. In this study, we describe detailed analysis of E. coli phylotypes, freshwater pathogens, land‐use, and host‐associated (human, ruminant and avian) MST markers obtained from freshwater samples collected as part of the New Zealand Ministry for the Environment's (MfE) Freshwater Pathogen Quantitative Microbial Risk Assessment (QMRA) studies as an update to the 1998–2000 Freshwater Pathogen QMRA study (Till et al., ). In parallel with bacterial and protozoal pathogen detection data, the objectives of this study were to (i) examine the prevalence of the recently described E. marmotae , E. ruysiae and E. whittamii species; (ii) describe any relationship of phylotypes with E. coli concentration and land‐use; and (iii) assess the relationship between naturalized E. coli phylotypes B1 and B2 with non‐recent or aged faecal sources and local land‐use.
Water sampling sites The water samples for this study originated from 2022 to 2023 large‐scale MfE Freshwater Pathogen study collected as part of a wider study to update the 1998–2000 QMRA (Till et al., ). Freshwater samples were taken as part of routine monitoring programs by Council (local New Zealand environmental agency) staff in late spring to early winter (October 2022–July 2023) at 41 sites around New Zealand (Figure ). Sampling was scheduled to provide national coverage of different land‐uses taking into account the logistics associated with long term sampling across nine different Councils at 41 sites. Catchment delineation and land‐use identification for freshwater sampling sites A hydrological sub‐catchment for each sampling site was generated using ArcGIS Pro (v.2.6.4). In each respective catchment, the sampling location was used as a pour point in the catchment delineation analysis. The pour point specifies where water should ‘drain’ from the catchment, thereby giving the total land surface draining to the sampling point. The catchment sizes ranged from approximately 3–8 km 2 . Land cover data (Table ) for each of the 41 sites was sourced from the Land Cover Database version 5.0, Mainland, New Zealand (Anon, ) to assist with identifying the dominant land‐use for site categorization. Sites with a history of elevated concentrations of E. coli were located across New Zealand and reflected different dominant land‐uses and impacts as designated by Councils: dairy ( n = 13); sheep and beef ( n = 10); mixed sheep and beef and dairy ( n = 2); urban ( n = 8); avian ( n = 4). Four sites were designated as low impact with lower potential for faecal contamination (exotic/natural forest dominated control sites). Sample sites were designated as ‘avian’ using prior observational information provided by Councils which noted large numbers of avian faecal contamination events caused by wildfowl flocks or bird roosts. Generally, sampling occurred between 6 AM and 1.30 PM New Zealand standard time. Samples were collected 200 mm below the water surface using aseptic methods. Most samples sites were selected from non‐tidal areas, however, if samples sites affected by tidal influences were identified, they were sampled around low tide. If the water was too deep, samples were taken from the bank using an extendable pole. After collection, samples were cooled and transported overnight with frozen packs to keep water samples chilled for FIB, MST and pathogen analyses to the Institute of Environmental Science and Research (ESR, Christchurch, New Zealand). Samples for protozoan analyses were sent overnight to MicroAquaTech, Massey University (New Zealand). Campylobacter isolation and detection For quantitative analysis of Campylobacter by Most Probable Number (MPN) enrichment, water volumes of 1 × 1000 ml, 3 × 10 ml, 3 × 1 ml and 3 × 0.1 ml were analysed (APHA, ; ISO, ; Medeiros & Hoffman, ) and cultured using Preston broth (Fort Richard Laboratories Ltd., Auckland, New Zealand) as described in the Data . Preston broth culture was plated onto modified charcoal‐cefoperazone‐deoxycholate agar (mCCDA) plates (Fort Richard) and incubated at 41.5 ± 1°C for 44 (±4) h, and then 2–4 putative Campylobacter colonies were transferred to Columbia Blood Agar (CBA) plates (Merck, Darmstadt, Germany) and incubated as before. Where putative Campylobacter colonies were observed on CBA, conventional PCR (Wong et al., ) was performed on DNA extracted from the original Preston broth culture to identify C. coli , C. jejuni and thermophilic Campylobacter . Salmonella isolation and detection For presence/absence analysis of Salmonella , 1 L of water was filtered through 0.45 μm (47 mm diameter) mixed cellulose ester membrane filters (Millipore, Merck) and placed into 25 ml of buffered peptone water (BPW) broth (Fort Richard Laboratories Ltd) and incubated at 37 ± 1°C for 18 ± 2 h as described in the Data . Isolates that were consistent with Salmonella were tested for the presence of Salmonella O‐ and H‐antigens by slide agglutination using polyvalent antisera (Remel, ThermoFisher Scientific, Vilnius, Lithuania) and metabolic characteristics (Microbact GNB 12A and 12B kits in combination, ThermoFisher Scientific). Cryptosporidium and giardia isolation and detection Protozoa were detected from 30 L water samples according to USEPA Methods (USEPA, ) as described in the Data . Immunomagnetic separation (IMS) using a Dynabeads GC‐Combo kit (ThermoFisher Scientific) and immunofluorescent staining for Cryptosporidium oocysts and Giardia cysts was performed using the EasyStain kit (BioPoint Pty Ltd.). Enumeration and storage of faecal indicator bacteria Escherichia coli were analysed in freshwater using Colilert Quanti‐Tray assays (100 ml of a 1 in 10 dilution of water sample, incubated at 35 ± 0.5°C for 24 to 28 h) (IDEXX Laboratories, Inc., Maine) (APHA, ). This provides a detection range of <10 Most Probable Number (MPN)/100 ml up to >24,200 MPN/100 ml. Wells that were yellow in colour and fluoresced when exposed to UV light, indicative of the presence of E. coli , were identified with a hand‐held UV unit (EA‐160/FE, Spectroline, New York). Between 3 October 2022 and 3 July 2023, positive Colilert 2000 trays (IDEXX, New Zealand) from 298 water samples stored at 4°C after testing was complete were transported (maintained at chilled temperature during overnight transit) in single weekly batches to AgResearch (Hopkirk Research Institute, Palmerston North) from ESR, Christchurch. For each individual Colilert tray, wells that fluoresced when exposed to UV light, indicative of the presence of E. coli were identified with a hand‐held UV unit (EA‐160/FE, Spectroline, New York), carefully opened using a sterile low‐gauge needle, and 150 μl (small wells) or 200 μl (large wells) of growth removed from each after pipetting up and down three times to homogenize the bacterial suspension in each well. Bacterial culture from the fluorescent wells of each individual Colilert plate was pooled and centrifuged at 5000× g for 10 min at 4°C using a Multifuge X3R (Thermo Fisher Scientific). The supernatant was discarded, the pellet resuspended in 1 ml EC broth and transferred to a 1.5 ml tube and centrifuged at 16,200× g for 2 min. The supernatant was discarded, the bacterial pellets were resuspended in 1 ml EC broth (Fort Richard Laboratories Ltd.) and added to cryovials containing glycerol (final concentration 30% v/v) and stored at −80°C. Colilert trays ( n = 298) were sub‐grouped to balance E. coli MPN/100 ml concentrations of <100 (low), 101 to 1000 (medium), and >1000 (high) in a 1:2:1 ratio across the 41 sample sites, where possible, to focus on samples requiring regulatory authority action in New Zealand: (i) 260 E. coli MPN/100 ml to spur water managers to identify sources of faecal contamination for mitigation purposes and (ii) 540 E. coli MPN/100 ml to inform the public that the site is unsuitable for primary contact recreational use (Anon, ). Subsequent isolation of E. coli was undertaken from a selection of 199 frozen glycerol stocks across all 41 sample sites on ECC CHROMagar plates (Fort Richard Laboratories Ltd.). Plates were incubated at 35°C for 18 to 21 h after which 20 well‐spaced, individual blue colonies (presumptive E. coli ) from each water enrichment (stored in glycerol) were selected and sub‐cultured onto MacConkey Agar plates (Fort Richard Laboratories Ltd.) and incubated as previously described. A crude DNA sample for further DNA analysis was prepared for each of the 3980 isolates by resuspending a single well‐spaced colony in 400 μl of sterile milliQ water, heated at 100°C for 10 min in a heating block and stored at −20°C. Another single well‐spaced colony was resuspended in 1.5 ml of sterile Brain heat infusion (BHI) broth (Oxoid) containing glycerol (30% v/v) and stored at −80°C. In total, 3980 presumptive E. coli were stored from 199 water samples. Multiplex PCRs for Escherichia species and E. Coli phylotypes Each of the boiled lysate preparations ( n = 3980) were used in individual PCR reactions to determine the E. coli phylotype or Escherichia cryptic Clades (I to V) using an extended quadruplex PCR phylotype assignment method (Clermont et al., , ). Allele specific multiplex PCR for the identification of phylotypes was carried out as described in the Data . Clade I to V PCRs were carried out according to Clermont et al . Real‐time PCR ( RT ‐ PCR ) for detection of diarrhoeagenic E. col i genes Real‐time multiplex PCR (RT‐PCR) assays were performed on an Applied Biosystems ViiA7 Real‐time PCR System (Life Technologies) targeting the virulence genes stx 1 and stx 2 from Shiga toxin‐producing E. coli (STEC), and intimin adherence ( eae ) gene from enteropathogenic E. coli (EPEC) and some STEC serogroups, as markers for the presence of pathogenic E. coli (Clarke, ; Nataro & Kaper, ), and the E. coli 16S rRNA gene as an internal control. Positive samples were described as those having quantification cycle thresholds (Cq) of <35. Additional metadata Rainfall data for water samples in the preceding 24, 48 and 72 h, was recorded by Council staff from each site and visit and was included in analysis. Average flow data (m 3 /s) from the preceding 24 h prior to sampling were provided by Council staff. Microbial source tracking markers Freshwater samples were filtered (in duplicate) through a 0.45 μm mixed cellulose ester membrane filters (Millipore, France) and DNA extracted as described in the Data . Quantitative PCR (qPCR) analysis was undertaken on a LightCycler 480 (Roche Diagnostics Ltd, CA). The PCR targets are presented in Table with PCR protocols and conditions outlined by Devane et al. and described in the Data . Each qPCR run included negative and positive controls, and standard curves of the appropriate target. The amplification efficiency of the PCR assays was considered acceptable at >90% for qPCR targets, and the coefficient of determination ( r 2 ) at ≥0.92 for assays. Statistical analysis Statistical analysis was performed using R version 3.6.2 (R Development Core Team, ), and ggpubr (Kassambara, ); figures were produced using the package ggplot2 (Wickham, ). Shannon diversity (total number of E. coli phylotypes and Escherichia species) per water sample was undertaken using the R package ‘vegan’ version 2.5‐6 (Oksanen et al., ). To test the effects of pathogens or pathogen‐associated genes ( Salmonella , Campylobacter , eae gene, stx gene, Cryptosporidium , Giardia , Any Bacteria, Any Protozoa, Any Pathogen), lme4 (Bates et al., ) was used to fit binomial generalized linear mixed effect models on the pathogen presence or absence data, to test associations with, for example, E. coli concentration MPN/100 ml (log 10 MPN) or E. coli phylotype (total 0–20) from each water sample determined using PCR. Individual “Site” was included in the model as a random effect. ‘Any Pathogen’ refers to the presence/absence of the Salmonella , Campylobacter , Cryptosporidium and Giardia using culture or microscopy methods, and the detection of stx (Shiga toxin) and eae (intimin) genes commonly found in diarrhoeagenic E. coli , using RT‐PCR. Previous study has shown that unlike other E. coli subtypes, phylotype B1 may persist and be stably maintained in freshwater (Berthe et al., ; Ratajczak et al., ; Touchon et al., ; Walk et al., ) but its presence may still have implications for the quality and safety of the affected area. Thus, the relative abundance of this ‘generalist’ E. coli was used for exploratory purposes to test the novel assumption where B1 was used as a proxy for non‐recent faecal contamination when identified in the presence of reduced numbers of the other E. coli phylotypes expected in fresh contamination. Based on this hypothesis, each individual water sample was grouped by abundance of phylotype B1 identified from the 20 isolates to represent low (<10 B1 isolates), intermediate (10–14 B1 isolates), and high (≥15 B1 isolates) for inclusion in models. Linear mixed effects models were also used to examine the relationship between for example, E. coli phylotypes/ Escherichia cryptic clades to examine the association with faecal source data. To assess whether there was a difference in phylotype profiles across samples by land‐use, a distance‐based analysis of variance (PERMANOVA) was performed including site as a random effect nested within land‐use (Anderson, ). p values of <0.05 were deemed as significant.
The water samples for this study originated from 2022 to 2023 large‐scale MfE Freshwater Pathogen study collected as part of a wider study to update the 1998–2000 QMRA (Till et al., ). Freshwater samples were taken as part of routine monitoring programs by Council (local New Zealand environmental agency) staff in late spring to early winter (October 2022–July 2023) at 41 sites around New Zealand (Figure ). Sampling was scheduled to provide national coverage of different land‐uses taking into account the logistics associated with long term sampling across nine different Councils at 41 sites.
A hydrological sub‐catchment for each sampling site was generated using ArcGIS Pro (v.2.6.4). In each respective catchment, the sampling location was used as a pour point in the catchment delineation analysis. The pour point specifies where water should ‘drain’ from the catchment, thereby giving the total land surface draining to the sampling point. The catchment sizes ranged from approximately 3–8 km 2 . Land cover data (Table ) for each of the 41 sites was sourced from the Land Cover Database version 5.0, Mainland, New Zealand (Anon, ) to assist with identifying the dominant land‐use for site categorization. Sites with a history of elevated concentrations of E. coli were located across New Zealand and reflected different dominant land‐uses and impacts as designated by Councils: dairy ( n = 13); sheep and beef ( n = 10); mixed sheep and beef and dairy ( n = 2); urban ( n = 8); avian ( n = 4). Four sites were designated as low impact with lower potential for faecal contamination (exotic/natural forest dominated control sites). Sample sites were designated as ‘avian’ using prior observational information provided by Councils which noted large numbers of avian faecal contamination events caused by wildfowl flocks or bird roosts. Generally, sampling occurred between 6 AM and 1.30 PM New Zealand standard time. Samples were collected 200 mm below the water surface using aseptic methods. Most samples sites were selected from non‐tidal areas, however, if samples sites affected by tidal influences were identified, they were sampled around low tide. If the water was too deep, samples were taken from the bank using an extendable pole. After collection, samples were cooled and transported overnight with frozen packs to keep water samples chilled for FIB, MST and pathogen analyses to the Institute of Environmental Science and Research (ESR, Christchurch, New Zealand). Samples for protozoan analyses were sent overnight to MicroAquaTech, Massey University (New Zealand).
For quantitative analysis of Campylobacter by Most Probable Number (MPN) enrichment, water volumes of 1 × 1000 ml, 3 × 10 ml, 3 × 1 ml and 3 × 0.1 ml were analysed (APHA, ; ISO, ; Medeiros & Hoffman, ) and cultured using Preston broth (Fort Richard Laboratories Ltd., Auckland, New Zealand) as described in the Data . Preston broth culture was plated onto modified charcoal‐cefoperazone‐deoxycholate agar (mCCDA) plates (Fort Richard) and incubated at 41.5 ± 1°C for 44 (±4) h, and then 2–4 putative Campylobacter colonies were transferred to Columbia Blood Agar (CBA) plates (Merck, Darmstadt, Germany) and incubated as before. Where putative Campylobacter colonies were observed on CBA, conventional PCR (Wong et al., ) was performed on DNA extracted from the original Preston broth culture to identify C. coli , C. jejuni and thermophilic Campylobacter .
For presence/absence analysis of Salmonella , 1 L of water was filtered through 0.45 μm (47 mm diameter) mixed cellulose ester membrane filters (Millipore, Merck) and placed into 25 ml of buffered peptone water (BPW) broth (Fort Richard Laboratories Ltd) and incubated at 37 ± 1°C for 18 ± 2 h as described in the Data . Isolates that were consistent with Salmonella were tested for the presence of Salmonella O‐ and H‐antigens by slide agglutination using polyvalent antisera (Remel, ThermoFisher Scientific, Vilnius, Lithuania) and metabolic characteristics (Microbact GNB 12A and 12B kits in combination, ThermoFisher Scientific).
Protozoa were detected from 30 L water samples according to USEPA Methods (USEPA, ) as described in the Data . Immunomagnetic separation (IMS) using a Dynabeads GC‐Combo kit (ThermoFisher Scientific) and immunofluorescent staining for Cryptosporidium oocysts and Giardia cysts was performed using the EasyStain kit (BioPoint Pty Ltd.).
Escherichia coli were analysed in freshwater using Colilert Quanti‐Tray assays (100 ml of a 1 in 10 dilution of water sample, incubated at 35 ± 0.5°C for 24 to 28 h) (IDEXX Laboratories, Inc., Maine) (APHA, ). This provides a detection range of <10 Most Probable Number (MPN)/100 ml up to >24,200 MPN/100 ml. Wells that were yellow in colour and fluoresced when exposed to UV light, indicative of the presence of E. coli , were identified with a hand‐held UV unit (EA‐160/FE, Spectroline, New York). Between 3 October 2022 and 3 July 2023, positive Colilert 2000 trays (IDEXX, New Zealand) from 298 water samples stored at 4°C after testing was complete were transported (maintained at chilled temperature during overnight transit) in single weekly batches to AgResearch (Hopkirk Research Institute, Palmerston North) from ESR, Christchurch. For each individual Colilert tray, wells that fluoresced when exposed to UV light, indicative of the presence of E. coli were identified with a hand‐held UV unit (EA‐160/FE, Spectroline, New York), carefully opened using a sterile low‐gauge needle, and 150 μl (small wells) or 200 μl (large wells) of growth removed from each after pipetting up and down three times to homogenize the bacterial suspension in each well. Bacterial culture from the fluorescent wells of each individual Colilert plate was pooled and centrifuged at 5000× g for 10 min at 4°C using a Multifuge X3R (Thermo Fisher Scientific). The supernatant was discarded, the pellet resuspended in 1 ml EC broth and transferred to a 1.5 ml tube and centrifuged at 16,200× g for 2 min. The supernatant was discarded, the bacterial pellets were resuspended in 1 ml EC broth (Fort Richard Laboratories Ltd.) and added to cryovials containing glycerol (final concentration 30% v/v) and stored at −80°C. Colilert trays ( n = 298) were sub‐grouped to balance E. coli MPN/100 ml concentrations of <100 (low), 101 to 1000 (medium), and >1000 (high) in a 1:2:1 ratio across the 41 sample sites, where possible, to focus on samples requiring regulatory authority action in New Zealand: (i) 260 E. coli MPN/100 ml to spur water managers to identify sources of faecal contamination for mitigation purposes and (ii) 540 E. coli MPN/100 ml to inform the public that the site is unsuitable for primary contact recreational use (Anon, ). Subsequent isolation of E. coli was undertaken from a selection of 199 frozen glycerol stocks across all 41 sample sites on ECC CHROMagar plates (Fort Richard Laboratories Ltd.). Plates were incubated at 35°C for 18 to 21 h after which 20 well‐spaced, individual blue colonies (presumptive E. coli ) from each water enrichment (stored in glycerol) were selected and sub‐cultured onto MacConkey Agar plates (Fort Richard Laboratories Ltd.) and incubated as previously described. A crude DNA sample for further DNA analysis was prepared for each of the 3980 isolates by resuspending a single well‐spaced colony in 400 μl of sterile milliQ water, heated at 100°C for 10 min in a heating block and stored at −20°C. Another single well‐spaced colony was resuspended in 1.5 ml of sterile Brain heat infusion (BHI) broth (Oxoid) containing glycerol (30% v/v) and stored at −80°C. In total, 3980 presumptive E. coli were stored from 199 water samples.
PCRs for Escherichia species and E. Coli phylotypes Each of the boiled lysate preparations ( n = 3980) were used in individual PCR reactions to determine the E. coli phylotype or Escherichia cryptic Clades (I to V) using an extended quadruplex PCR phylotype assignment method (Clermont et al., , ). Allele specific multiplex PCR for the identification of phylotypes was carried out as described in the Data . Clade I to V PCRs were carried out according to Clermont et al .
PCR ( RT ‐ PCR ) for detection of diarrhoeagenic E. col i genes Real‐time multiplex PCR (RT‐PCR) assays were performed on an Applied Biosystems ViiA7 Real‐time PCR System (Life Technologies) targeting the virulence genes stx 1 and stx 2 from Shiga toxin‐producing E. coli (STEC), and intimin adherence ( eae ) gene from enteropathogenic E. coli (EPEC) and some STEC serogroups, as markers for the presence of pathogenic E. coli (Clarke, ; Nataro & Kaper, ), and the E. coli 16S rRNA gene as an internal control. Positive samples were described as those having quantification cycle thresholds (Cq) of <35.
Rainfall data for water samples in the preceding 24, 48 and 72 h, was recorded by Council staff from each site and visit and was included in analysis. Average flow data (m 3 /s) from the preceding 24 h prior to sampling were provided by Council staff.
Freshwater samples were filtered (in duplicate) through a 0.45 μm mixed cellulose ester membrane filters (Millipore, France) and DNA extracted as described in the Data . Quantitative PCR (qPCR) analysis was undertaken on a LightCycler 480 (Roche Diagnostics Ltd, CA). The PCR targets are presented in Table with PCR protocols and conditions outlined by Devane et al. and described in the Data . Each qPCR run included negative and positive controls, and standard curves of the appropriate target. The amplification efficiency of the PCR assays was considered acceptable at >90% for qPCR targets, and the coefficient of determination ( r 2 ) at ≥0.92 for assays.
Statistical analysis was performed using R version 3.6.2 (R Development Core Team, ), and ggpubr (Kassambara, ); figures were produced using the package ggplot2 (Wickham, ). Shannon diversity (total number of E. coli phylotypes and Escherichia species) per water sample was undertaken using the R package ‘vegan’ version 2.5‐6 (Oksanen et al., ). To test the effects of pathogens or pathogen‐associated genes ( Salmonella , Campylobacter , eae gene, stx gene, Cryptosporidium , Giardia , Any Bacteria, Any Protozoa, Any Pathogen), lme4 (Bates et al., ) was used to fit binomial generalized linear mixed effect models on the pathogen presence or absence data, to test associations with, for example, E. coli concentration MPN/100 ml (log 10 MPN) or E. coli phylotype (total 0–20) from each water sample determined using PCR. Individual “Site” was included in the model as a random effect. ‘Any Pathogen’ refers to the presence/absence of the Salmonella , Campylobacter , Cryptosporidium and Giardia using culture or microscopy methods, and the detection of stx (Shiga toxin) and eae (intimin) genes commonly found in diarrhoeagenic E. coli , using RT‐PCR. Previous study has shown that unlike other E. coli subtypes, phylotype B1 may persist and be stably maintained in freshwater (Berthe et al., ; Ratajczak et al., ; Touchon et al., ; Walk et al., ) but its presence may still have implications for the quality and safety of the affected area. Thus, the relative abundance of this ‘generalist’ E. coli was used for exploratory purposes to test the novel assumption where B1 was used as a proxy for non‐recent faecal contamination when identified in the presence of reduced numbers of the other E. coli phylotypes expected in fresh contamination. Based on this hypothesis, each individual water sample was grouped by abundance of phylotype B1 identified from the 20 isolates to represent low (<10 B1 isolates), intermediate (10–14 B1 isolates), and high (≥15 B1 isolates) for inclusion in models. Linear mixed effects models were also used to examine the relationship between for example, E. coli phylotypes/ Escherichia cryptic clades to examine the association with faecal source data. To assess whether there was a difference in phylotype profiles across samples by land‐use, a distance‐based analysis of variance (PERMANOVA) was performed including site as a random effect nested within land‐use (Anderson, ). p values of <0.05 were deemed as significant.
An extended longitudinal study was undertaken over 39 weeks from October 2022 to July 2023. Visits were made to 41 sites (4 avian, 13 dairy, 4 low impact, 2 mixed—sheep, beef and dairy, 10 sheep and beef and 8 urban). On average, sites were visited on five separate occasions (min 1, max 9); two sites were visited only once as replacements for inaccessible sites due to the impacts of Cyclone Gabrielle (13 February 2023), which temporarily restricted access to the original selected sites. Following selection across low/medium/high E. coli concentrations (at 1:2:1 ratio) and across sample sites, 199 Colilert trays were taken forward for further analysis of 20 E. coli isolates from each tray where 45 (22.6%) were low, 106 (53.3%) medium and 48 (24.1%) high E. coli MPN/100 ml. E. coli were detected from all 199 water samples analysed in detail at a geometric mean of 379 MPN/100 ml (median 330 MPN/100 ml) and a geometric mean of 349 MPN/100 ml for all 298 samples received (median 315 MPN/100 ml); urban sites had the highest average E. coli contamination rates (geometric mean 813MPN/100 ml). Compared to low impact sites (geometric mean 91 MPN/100 ml), there was no significant difference in average E. coli concentrations for avian (geometric mean 166 MPN/100 ml, p = 0.43) and mixed impact sites (geometric mean 259 MPN/100 ml, p = 0.27), but average E. coli contamination rates for dairy (geometric mean 597 MPN/100 ml, p = 0.004), sheep and beef (geometric mean 318 MPN/100 ml, p = 0.046) and urban ( p = 0.001) were all significantly higher. The 199 Colilert trays representative of freshwater samples from avian ( n = 24), dairy ( n = 48), low impact ( n = 20), mixed ( n = 12), sheep and beef ( n = 44) and urban ( n = 51) land‐uses, were used for further analysis of 20 E. coli isolates from each tray. Molecular typing of the 3980 isolates indicated that B1 was the most common phylotype (1912/3980, 48.04%) followed by almost equal numbers of A (587, 14.75%) and B2 (592, 14.87%) (Figure ). Other E. coli phylotypes were less frequently isolated including C ( n = 67, 1.68%), D ( n = 409, 10.3%), E ( n = 319, 8.02%), F ( n = 24, 0.60%), G ( n = 34, 0.85%) and Clade I ( n = 9, 0.23%). Only four isolates were unable to be assigned a phylotype following PCR. Cryptic Escherichia were rare, with only one E. ruysiae /Clade IV (0.03%) and 22 E. marmotae /Clade V (0.55%) identified from 14 sites (1 mixed, 1 avian, 2 dairy, 2 low impact, 8 urban). Phylotype B1 (average 9.6 per sample set of 20 isolates per water sample) isolates were detected at a higher rate than A (average 2.95) and B2 (2.97). Overall, there was more variation of each phylotype (identified from the 20 isolates collected per water sample) related to the number of visits per site compared with variation between the sites with the same land‐use (Figure ). Overall phylotypes A and B1 were more abundant in the water samples sourced from avian, dairy, mixed, sheep and beef and urban land‐uses whereas the opposite was observed in phylotype B2 which was scarce in the avian, dairy, mixed, sheep and beef and urban land‐uses but much more common in water samples from the low impact sites (Figure ). The association between each individual phylotype and land‐use was examined compared to the ‘low impact’ land‐use, where ‘Site’ was included as a random effect. For phylotype A there was a much stronger association with the ‘dairy’ and ‘urban’ land‐use, and B1 across all land‐uses except low impact (Table ). In contrast, phylotype D, and especially B2 were much less associated with all land‐uses compared to ‘low impact’ (Table ). Phylotype C was associated with avian, whilst there was no association ( p > 0.05) between phylotype E and land‐use (Table ). There was evidence for a difference in phylotype profile by land‐use (F_{pseudo} = 5.01, p = 0.0003) and between sites within land‐use (F_{pseudo} = 1.24, p = 0.03), with land‐use differences explaining 15.5% of the variation in phylotype profile and site within land‐use explaining 18.2% of variation, leaving 66.2% variation due to visit within site. When phylotype and E. coli log 10 MPN were examined, phylotype A was positively correlated with E. coli concentration (log 10 MPN/100 ml) ( p < 0.0001, OR 1.26, 95% CI 1.17–1.36) for each water sample, but for B2 ( p = 0.0007, OR 0.86, 95% CI 0.79–0.84) and D ( p < 0.0001, OR 0.82, 95% CI 0.75–0.90) there was a negative correlation (Table ). All other phylotypes (B1, E, F and G) were not correlated with E. coli concentrations ( p > 0.05) (Table ). In addition to culture‐based methods for the detection of Campylobacter and Salmonella , and microscopy to detect Cryptosporidium and Giardia , an RT‐PCR‐based approach was undertaken to examine the prevalence of stx and eae genes in DNA extractions obtained from culture enrichments. Overall Campylobacter species were the most common pathogen detected (62.8%, 125 of 199 water samples), followed by the eae gene (62.3%, 124 of 199), Giardia oocysts (59.8%, 119 of 199) Cryptosporidium oocysts (44.2%, 88 of 199), Salmonella (24.6%, 49 of 199) and stx genes (17.6%, 35 of 199). Although C. jejuni (61.3%, 122 of 199) and C. coli (18.6%, 37 of 199) were identified separately by PCR, most water samples where C. coli was identified (91.4%, 32 of 35) also contained C. jejuni , therefore these data were combined to describe the presence of generic Campylobacter . At least one of the six pathogen types was detected in 90.0% (179/199) of the water samples; bacteria in 80.9% ( n = 161) and protozoa in 71.4% ( n = 142). The persistence of E. coli phylotype B1 has been suggested as an indicator of the age of faecal inputs to a waterbody (Berthe et al., ; Ratajczak et al., ; Touchon et al., ; Walk et al., ). The age of faecal contamination was evaluated based on the detection frequency of B1 isolates in each water sample. Recent faecal inputs generally contain a higher diversity of E. coli phylotypes and strains (Bergholz et al., ; Gordon et al., ; Son et al., ). To explore the validity of this association between numbers of B1 isolates in a water sample and the age of faecal contamination, samples were grouped to represent low (<10 B1 isolates/20), intermediate (10 to 14 B1 isolates/20), and high (≥15 B1 isolates/20) phylotype B1. Overall, there was no difference of the geometric mean E. coli log 10 MPN/100 ml for low (average 344 MPN/100 ml), intermediate (average 557 MPN/100 ml) and high (average 254 MPN/100 ml) B1 isolates ( p = 0.065). Importantly, increased numbers of individual pathogens (i.e., 1–6) detected were associated with low and intermediate B1 compared to high B1 ( p < 0.0001). Where B1 were present at <10, 10 to 14, or ≥ 15 isolates, pathogens were present, 88.0% (low B181/92 samples), 98.5% (intermediate B165/66 samples) and 80.5% (high B133/41 samples), respectively ( p = 0.011). However, although high numbers (≥15 B1 isolates) of persistent phylotype B1 had the lowest number of pathogens on average supporting the hypothesis that persistent high numbers of phylotype B1 could be an indicator of non‐recent, aged contamination events. In addition, a high proportion of these samples with low phylotype B1 had pathogens detected (80.5%) as per the prevalence of ‘Any pathogen’. The presence of Salmonella , eae gene, Giardia , ‘Any bacteria’, and ‘Any protozoa’ were more associated with water samples with low and/or intermediate age B1 levels compared to high (Table ). Interestingly, ‘Any pathogen’ was associated with intermediate B1 but not low when compared to high numbers (≥15 B1 isolates) of phylotype B1 indicative of non‐recent contamination (Table ). Neither Cryptosporidium , Campylobacter , nor stx toxin gene presence correlated with the three groupings of B1 persistence (Table ). The Shannon diversity of phylotypes (per 20 isolates from each water sample) was closely related to E. coli log 10 MPN/100 ml ( p < 0.00001) and compared to high (≥15) phylotype B1, there was increased Shannon diversity in water samples with intermediate ( p < 0.0001) and low ( p < 0.0001) B1 (Figure ). Water samples containing increased phylotype diversity were associated with low (<10) phylotype B1 (Figure ), but in contrast, water samples from low impact sites where fewer faecal sources would be anticipated were anomalous and characterized by low numbers of phylotype B1 (i.e., <10 B1 isolates/20 isolates) and low phylotype diversity due to increased phylotype B2. Compared to low impact, there was also increased phylotype diversity associated with all other land‐uses ( p < 0.01). These data suggest that increased E. coli concentration (log 10 MPN/ml) increases phylotype diversity, and even after accounting for E. coli concentration effects, land‐use and phylotype B1 groupings may also have an effect. Any link between pathogen detection and phylotype was assessed and for analysis of phylotypes B1, B2, C, D, E, F or G there was no interaction with pathogens (Table ). However, for phylotype A, there was a positive association with the eae gene ( p = 0.008) and Salmonella ( p = 0.007) (Table ). Furthermore, E. coli concentrations (log 10 MPN/100 ml) were widely associated with bacterial pathogens and diarrhoeagenic E. coli genes (Table ). No interactions were seen with Protozoa, and no negative associations were observed (Table ). Detection of pathogens occurred more often from urban and dairy land‐uses compared to low impact sites (Table ); for avian sites, only the detection of Salmonella was more significant from water samples compared to low impact sites ( p = 0.009). For the stx toxin gene, Giardia and Cryptosporidium , there was no significant association with land‐use compared to low‐impact sites (Table ). Three MST markers (Human, CrAssphage, 1.7–6.08 log 10 Gene Copies [GC]/100 ml; Ruminant, BacR, 1.26–6.46 log 10 GC/100 ml; Avian, GFD, 1.18–4.15 log 10 GC/100 ml) were examined in more detail to identify dominant faecal sources associated with land‐use (Figure ). Most water samples (68.63%, 35 of 51) from urban sites had Human as the dominant faecal source; most from dairy, mixed and sheep and beef sites had Ruminant faecal sources (74.04%, 77 of 104); 33.33% of avian sites (8 of 24) had Avian faecal sources; whereas 35.0%, low impact (7 of 20) had no, or low concentrations, of the faecal sources that were tested, detected. Although no faecal sources were identified in water samples (18 of 199, 9.04%) with E. coli concentrations of between 63 and540 MPN/100 ml, some (10 of 199, 5.02%) had at least one, and up to four different pathogens identified in these water samples. Avian, Human and Ruminant MST markers (log 10 , GC/100 ml) were examined in more detail to identify associations with land‐use (Table ), with E. coli phylotypes (Table ) and pathogens (Table ). Three human MST markers were initially examined, and in subsequent analyses, CrAssphage concentration was used as the human source indicator when affirmed by detection of at least one other human marker detected at the LOQ. In previous study, crAssphage has shown it has a high level of sensitivity and specificity, and persistence in freshwater studies (Ahmed et al., ; Ballesté et al., ; Gyawali et al., ). When compared to low impact areas, faecal source data (log 10 , GC/100 ml) were generally concomitant with land‐use (Table ); for example, Human MST marker and urban land‐use ( p < 0.0001), and Ruminant MST marker with dairy land‐use ( p = 0.004). The Avian MST microbial marker was ubiquitous being found across all land‐uses. Escherichia coli phylotype associations with MST markers reflected phylotype and land‐use observations (Table ). Phylotype A, for example, was more associated with the Avian MST markers ( p < 0.0001), B1 positively associated with ruminant ( p = 0.0004), but negatively with Avian ( p = 0.016) and Human ( p = 0.005), and B2 negatively with the Ruminant MST marker ( p = 0.002). Phylotype E was also more commonly associated with the Avian MST marker ( p = 0.0003), and E. marmotae less associated with the Ruminant MST marker ( p = 0.036). Other phylotypes were not associated with the MST markers (Table ). Both bacterial and protozoal pathogens were associated with the three MST markers examined in more detail (Table ). Despite Giardia being associated with the Avian MST marker ( p = 0.026), there was no association of Cryptosporidium with any of the faecal source markers suggesting that oocysts from this protozoan species are equally prevalent and/or ubiquitous with all three MST faecal sources, that is, no discrimination. Notably the zoonotic pathogen Campylobacter and the diarrhoeagenic E. coli ‐associated virulence factors stx and eae had the highest odds ratio for association with the ruminant faecal source marker (Table ). The highest odds ratio across all three faecal source markers was for ‘Any bacteria’ with the Avian MST marker ( p = 0.002, OR 4.43, 95% CI 1.7–11.5). The qPCR 16S rRNA internal control (GenBac3 1.28–7.99 log 10 , GC/100 ml) data was not associated with any of the pathogens examined (Table ). The significance of increased rainfall on E. coli concentrations (log 10 MPN/100 ml) peaked at 48 h, (24 h, p = 0.06; 48 h, p = 0.001; 72 h, p = 0.09), and with increased flow ( p = 0.0002). Rainfall was also positively associated with the presence of the eae gene (0–24 h: OR 1.18, 95% CI 1.04–1.35, p = 0.01), and stx genes (0–24 h: OR 1.11, 95% CI 1.04–1.18, p = 0.002). Higher flow (preceding 24 h) was significantly associated with increased E. coli concentrations (log 10 MPN/100 ml), and there was also a positive association between Salmonella (OR 1.04, 95% CI 1.01–1.07, p = 0.005) with water flow but no association between ‘Any pathogen’ or the other pathogens detected ( p > 0.05).
E. coli is one of the most‐extensively studied gut bacteria but host/habitat niche specialization (Cookson et al., ; Petit et al., ; Touchon et al., ) of different subtypes has only become apparent through the use of molecular (Clermont et al., ; Clermont et al., ) and genomic methods (Cookson et al., ; Luo et al., ; Nowicki et al., ) to sub‐type isolates obtained from diverse environments. In parallel, the realization that E. coli may also persist in water, soil and sediment has provided new impetus to investigate the maintenance and survival of newly described environmental E. coli ‐like Escherichia spp. (Gilroy et al., ; Liu et al., ; van der Putten et al., ), isolated from terrestrial and aquatic habitats (Koh et al., ; Mire et al., ; Moinet et al., ). The advances in molecular methods to subtype E. coli and identify E. marmotae and E. ruysiae should allow new insights into their overall abundance and association with enteric pathogen species. This study made use of the routine Colilert (IDEXX) culture enrichments from water samples to provide a comprehensive analysis of E. coli phylotypes and recently described Escherichia species. Overall, the non‐ E. coli Escherichia comprised only a minor proportion of the isolates subtyped using PCR (0.6%, 24/3980), with 23 E. marmotae and 1 E. ruysiae identified from 14 sites (Figure ). Previous study from New Zealand has indicated that E. marmotae and E. ruysiae can be isolated from avian faeces, are present in wildlife and pristine bush habitats, and have fewer virulence factors compared to E. coli (Cookson et al., ; Moinet et al., ). Together with data from this study for example, where E. marmotae were negatively associated with the Ruminant MST marker (Table ), these lines of information from recent studies, provide important evidence to water managers that despite being indistinguishable from FIB using standard culture‐based enumeration methods, the recently characterized Escherichia species are not associated with waterborne pathogens and represent a lower likelihood of potential health risk to recreational users of water. Furthermore, the low prevalence of these non‐ E. coli Escherichia species was considerably less than the error of the Quanti‐Tray MPN enumeration method used with the Colilert media (Muirhead & Meenken, ). A potential limitation of our experimental approach analysing Colilert enrichments is the impact of numerical dominance of specific E. coli clones after incubation and transport (chilled), concomitant with any contrasting competitive exclusion and survival characteristics between different E. coli clones in individual Colilert wells. In future studies, more labour intensive, direct recovery of E. coli on membrane filters could be employed to assess phylotype variation without sample enrichment. However, we have shown that non‐ E. coli Escherichia species are unlikely to contribute frequently to FIB exceedances when using Colilert analysis for microbial water quality monitoring. Here, water samples ( n = 199) were selected to cover a broad range of E. coli concentrations with selection of 50% of samples having E. coli concentrations covering the relevant concentrations for recreational water quality guidelines (100–1000 MPN/100 ml). This strategy allowed us to confirm our hypothesis that increased E. coli phylotype diversity in each water sample was most likely associated with recent faecal contamination and increased E. coli concentrations (Figure ), however, this was in contrast to a prior study undertaken in an urban area where a negative relationship was observed between E. coli concentration and phylotype diversity (Saraceno et al., ), likely due to fewer diverse land‐uses and no characterization of the age of faecal contamination events being examined. What constitutes an aged faecal source and what microbial metrics can be used to help define such a parameter remains unclear. Thus, the extent of this dataset including prevalence of pathogens and breadth of sampling sites provided an opportunity to explore the novel assumption of a candidate microbial metric for faecal age evaluation: high numbers of phylotype B1 in a water sample as suggestive of aged faecal contamination, compared with higher prevalence of other E. coli phylotypes (low B1) associated with recent faecal contamination. Interestingly, water samples dominated by phylotype B1, (potentially) indicative of aged faecal sources, were virtually absent in low impact sites dominated by phylotype B2, likely due to the reduced faecal contamination sources overall (Figure ). Where low phylotype B1 (potentially) indicative of recent faecal sources were encountered in water from low impact sites, these samples were associated with low E. coli concentration. In water sampling sites impacted by reduced faecal sources and with low E. coli concentrations, the congruence of low phylotype diversity and the dominance of phylotype B1 likely supports the candidate faecal age metric and reflects the die‐off of other phylotypes less resilient and more unsuited to environmental persistence compared to B1 (Figure ). Furthermore, water samples impacted by low and/or intermediate B1 levels as a proxy for more recent faecal inputs were associated with an increasing number of bacterial and protozoal pathogen types compared to waters with putative aged sources (high B1). However, this last point needs to be viewed in the context of a high proportion of all samples (≥ 80.5%) containing at least one pathogen. The generalist nature of the naturalized phylotype B1 and its enhanced environmental resilience (Nowicki et al., ; Petit et al., ; Touchon et al., ) may influence its non‐association with pathogens from more recent faecal contamination events identified in this study (Table ), but this observation is somewhat confounded by its high prevalence in all land‐uses except low impact. Nevertheless, the low abundance of B1 in low impact areas provides some confidence that these sites were less impacted by faecal sources compared to other land‐uses (Table ). In the same way, the reduced environmental persistence of phylotype A clearly contrasted with the increased phylotype prevalence data from contrasting land‐uses (e.g., low impact and dairy) (Table ). Along with B1, phylotype A is considered a generalist often associated with human and animal faecal samples (Johnson et al., ; Saraceno et al., ) and wastewater (Behruznia & Gordon, ; Tavares et al., ), and was the only E. coli phylotype indicative of recent faecal contamination through a positive association with E. coli concentration, bacterial pathogens, but not protozoa. Other studies have examined the relationship between E. coli concentration and pathogens (Hörman et al., ; Till et al., ; Weller et al., ), but none have undertaken a phylotype‐specific approach to examine interactions with common freshwater pathogens such as Campylobacter , Salmonella , or protozoa ( Cryptosporidium and Giardia ). A previous study in Finland failed to identify a significant correlation between E. coli and pathogens in water samples taken from rivers and lakes (Hörman et al., ) and results may have been impacted by more extreme seasonal temperatures and less variation in the age of faecal sources. In the current study, phylotype B2 was overrepresented in low impact sites (Figure and Table ), which aligns with our previous observations of its increased abundance in pristine bush environments compared to downstream sites impacted by pastoral farming (Cookson et al., ). Intriguingly, phylotype B2 has also been identified as a human‐associated E. coli phylotype, commonly isolated from urinary tract infections and associated with the extended‐spectrum beta‐lactamase resistant antibiotic resistance phenotype (Toombs‐Ruane et al., ). Therefore, we hypothesise that there are likely to be multiple pathotypes represented by phylotype B2, including those from forested sites (Petit et al., ), wildlife (Moinet et al., ) and humans with contrasting pathotype‐specific genetic characteristics (Johnson et al., ; Smati et al., ) which may influence their survival and persistence in the environment. The abundance of B2 in low impact sites less affected by faecal sources in this study contrasts with other studies where B2 were identified as lacking survival‐associated functions and were shown to survive for shorter periods in secondary environments, such as beach sand (Rumball et al., ; Rumball et al., ). Thus, for environmental persistence studies external to the host, the use of human‐associated phylotype B2 sourced from urban sites or extra‐intestinal E. coli pathotypes, may not reflect the survival of B2 sourced from pristine forested sites (Cookson et al., ; Petit et al., ). Future whole genome sequence analysis and identification of human‐ or environmental‐associated genes from phylotype B2 isolates recovered from contrasting land‐uses in this study will further delineate intra‐phylotype variations and host/niche‐specific characteristics. In New Zealand, human gastrointestinal disease and notification of infections due to the protozoal pathogens Cryptosporidium and Giardia is often associated with environmental exposure with recreational water contact being a common risk factor (2020 data: Cryptosporidium , 22.5%; Giardia , 28.4%; Anon, ). Notably, neither protozoal pathogen was associated with E. coli concentration (log 10 MPN/100 ml) (Table ) or E. coli phylotype (Table ), but ‘Any protozoa’ was positively associated with water samples obtained from dairy land‐use (Table ), with Giardia strongly associated with low B1 (putative recent faecal inputs), and ‘Any protozoa’ with intermediate and low B1 (putative mixed and recent faecal inputs) (Table ). Both protozoan species have complex life cycles where the infectious oocysts can survive for extensive periods in a freshwater environment prior to subsequent infection of a new host and subsequent waterborne outbreaks of diarrhoeal disease (Devane et al., ; Efstratiou et al., ). In this study, both Cryptosporidium and Giardia oocysts were identified from water samples collected from low impact sites, characterized by low E. coli concentrations (<50 MPN/100 ml) and suggestive of aged faecal contamination events but which, however, did not match our definition based on B1 abundance (Figure ). Protozoa, especially Cryptosporidium , represent a significant burden of enteric disease in New Zealand with some evidence of seasonal prevalence in waterways (Phiri et al., ) corresponding somewhat with human cryptosporidiosis infections (Lake et al., ). Despite protozoa detection rates of 71.36% (142/199 water samples), there were fundamental disparities between the association of bacterial pathogens and protozoal oocysts in water column samples and exploratory variables included in this study (e.g., E. coli concentration MPN/100 ml, E. coli phylotype, faecal age, rainfall, water flow etc.). These observations may be influenced by contrasting kinetics and/or settling velocity deposition through sedimentation (Searcy et al., ) for the two microbial types and the contrasting sizes of individual protozoal (oo)cysts (≥5 μm) (Robertson & Lim, ; Ryan & Xiao, ) and generic E. coli cells (1–2 μm). Alternatively, low concentrations in faecal samples (Moriarty et al., ) and inability to grow outside hosts, predation of (oo)cysts by ciliated protozoan species (Siqueira‐Castro et al., ) or effect of ambient sunlight (Ahmed et al., ; King et al., ) on oocysts may also influence these disparities. The use of MST to provide evidence of specific faecal sources provides additional evidence for water managers to apply targeted mitigation strategies to reduce contamination (Devane et al., ; Harwood et al., ; Tran et al., ; Weller et al., ). Although dominant Avian faecal sources were widespread in water samples from all land‐uses including low impact (Table ) and in previous study (Flores et al., ), other research using the same avian qPCR (GFD) detected avian faecal sources only rarely (Weller et al., ). Comparison of MST markers and E. coli phylotype B1 (Table ) also correlated with the phylotype association with land‐use noted before (Table ) where phylotype B1 was associated with ruminants (de Castro et al., ; Johnson et al., ; Massot et al., ). Similarly, phylotype A has been found in high proportions in highly polluted urban streams (Saraceno et al., ) and was also associated with urban land‐use in this study, but there was no association with the Human MST marker (Table ). There was a strong, positive association between bacteria ( Campylobacter , pathogenic E. coli stx and eae genes) and the Ruminant MST marker (Table ). Both Campylobacter , such as C. jejuni and C. coli , and STEC are zoonotic pathogens that commonly inhabit the gut of cattle and sheep as animal reservoirs (Cookson et al., ; Irshad et al., ). Positive interactions between pathogenic E. coli markers and ruminant faecal source markers were noted in previous studies from USA (Weller et al., ) and France (Petit et al., ), however unlike the study from USA, eae was also associated with the Human MST marker ( p = 0.0(46). Although the stx and eae genes can be identified together in STEC, these pathogenic E. coli markers may also be present separately in STEC/EPEC isolates, including those from freshwater sites (Koczura et al., ; Marucci et al., ). However, the pathogenicity of water isolates with uncommon stx ‐subtypes remains to be determined (Cookson et al., ) and the eae gene has been associated with prolonged EPEC survival in Cladophora (Byappanahalli et al., ). Furthermore, although Campylobacter was associated with the Ruminant ( p = 0.006) and Avian ( p = 0.023) markers, there was no interaction with the Human MST marker (Table ). This observation may reflect the dominance of pastoral landscapes in the New Zealand environment and the widespread impact of ruminant faecal sources, but that in some extreme cases Campylobacter may be associated with human pollution sources in urban areas (Devane et al., ). Interestingly Salmonella was associated with avian and urban land‐uses but not Human and Avian faecal sources (Table and Table ). Several pathogenic Salmonella serovars have been identified as the zoonotic agent associated with local human disease (Bloomfield et al., ; Ford et al., ; Fu et al., ), and further study is required to establish their prevalence in New Zealand bird species (Moriarty, Karki, et al., ) and links with waterborne outbreaks of disease. Overall, there was some association of pathogens with land‐use that was congruent with MST markers; for example, Campylobacter and the eae gene were associated with dairy land‐use (Table ) and the Ruminant MST marker (Table ). Freshwater environments and associated E. coli concentrations are highly dynamic being influenced by water flow rate and weather events in addition to land‐use (Muirhead & Meenken, ). Rainfall events and soil saturation significantly influence the overland flow processes whereby pathogens move across land to water (Muirhead et al., ). However, these hydrological events and their impact on the movement of pathogens from soil and faecal material are not uniform, which results in the exceptional variability in multiple factors measured from any particular site in this study. This was illustrated where visit/sampling event was more variable than site in terms of phylotypes identified (Figure ) and PERMANOVA data where increased phylotype variation was due to visit within site. Combined, these variables interact to significantly influence the E. coli concentration, phylotype composition, types and ages of faecal sources, and pathogens detected across different land‐uses. Thus, the composition and metadata associated with every water sample is unique. For example, recent rainfall (48 h preceding sampling) had a significant impact on freshwater E. coli concentrations (MPN/100 ml), and the presence of eae and stx genes as a proxy for pathogenic E. coli . Interestingly, the eae and stx genes were only significantly associated with rainfall in the preceding 24 h of sampling. The contrasting duration of Campylobacter (<24 h, p = 0.057) and pathogenic E. coli after rainfall is likely to reflect both the increased persistence of E. coli strains in the environment compared to the more rapid die‐off of Campylobacter , such as C. jejuni (Moriarty et al., ; Moriarty, Mackenzie, et al., ), and the higher concentration of E. coli contamination that may occur from multiple animal inputs (Moriarty et al., ). Flow measurements were also associated with increased freshwater E. coli concentrations (MPN/100 ml), but of the pathogens only Salmonella , was associated with increased flow measurements suggesting short‐lived persistence in waterways. This study took a spatial approach to identify the dominant land‐use associated with freshwater sampling sites, but geo‐spatial mapping often identified multiple land‐use designations for each site (Table ). Because individual sites are influenced by multiple land‐uses and are likely impacted by multiple faecal sources on any individual water sampling occasion, it is unclear what the relative contribution of land‐use is on pathogen prevalence. However, despite the increased overall E. coli MPN/100 ml contamination for dairy (1368 MPN/100 ml) and urban (3347 MPN/100 ml) the prevalence of ‘Any pathogen’ was associated with dairy ( p = 0.039) but not urban land‐uses ( p = 0.138) (Table ). This is supported by another study where E. coli and Campylobacter were more associated with New Zealand agricultural sites, and protozoa more prevalent in waterways with higher numbers of ruminants in the catchment and lower water quality (Phiri et al., ). Whilst none of the faecal sources monitored were identified from 18 of the water samples, 10 contained at least one and up to four different pathogens. These data clearly indicate that potential recreational health risk remains from, for example, low impact sites, where water samples occur with low E. coli concentrations, and where Ruminant, Human or Avian faecal sources are absent. However, a risk assessment would be required to determine if pathogens are present at concentrations likely to affect human health. Analysis with additional MST markers such as cat, dog or possum ( Trichosurus vulpecula , a prevalent introduced pest) (Moinet et al., ) may provide clues on other faecal sources. The influence of sediments on microbial quality in the overlying water column has attracted increasing scrutiny with the proposal that sediments in water bodies can act as reservoirs for both FIB and pathogens (Devane et al., ; Devane et al., ; Petit et al., ). This reservoir effect from non‐recent faecal inputs may be one explanation for the disconnection between MST markers, FIB and pathogen presence in water samples. Persistent faecal microbes may be entrained from sediments into the water column even under base flow conditions. These factors suggest that broad understanding and detailed information regarding catchment dynamics, including temporal and spatial sampling events, are required to characterize the full range of (intermittent) faecal sources likely to affect informed decisions on mitigations.
By taking a molecular approach to delineate E. coli and closely related Escherichia species ( E. marmotae and E. ruysiae ), this study has provided phylotype‐specific associations with dominant land‐use, faecal inputs into a water body and links with bacterial pathogens. Initial observations exploring the E. coli population across a wide range of both land‐uses and E. coli concentrations, suggests (i) that E. marmotae , E. ruysiae and E. whittamii are rare and not associated with waterborne pathogens, (ii) a clear role for E. coli concentrations and phylotype A as an indicator for bacterial freshwater pathogens, (iii) that phylotype B2 is an occupier of low impact sites dominated by exotic/native forest and (iv) that the prevalence of phylotype B1 as a potential metric for faecal age is worthy of additional investigation. Furthermore, compared to studies focussing on highly impacted sites with chronic faecal contamination alone, pathogens were detected from some low impact sites, with low E. coli concentrations and an absence of ruminant, avian and human microbial faecal source markers, indicative of potential human health risk. Implementation of new guidelines including these data and further data from the Freshwater Pathogen QMRA study will assist with understanding how many sampling events may be required to understand the dominant faecal sources likely to (intermittently) impact a site. Furthermore, the QMRA modelling will incorporate pathogen concentrations, providing an improved understanding of the potential human health risk associated with recreational water contact.
Adrian L. Cookson: Conceptualization; investigation; funding acquisition; writing – original draft; supervision; data curation; project administration; writing – review and editing; methodology; validation; visualization; formal analysis. Meg Devane: Conceptualization; investigation; funding acquisition; methodology; data curation; formal analysis; validation; visualization; writing – original draft; writing – review and editing; supervision; project administration. Jonathan C. Marshall: Software; formal analysis; conceptualization; investigation; funding acquisition; writing – review and editing; methodology; validation; visualization; data curation. Marie Moinet: Investigation; methodology; formal analysis; project administration; data curation; supervision; writing – review and editing; visualization; validation. Amanda Gardner: Investigation; methodology; supervision; writing – review and editing; data curation. Rose M. Collis: Investigation; methodology; writing – review and editing; data curation; formal analysis. Lynn Rogers: Data curation; formal analysis; methodology; writing – review and editing; investigation. Patrick J. Biggs: Writing – review and editing; methodology; investigation; conceptualization; validation; visualization; software. Anthony B. Pita: Methodology; writing – review and editing; formal analysis; data curation; investigation. Angela J. Cornelius: Methodology; writing – review and editing; formal analysis; data curation; supervision; investigation; validation. Iain Haysom: Supervision; resources; writing – review and editing; formal analysis; project administration; data curation; methodology; validation. David T. S. Hayman: Writing – review and editing; methodology; supervision; resources; investigation; visualization. Brent J. Gilpin: Writing – review and editing; funding acquisition; methodology; validation; project administration; data curation; supervision; resources. Margaret Leonard: Funding acquisition; resources; supervision; data curation; project administration; writing – review and editing; methodology; validation.
The authors declare no competing financial interests.
DATA S1. Supporting Information. TABLE S1. Catchment delineation and land‐use identification for freshwater sampling sites. Land cover data for each of the 41 sites was sourced from the Land Cover Database version 5.0, Mainland, New Zealand1. Land‐use Av, avian; D, Dairy; Lm, low impact; Mix, mixed sheep, beef and dairy; Sh, sheep and beef; U, urban.
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ALK-positive large B-cell lymphoma (ALK + LBCL) with aberrant CD3 expression | 09e02e21-0fba-415f-93a6-f0e1ff42b7f6 | 11127819 | Anatomy[mh] | Since the discovery of the oncogenic anaplastic kinase (ALK) gene in anaplastic large cell lymphoma , the number of ALK-positive neoplasms has expanded and now includes ALK + LBCL, a rare subtype of aggressive diffuse large B cell lymphoma . Architecturally, in lymph nodes, the neoplastic proliferation is diffuse with at least a partial sinusoidal pattern. Cell morphology shows plasmablastic or immunoblastic differentiation. Characteristically, immunohistochemistry in ALK + LBCL expresses plasmacytic markers including CD38, CD138, and MUM1. B cell and T cell markers are generally negative. ALK expression generally shows a cytoplasmic granular pattern. Although lineage-specific T cell markers are generally negative, up to 50% can show CD4 positivity . Only rare cases are reported to express CD3 . Rare cases also have been reported to be EBV positive . The ALK protein is a receptor tyrosine kinase, encoded by the ALK gene on chromosome 2p23 . Aberrant kinase activity associated with ALK can be seen with gene rearrangement, amplification, and point mutations of the ALK gene ; however, fusion proteins with ALK are seen most commonly in ALK + LBCL. ALK is rearranged in ALK + LBCL, most commonly CLTC::ALK , but other translocations have been reported involving the ALK gene . Multiple signaling pathways are stimulated in response to ALK, including Ras/ERK, JAK/STAT, PI3k/Ark, and PLCγ pathways . Activation of STAT3 and STAT5 is reported in ALK + LBL with STAT3 expression reported in some cases .
A 58-year-old man presented with cervical adenopathy. The specimen was noted to be involved by a large cell neoplasm, which was favored to be hematopoietic, and referred to our institution. There is no other available past medical history or follow-up. Flow cytometry from an outside laboratory at the time of excision showed “no monotypic B-cell, phenotypically aberrant T-cell or blast cell population with identified.”
Immunohistochemistry Immunohistochemistry was performed according to standardized automated operating protocols. Immunohistochemistry included the following antibodies: CD45, ALK1, CD138, CD3, MYC, MUM1, EMA PAX5, CD79a, BCL6, CD10, CD21, BCL2, CD30, CD3, CD2, CD5, CD7, CD4, CD8, cyclin D1, CD56, HHV-8, P24 (HIV), AE1/AE3, MART-1, and in situ staining for Kappa, lambda, and EBER. CD3 performed at the outside institution was repeated at our institution using DAKO (A0452). T cell receptor (TCR) and immunoglobulin heavy (IGH) chain rearrangement PCR-based detection for clonal TCR gamma and IGH was performed. Extracted DNA from the paraffin-embedded tissue using a modified version of QIAGEN QIAmp DNA purification protocol and the PCR-based BIOMED-2 assay (Invivoscribe, San Diego, CA) was performed. The manufacturer’s instructions were strictly followed in interpreting this assay. TCR beta was not performed. FISH Deparaffinized tissue sections were hybridized using the manufacturer’s recommendations with the following probes Vysis LSI MYC Dual Color Breakapart DNA probe, Vysis IGH/BCL2 Dual Color Fusion DNA translocation probe, Vysis LSI BCL6 (ABR) Dual Color Breakapart DNA probe, and Vysis LSI ALK Dual Color Breakapart DNA probe.
Immunohistochemistry was performed according to standardized automated operating protocols. Immunohistochemistry included the following antibodies: CD45, ALK1, CD138, CD3, MYC, MUM1, EMA PAX5, CD79a, BCL6, CD10, CD21, BCL2, CD30, CD3, CD2, CD5, CD7, CD4, CD8, cyclin D1, CD56, HHV-8, P24 (HIV), AE1/AE3, MART-1, and in situ staining for Kappa, lambda, and EBER. CD3 performed at the outside institution was repeated at our institution using DAKO (A0452).
PCR-based detection for clonal TCR gamma and IGH was performed. Extracted DNA from the paraffin-embedded tissue using a modified version of QIAGEN QIAmp DNA purification protocol and the PCR-based BIOMED-2 assay (Invivoscribe, San Diego, CA) was performed. The manufacturer’s instructions were strictly followed in interpreting this assay. TCR beta was not performed.
Deparaffinized tissue sections were hybridized using the manufacturer’s recommendations with the following probes Vysis LSI MYC Dual Color Breakapart DNA probe, Vysis IGH/BCL2 Dual Color Fusion DNA translocation probe, Vysis LSI BCL6 (ABR) Dual Color Breakapart DNA probe, and Vysis LSI ALK Dual Color Breakapart DNA probe.
Hematoxylin and eosin staining demonstrated lymph node tissue showing predominantly diffuse effacement of the architecture (Fig. A) with a focal sinusoidal pattern. The sections show a proliferation of large, atypical cells with round nuclear contours, prominent central nucleoli, and plasmablastic features (Fig. B). Frequent mitotic figures and focal necrosis are present. Reported flow cytometry showed “no monotypic B-cell or aberrant T-cell population.” Immunohistochemistry shows the neoplastic cells are positive for CD45, ALK1 (granular cytoplasmic staining) (Fig. C), CD138 (Fig. D), focal CD3 (Fig. E; star and 1F), MYC (Fig. G), MUM1 (Fig. H), and EMA (not shown). CD3 shows an unusual focal cytoplasmic and granular staining pattern in both the stain submitted by the outside institution and the repeated stain. In situ hybridization studies of kappa and lambda light chains demonstrate kappa restriction (Fig. I). Neoplastic cells are negative for CD20, PAX5, CD79a, BCL6 (weak to negative), CD10, CD21, BCL2, CD30, CD2, CD5, CD7, CD4, CD8, cyclin D1, CD56, HHV-8, P24 (HIV), AE1/AE3, MART-1, and in situ staining for EBER (not shown). Fluorescence in situ hybridization (FISH) reveals intact MYC , BCL6 genes, and negative IGH::BCL2 . ALK is disrupted (Fig. D). Clonal IGH and TCR gamma chain gene rearrangements are present (Fig. A–C). The TCR gamma shows a prominent peak in an otherwise polyclonal background.
ALK + LBCL is a distinct, rare, and aggressive type of large B cell lymphoma, which typically presents in lymph nodes, most commonly the cervical ; however, extranodal sites are also affected. There is a wide age range at presentation with a median of 35–38 years and a male predominance. Patients present with high stage disease in most cases with median survival of 11–24 months . There is no known association with immunodeficiency or viral association including Epstein Barr virus, HHV8, or HIV. Architecture of the lymph node is diffusely effaced but often shows a partial sinusoidal pattern. Cellular morphology appears immunoblastic and/or plasmablastic . Rarely large Reed-Sternberg-like cells can be present . There is increased mitotic activity and can be necrosis. Our case showed effaced architecture with the neoplastic cells showing a plasmablastic appearance. Immunohistochemistry evaluation of the neoplastic cells in ALK + LBCL shows expression of plasmacytic markers including CD138, MUM1, CD38, and VS38. CD20 is often negative or weakly positive in a minority of cases. Other B cell antibodies including CD19 and CD22 are negative. PAX5 and CD79a are seen in a minority of cases. Other stains, which may be positive, include EMA, BOB-1, OCT-2, and CD45RB (may be weak). Cytoplasmic immunoglobulin most often IgA can be positive. CD10, MYC, and STAT3 can be positive. Lineage-specific T cell antigens and T cell-associated antigens are generally negative; however, CD4, CD57, and CD43 expression is reported in approximately half of cases. Immunohistochemistry in our case was compatible with ALK + LBCL, but the cells demonstrated partial CD3 expression, a finding that has been reported in only rare cases . EBV is almost uniformly negative, although rare cases have been positive . CD30 is negative in the majority of cases. All cases express ALK protein or contain the ALK translocation . The staining pattern of ALK is most often in a cytoplasmic granular pattern; this expression pattern is the result of the lymphomagenic translocation ALK::CLTC [t(2;17)(p23;q23)] in the majority of cases. Other translocation partners include NPM1 , SEC31A , SQSTM1 , RANBP2 GORASP2 , EML4 , and IGL ; these translocations can give distinct staining patterns . Our case shows cytoplasmic granular ALK staining. ALK by FISH was disrupted in our case; however, the partner gene cannot be determined in the FISH assay and NGS was not performed. FISH for BCL2 , BCL6 , and MYC were intact. Aberrant T cell-specific markers expressed on LBCL have been reported, most notably CD5, but other markers including CD2, CD4, CD7, and CD8 have also been reported . Aberrant CD3 on LBCL is unusual but is reported sporadically . Some cases with CD3 expression on LBCL have been associated with EBV leading to speculation that EBV may promote linage infidelity; however, EBV is not uniformly present, and our case does not express EBER. Expression of more than one T cell-specific or associated marker is seen in rare cases of LBCL. The biologic significance is unknown in cases of LCBL with aberrant CD3 due to their rarity . ALK + LBCL has not been studied extensively with IGH and TCR previously, but in studies where IGH and TCR rearrangements were performed, no TCR clonal rearrangement was identified; however, no case showed CD3 expression in these studies . Coexisting IGH and TCR clones in gene rearrangement studies is not previously reported, but there are limited studies looking at TCR rearrangements in ALK + LBCL. Although our case may be considered “borderline” in some laboratories due to the apparent polyclonal background, the TCR assay was read as clonal following the manufacturer’s instructions. The source of this peak may derive from the focal neoplastic cells, which express aberrant CD3 or conversely, the peak may derive background T cells reacting to the neoplastic B cells. It has also been documented that false positive TCR rearrangements can occur in B cell lymphomas and reactive processes using BIOMED-2 assays . Although theoretically TCR gamma and TCR beta assays together could be used to confirm the clone, both may sometimes be clonal in the same reactive process . Although the source and significance of the T cell peak in our case is uncertain, the utility of BIOMED-2 assays is well documented, and this assay is standard worldwide but caution in reading the assay should be observed. In conclusion, we present a case of ALK + LBCL with aberrant expression of CD3 and gene rearrangement of both IGH and TCR . A panel of immunohistochemistry markers including multiple T cell, B cell, and plasmacytic markers was necessary to characterize the lymphoma, which is recommended when lineage-specific antigen expression is ambiguous. Because aberrant expression of CD3 is rare in ALK + LBCL, we performed IGH and TCR to help delineate lineage of this ALK + LBCL, testing which may be helpful particularly if aberrant T cell markers are expressed. However, as this case illustrates, gene rearrangement studies should be interpreted with caution and considered within the clinical and pathologic context. The clinical behavior of our case of ALK + LBCL is uncertain due to the rarity of aberrant CD3 in this entity and the lack of clinical follow-up information. The clinical behavior may not differ from conventional cases of ALK + LBCL; however, further study is necessary.
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Methanogenesis coupled hydrocarbon biodegradation enhanced by ferric and sulphate ions | 3642529f-e762-4c34-9604-8e720d2ec855 | 11362221 | Microbiology[mh] | Crude oil and its derivatives still supply a significant part of the energy worldwide. The extensive usage leads to oil spills, which contaminate the environment. Certain hydrocarbon compounds such as PAHs, BTEX and volatile alkanes imply a serious threat to human and animal health as well (Davidson et al. ; Sun et al. ; Halios et al. ). Larger spills such as the consequence of the BP Deepwater Horizon catastrophe in 2010 can also inflict significant damage on the economy of the affected area (Buckingham-Howes et al. ; Keating et al. ). The negative impact of hydrocarbons on the environment, health and economy is indisputable, and rapid intervention is essential. Bioremediation can be an environmentally safe and cost-effective solution for the removal of hydrocarbons (da Silva et al. ). Many bacterial groups are capable of hydrocarbon biodegradation either under aerobic or anaerobic conditions (Abbasian et al. ; Laczi et al. ). The aerobic pathways are faster and more effective than their anaerobic counterparts, and they are preferred during bioremediation. However, hydrocarbons can seep into deeper soil layers or sediments where the environment is anoxic and hinders biodegradation. Bacteria developed enzymes including succinate synthases to activate hydrocarbons (Fuchs et al. ; Boll et al. ). Such enzymes, e.g. alkyl/benzyl-succinate synthases, occur in Smithella sp., Thauera aromatica and Azoarcus sp. (Leuthner and Heider ; Achong et al. ; Tan et al. ). Similar enzymes in the Asgard group of archaea were recently found, indicating their ability to utilise hydrocarbons (Zhang et al. ). The anaerobic mineralisation of aromatic compounds consists of a peripheral and a central pathway (Fuchs et al. ). Alternative activation pathways of the aromatic compounds, such as carboxylation and hydroxylation, are also known (Boll et al. ). The central pathway utilises benzoyl-CoA as the central metabolite of aromatics biodegradation. It can occur in an ATP-dependent and ATP-independent manner catalysed by Class I and Class II benzoyl-CoA reductases (Rabus et al. ; Tiedt et al. ; Anselmann et al. ). Alternative terminal electron acceptors (TEA) including nitrate, sulphate and ferric ions sustain anaerobic respiration. Still, their presence can be scarce in deep soil or sediments as their concentration decreases with depth (Li et al. ). Anaerobic hydrocarbon biodegradation can also couple with methanogenesis. The methane produced from hydrocarbon biodegradation can be utilised as an energy carrier, partially recovering the energy of the oil spill (Parkes ; Xia et al. ; Suda et al. ). Microbially enhanced energy recovery can be used to decrease the costs of remediation. In this study, we performed laboratory-scale batch fermentation experiments with hydrocarbon-contaminated soil samples derived from the B4 sampling site of the NATO base in Taszár, Hungary, from a depth of 7.5 m described earlier (Molnár et al. ). We examined the effect of sulphate and ferric ions on hydrocarbon biodegradation and biomethane production. We also determined the microbial community composition and recovered metagenome-assembled genomes (MAGs) from sequencing data.
Sampling The sample for the enrichment culture was taken from the B4 sampling site of the military base of Taszár from 7.5 to 8.0 m (Molnár et al. ). The hydrocarbon-contaminated soil was collected into 500-mL borosilicate bottles (Teqler, Wecker, Luxembourg) and sealed with a silicone septum. The bottle was flushed with nitrogen (purity: 5.0, Messer Hungarogas Ltd. Budapest, Hungary). Soil (30 g) was kept anaerobically and was immediately frozen at − 80 °C for later metagenomic analysis as the samples arrived at the lab. Starter enrichment culture Enrichment cultures were prepared under anaerobic conditions in a Bactron IV™ Anaerobic Chamber (Sheldon Manufacturing Inc., Cornelius, OR, USA). The chamber’s atmosphere was set with a 5% hydrogen in 95% nitrogen gas mixture (Linde Gas Hungary Co CLtd., Budapest, Hungary). Fresh soils (30 g) were weighed into 160-mL serum bottles (Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 30-mL heat-sterilised distilled water. The vials were sealed with butyl-PTFE septa (Sigma-Aldrich). The vials were flushed with nitrogen (purity: 5.0) and then incubated at 30 °C in an incubator without shaking. Head-space gas composition was measured weekly. Starter cultures were utilised after 6 weeks, where methane levels were stabilised in the head space. Preparation of the samples Samples were prepared in a Bactron IV™ Anaerobic Chamber. The three starter cultures were mixed, and 0.5-mL suspensions were aliquoted into serum bottles (full volume: 38 mL headspace: 30 mL), and then 5 g of the original soil was added to the samples. All samples were supplemented with a 4.5-mL minimal salt medium containing various electron acceptors (Fig. ) to facilitate hydrocarbon biodegradation. Table summarises the compositions of media used during the experiments. All samples were sealed with butyl-PTFE septa and flushed with nitrogen (purity: 5.0) for 5 min. After flushing, 6-mL nitrogen was removed from the CO 2 -amended samples with a syringe and an equal volume of food industrial CO 2 (Gourmet C, Messer Hungarogas Ltd.) was added. This resulted in a concentration of 20% v/v in the headspace. Three types of carbon sources were added to each electron acceptor containing samples using a Hamilton glass syringe. An aliphatic mixture (61.6-mg n-hexadecane, 3.9-mg pristane, 3.9-mg cyclohexane, 3.9-mg methyl-cyclohexane and 3.6-mg n-decane) and an aromatic mixture (90-mg methyl-naphthalene, 4.3-mg toluene, 4.3-mg ethyl-benzene, 2.7-mg naphthalene and 2.5-mg phenanthrene). A sample group without an additional carbon source was prepared as a biotic control for each electron acceptor. Abiotic controls for GC/MS measurements were prepared as follows. A total of 5 g of original soils were heat sterilised with the Tyndall method with three sterilisation cycles. The heat-sterilised soils were mixed with a minimal salt medium lacking externally added electron acceptors. The samples were incubated at 30 °C in a stationary incubator. All samples were prepared in sextuplicates except for the biotic and abiotic control samples, which were only triplicated. Analytical methods Determination of methane concentration in the headspace Methane concentration was measured with an Agilent 6890 gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a split/splitless (S/SL) injector and a thermal conductivity detector (TCD). The analytical column was an HP-Molsieve (30 m × 0.53 mm × 25 μm) with a 5-Å pore size. The carrier gas was argon (purity 5.0, Messer Hungarogas Ltd.). Column flow was 1.1 mL/min, and the oven temperature was set to 47 °C. The injector was operated in split mode with a split ratio of 9:1. Samples were injected manually with a gastight syringe (Hamilton 1725 RN, Hamilton Co., Reno, NV, USA). Methane calibration was done with a gas mixture containing 5% (v/v) methane, 5% (v/v) carbon dioxide and 5% (v/v) hydrogen (Linde Gas Hungary Co. Cltd.). Calibration samples were on atmospheric pressure. The molarity of CH 4 was calculated based on the calibration curve with the ideal gas law using atmospheric pressure. The energy content of the methane was determined by first calculating the mass of the produced gas based on its molarity and then multiplying this mass by the calorific value of methane, which is 55.5 MJ/kg. With this method, hydrogen and oxygen can also be measured from the sample headspace simultaneously with methane. Determination of carbon dioxide concentration in sample headspace Carbon dioxide was measured with a Shimadzu 2010 gas chromatograph equipped with an S/SL injector and a TCD. The analytical column was an HP-PLOTQ (30 m × 0.32 mm × 20 μm) capillary column. The carrier gas was nitrogen (purity 5.0, Messer Hungarogas Ltd.). The column flow was set to 1.25 mL/min, and the split ratio was 4:1. Samples were injected manually with a gastight syringe (1702 RN, Hamilton Co.) Calibration was performed with the same gas mixture as in the case of methane. Determination of hydrocarbon concentration in the samples Half of the samples (3–3 parallel from each set) and the abiotic controls were extracted with 5-mL diethyl-ether overnight at room temperature with shaking at 200 rpm on a Biosan OS-20-orbitary shaker (SIA Biosan, Riga, Latvia). The organic phase was sampled with syringes to avoid opening the vials. The hydrocarbon contents of the samples were measured with an Agilent 7890B gas chromatograph mounted with an Agilent 5975C VL-MSD mass spectrometer and an Agilent 7683B autosampler (Agilent Technologies Inc.). The gas chromatograph was also equipped with an S/SL injector. The analytical column was an HP-5 ms Ultra Inert (30 m × 0.25 mm × 0.25 μl) capillary column, and the carrier gas was helium (purity: 6.0, Messer Hungarogas Ltd.). From all samples, 1 μl was injected into the inlet. The energy of the electron beam was set to 70 eV in the electron impact chamber, and the mass spectrometer was run in single ion monitoring mode. The gas flow rate, the GC’s heating profile and the selected ions varied depending on the utilised carbon source as follows. Aliphatic mixture The gas flow in the column was set to 3.0 mL/min, and the split ratio was 29:1. The initial column temperature was 40 °C kept for 5 min. The heating ramp was set to 5 °C/min with a final temperature of 220 °C sustained for 3 min. The analysed ions were the following: m/z = 57, 83 and 84. Aromatic mixture The GC programme runs with a 1.2 mL/min column gas flow and a 19:1 split ratio. The initial oven temperature was 45 °C, held up for 4 min. The heating ramp was adjusted to 15 °C/min, and the final temperature was 230 °C kept for 5 min. The measured ions were m/z = 91, 128, 142 and 178. Preparation of DNA samples DNA preparations were done from 5 mL of the cultures. In the case of the original soil sample, 2.5 g of soil was used. Samples were with the QiaGen PowerBead Tubes derived from the RNeasy PowerSoil Mini Kit (QIAGEN N.V. Venlo, the Netherlands). The kit’s protocol was followed until the first precipitation of nucleic acids. The precipitated nucleic acids were dissolved in 50-µL AccuGENE™ molecular biology water (Lonza, Basel, Switzerland) and purified by 1% agarose gel electrophoresis to eliminate contaminants entirely. Gel slices containing genomic DNA were cut with sterile blades and put into 0.5-mL PCR tubes. The bottom of the tubes was previously lined up with a thin layer of glass wool and heat sterilised. The gel slices were deep-freezed at − 80 °C for 2 h. Afterwards, the bottom of the tubes was perforated with a hot steel needle, and the liquid content of the gel slices was centrifuged (900 × g, 10 min) into a 1.5-mL microcentrifuge tube. The samples were treated with phenol:chloroform:isoamyl alcohol (25:24:1, pH = 6.6; Ambion, Austin, TX, USA), and then nucleic acids were precipitated with ice-cold isopropanol for 30 min at − 20 °C. After centrifugation (15,000 × g, 30 min), the pellets were washed with ice-cold 70% ethanol. DNA was dissolved in AccuGENE™ molecular biology water and sent for whole-metagenome sequencing. Whole metagenome sequencing Sequencing was performed by Delta Bio 2000 Ltd. (Szeged, Hungary). Briefly, the DNA library was prepared with Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA) and sequenced on a NextSeq 550 (Illumina Inc.) with a NextSeq 500/550 Mid Output Kit v2.5 through 300 cycles as it was instructed by the manufacturer. Bioinformatical methods The GNU parallel (Tange ) and homemade bash scripts were used to automate processes on multiple samples. Visualisation of the data was performed with Microsoft Excel and the seaborn (Waskom ), Pandas (McKinney ) and Matplotlib (Hunter ) modules of Python 3. For cluster map generation, the distance matrices were calculated with the Bray–Curtis method built in the seaborn module. Quality check and assessment of the reads Read quality was summarised with FastQC (Andrews ), followed by MultiQC (Ewels et al. ) analysis. Quality trimming was performed with Trimmomatic (Bolger et al. ) with the following parameters: HEADING:2, TRAILING:3, SLIDINGWINDOW:8:20 and MINLEN:100. Only surviving paired reads were considered in the subsequent analysis. Read-based taxonomy The taxonomic composition of the samples was determined with Kaiju (Menzel et al. ). Unassigned reads were omitted from further analysis, and taxons that did not reach 1% of the total read count in the particular sample were also not considered. Read abundance was normalised as a percentage of the assigned read number for the given sample. Whole metagenome assembly and MAG recovery The quality trimmed samples were assembled into one large co-assembly with MEGAHIT (Li et al. ). The assembly statistics were generated by QUAST (Gurevich et al. ). MetaBat 2 (Kang et al. ), MaxBin 2 (Wu et al. ) and Concoct (Alneberg et al. ) were used for binning of individual MAGs. Bin refinement of the MAGs was carried out with MetaWrap (Uritskiy et al. ). The Quant_bins module of MetaWrap was used to estimate the abundance of the individual MAGs through the samples. The MAG abundance results were expressed in genome copies per million reads (GCPM). Log 2 GCPM values were visualised with the cluster map module of seaborn. The reassembled bins were identified with GTDB-tk (Chaumeil et al. ), and their functional analysis was performed with Prokka (Seemann ) and eggnog-mapper (Huerta-Cepas et al. ). Individual genes were identified with blastx and blastp algorithms using the MAGs and their deduced proteins against known proteins as a query. Statistical analysis Statistical analysis was performed in SigmaPlot using one-way analysis of variance (ANOVA; p ≤ 0.05) followed by Duncan’s multiple range test.
The sample for the enrichment culture was taken from the B4 sampling site of the military base of Taszár from 7.5 to 8.0 m (Molnár et al. ). The hydrocarbon-contaminated soil was collected into 500-mL borosilicate bottles (Teqler, Wecker, Luxembourg) and sealed with a silicone septum. The bottle was flushed with nitrogen (purity: 5.0, Messer Hungarogas Ltd. Budapest, Hungary). Soil (30 g) was kept anaerobically and was immediately frozen at − 80 °C for later metagenomic analysis as the samples arrived at the lab.
Enrichment cultures were prepared under anaerobic conditions in a Bactron IV™ Anaerobic Chamber (Sheldon Manufacturing Inc., Cornelius, OR, USA). The chamber’s atmosphere was set with a 5% hydrogen in 95% nitrogen gas mixture (Linde Gas Hungary Co CLtd., Budapest, Hungary). Fresh soils (30 g) were weighed into 160-mL serum bottles (Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 30-mL heat-sterilised distilled water. The vials were sealed with butyl-PTFE septa (Sigma-Aldrich). The vials were flushed with nitrogen (purity: 5.0) and then incubated at 30 °C in an incubator without shaking. Head-space gas composition was measured weekly. Starter cultures were utilised after 6 weeks, where methane levels were stabilised in the head space.
Samples were prepared in a Bactron IV™ Anaerobic Chamber. The three starter cultures were mixed, and 0.5-mL suspensions were aliquoted into serum bottles (full volume: 38 mL headspace: 30 mL), and then 5 g of the original soil was added to the samples. All samples were supplemented with a 4.5-mL minimal salt medium containing various electron acceptors (Fig. ) to facilitate hydrocarbon biodegradation. Table summarises the compositions of media used during the experiments. All samples were sealed with butyl-PTFE septa and flushed with nitrogen (purity: 5.0) for 5 min. After flushing, 6-mL nitrogen was removed from the CO 2 -amended samples with a syringe and an equal volume of food industrial CO 2 (Gourmet C, Messer Hungarogas Ltd.) was added. This resulted in a concentration of 20% v/v in the headspace. Three types of carbon sources were added to each electron acceptor containing samples using a Hamilton glass syringe. An aliphatic mixture (61.6-mg n-hexadecane, 3.9-mg pristane, 3.9-mg cyclohexane, 3.9-mg methyl-cyclohexane and 3.6-mg n-decane) and an aromatic mixture (90-mg methyl-naphthalene, 4.3-mg toluene, 4.3-mg ethyl-benzene, 2.7-mg naphthalene and 2.5-mg phenanthrene). A sample group without an additional carbon source was prepared as a biotic control for each electron acceptor. Abiotic controls for GC/MS measurements were prepared as follows. A total of 5 g of original soils were heat sterilised with the Tyndall method with three sterilisation cycles. The heat-sterilised soils were mixed with a minimal salt medium lacking externally added electron acceptors. The samples were incubated at 30 °C in a stationary incubator. All samples were prepared in sextuplicates except for the biotic and abiotic control samples, which were only triplicated.
Determination of methane concentration in the headspace Methane concentration was measured with an Agilent 6890 gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a split/splitless (S/SL) injector and a thermal conductivity detector (TCD). The analytical column was an HP-Molsieve (30 m × 0.53 mm × 25 μm) with a 5-Å pore size. The carrier gas was argon (purity 5.0, Messer Hungarogas Ltd.). Column flow was 1.1 mL/min, and the oven temperature was set to 47 °C. The injector was operated in split mode with a split ratio of 9:1. Samples were injected manually with a gastight syringe (Hamilton 1725 RN, Hamilton Co., Reno, NV, USA). Methane calibration was done with a gas mixture containing 5% (v/v) methane, 5% (v/v) carbon dioxide and 5% (v/v) hydrogen (Linde Gas Hungary Co. Cltd.). Calibration samples were on atmospheric pressure. The molarity of CH 4 was calculated based on the calibration curve with the ideal gas law using atmospheric pressure. The energy content of the methane was determined by first calculating the mass of the produced gas based on its molarity and then multiplying this mass by the calorific value of methane, which is 55.5 MJ/kg. With this method, hydrogen and oxygen can also be measured from the sample headspace simultaneously with methane. Determination of carbon dioxide concentration in sample headspace Carbon dioxide was measured with a Shimadzu 2010 gas chromatograph equipped with an S/SL injector and a TCD. The analytical column was an HP-PLOTQ (30 m × 0.32 mm × 20 μm) capillary column. The carrier gas was nitrogen (purity 5.0, Messer Hungarogas Ltd.). The column flow was set to 1.25 mL/min, and the split ratio was 4:1. Samples were injected manually with a gastight syringe (1702 RN, Hamilton Co.) Calibration was performed with the same gas mixture as in the case of methane. Determination of hydrocarbon concentration in the samples Half of the samples (3–3 parallel from each set) and the abiotic controls were extracted with 5-mL diethyl-ether overnight at room temperature with shaking at 200 rpm on a Biosan OS-20-orbitary shaker (SIA Biosan, Riga, Latvia). The organic phase was sampled with syringes to avoid opening the vials. The hydrocarbon contents of the samples were measured with an Agilent 7890B gas chromatograph mounted with an Agilent 5975C VL-MSD mass spectrometer and an Agilent 7683B autosampler (Agilent Technologies Inc.). The gas chromatograph was also equipped with an S/SL injector. The analytical column was an HP-5 ms Ultra Inert (30 m × 0.25 mm × 0.25 μl) capillary column, and the carrier gas was helium (purity: 6.0, Messer Hungarogas Ltd.). From all samples, 1 μl was injected into the inlet. The energy of the electron beam was set to 70 eV in the electron impact chamber, and the mass spectrometer was run in single ion monitoring mode. The gas flow rate, the GC’s heating profile and the selected ions varied depending on the utilised carbon source as follows. Aliphatic mixture The gas flow in the column was set to 3.0 mL/min, and the split ratio was 29:1. The initial column temperature was 40 °C kept for 5 min. The heating ramp was set to 5 °C/min with a final temperature of 220 °C sustained for 3 min. The analysed ions were the following: m/z = 57, 83 and 84. Aromatic mixture The GC programme runs with a 1.2 mL/min column gas flow and a 19:1 split ratio. The initial oven temperature was 45 °C, held up for 4 min. The heating ramp was adjusted to 15 °C/min, and the final temperature was 230 °C kept for 5 min. The measured ions were m/z = 91, 128, 142 and 178.
Methane concentration was measured with an Agilent 6890 gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a split/splitless (S/SL) injector and a thermal conductivity detector (TCD). The analytical column was an HP-Molsieve (30 m × 0.53 mm × 25 μm) with a 5-Å pore size. The carrier gas was argon (purity 5.0, Messer Hungarogas Ltd.). Column flow was 1.1 mL/min, and the oven temperature was set to 47 °C. The injector was operated in split mode with a split ratio of 9:1. Samples were injected manually with a gastight syringe (Hamilton 1725 RN, Hamilton Co., Reno, NV, USA). Methane calibration was done with a gas mixture containing 5% (v/v) methane, 5% (v/v) carbon dioxide and 5% (v/v) hydrogen (Linde Gas Hungary Co. Cltd.). Calibration samples were on atmospheric pressure. The molarity of CH 4 was calculated based on the calibration curve with the ideal gas law using atmospheric pressure. The energy content of the methane was determined by first calculating the mass of the produced gas based on its molarity and then multiplying this mass by the calorific value of methane, which is 55.5 MJ/kg. With this method, hydrogen and oxygen can also be measured from the sample headspace simultaneously with methane.
Carbon dioxide was measured with a Shimadzu 2010 gas chromatograph equipped with an S/SL injector and a TCD. The analytical column was an HP-PLOTQ (30 m × 0.32 mm × 20 μm) capillary column. The carrier gas was nitrogen (purity 5.0, Messer Hungarogas Ltd.). The column flow was set to 1.25 mL/min, and the split ratio was 4:1. Samples were injected manually with a gastight syringe (1702 RN, Hamilton Co.) Calibration was performed with the same gas mixture as in the case of methane.
Half of the samples (3–3 parallel from each set) and the abiotic controls were extracted with 5-mL diethyl-ether overnight at room temperature with shaking at 200 rpm on a Biosan OS-20-orbitary shaker (SIA Biosan, Riga, Latvia). The organic phase was sampled with syringes to avoid opening the vials. The hydrocarbon contents of the samples were measured with an Agilent 7890B gas chromatograph mounted with an Agilent 5975C VL-MSD mass spectrometer and an Agilent 7683B autosampler (Agilent Technologies Inc.). The gas chromatograph was also equipped with an S/SL injector. The analytical column was an HP-5 ms Ultra Inert (30 m × 0.25 mm × 0.25 μl) capillary column, and the carrier gas was helium (purity: 6.0, Messer Hungarogas Ltd.). From all samples, 1 μl was injected into the inlet. The energy of the electron beam was set to 70 eV in the electron impact chamber, and the mass spectrometer was run in single ion monitoring mode. The gas flow rate, the GC’s heating profile and the selected ions varied depending on the utilised carbon source as follows. Aliphatic mixture The gas flow in the column was set to 3.0 mL/min, and the split ratio was 29:1. The initial column temperature was 40 °C kept for 5 min. The heating ramp was set to 5 °C/min with a final temperature of 220 °C sustained for 3 min. The analysed ions were the following: m/z = 57, 83 and 84. Aromatic mixture The GC programme runs with a 1.2 mL/min column gas flow and a 19:1 split ratio. The initial oven temperature was 45 °C, held up for 4 min. The heating ramp was adjusted to 15 °C/min, and the final temperature was 230 °C kept for 5 min. The measured ions were m/z = 91, 128, 142 and 178.
The gas flow in the column was set to 3.0 mL/min, and the split ratio was 29:1. The initial column temperature was 40 °C kept for 5 min. The heating ramp was set to 5 °C/min with a final temperature of 220 °C sustained for 3 min. The analysed ions were the following: m/z = 57, 83 and 84.
The GC programme runs with a 1.2 mL/min column gas flow and a 19:1 split ratio. The initial oven temperature was 45 °C, held up for 4 min. The heating ramp was adjusted to 15 °C/min, and the final temperature was 230 °C kept for 5 min. The measured ions were m/z = 91, 128, 142 and 178.
DNA preparations were done from 5 mL of the cultures. In the case of the original soil sample, 2.5 g of soil was used. Samples were with the QiaGen PowerBead Tubes derived from the RNeasy PowerSoil Mini Kit (QIAGEN N.V. Venlo, the Netherlands). The kit’s protocol was followed until the first precipitation of nucleic acids. The precipitated nucleic acids were dissolved in 50-µL AccuGENE™ molecular biology water (Lonza, Basel, Switzerland) and purified by 1% agarose gel electrophoresis to eliminate contaminants entirely. Gel slices containing genomic DNA were cut with sterile blades and put into 0.5-mL PCR tubes. The bottom of the tubes was previously lined up with a thin layer of glass wool and heat sterilised. The gel slices were deep-freezed at − 80 °C for 2 h. Afterwards, the bottom of the tubes was perforated with a hot steel needle, and the liquid content of the gel slices was centrifuged (900 × g, 10 min) into a 1.5-mL microcentrifuge tube. The samples were treated with phenol:chloroform:isoamyl alcohol (25:24:1, pH = 6.6; Ambion, Austin, TX, USA), and then nucleic acids were precipitated with ice-cold isopropanol for 30 min at − 20 °C. After centrifugation (15,000 × g, 30 min), the pellets were washed with ice-cold 70% ethanol. DNA was dissolved in AccuGENE™ molecular biology water and sent for whole-metagenome sequencing.
Sequencing was performed by Delta Bio 2000 Ltd. (Szeged, Hungary). Briefly, the DNA library was prepared with Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA) and sequenced on a NextSeq 550 (Illumina Inc.) with a NextSeq 500/550 Mid Output Kit v2.5 through 300 cycles as it was instructed by the manufacturer.
The GNU parallel (Tange ) and homemade bash scripts were used to automate processes on multiple samples. Visualisation of the data was performed with Microsoft Excel and the seaborn (Waskom ), Pandas (McKinney ) and Matplotlib (Hunter ) modules of Python 3. For cluster map generation, the distance matrices were calculated with the Bray–Curtis method built in the seaborn module.
Read quality was summarised with FastQC (Andrews ), followed by MultiQC (Ewels et al. ) analysis. Quality trimming was performed with Trimmomatic (Bolger et al. ) with the following parameters: HEADING:2, TRAILING:3, SLIDINGWINDOW:8:20 and MINLEN:100. Only surviving paired reads were considered in the subsequent analysis.
The taxonomic composition of the samples was determined with Kaiju (Menzel et al. ). Unassigned reads were omitted from further analysis, and taxons that did not reach 1% of the total read count in the particular sample were also not considered. Read abundance was normalised as a percentage of the assigned read number for the given sample.
The quality trimmed samples were assembled into one large co-assembly with MEGAHIT (Li et al. ). The assembly statistics were generated by QUAST (Gurevich et al. ). MetaBat 2 (Kang et al. ), MaxBin 2 (Wu et al. ) and Concoct (Alneberg et al. ) were used for binning of individual MAGs. Bin refinement of the MAGs was carried out with MetaWrap (Uritskiy et al. ). The Quant_bins module of MetaWrap was used to estimate the abundance of the individual MAGs through the samples. The MAG abundance results were expressed in genome copies per million reads (GCPM). Log 2 GCPM values were visualised with the cluster map module of seaborn. The reassembled bins were identified with GTDB-tk (Chaumeil et al. ), and their functional analysis was performed with Prokka (Seemann ) and eggnog-mapper (Huerta-Cepas et al. ). Individual genes were identified with blastx and blastp algorithms using the MAGs and their deduced proteins against known proteins as a query.
Statistical analysis was performed in SigmaPlot using one-way analysis of variance (ANOVA; p ≤ 0.05) followed by Duncan’s multiple range test.
Methane and carbon dioxide evolution of the cultures We have followed methane evolution during the entire length of the incubation period. The measurements were performed weekly. All samples produced methane during the 8 weeks except for the samples amended with the aromatics mixture (FAR, MAR, MCAR, SAR) and the abiotic controls (KA, KAR, KK) (Fig. ). In samples, where the methanogenesis was not inhibited, the methane concentration increased gradually as time passed. The fastest methane production was reached in the FK and FA samples. These samples produced 37.7 ± 4.1 µmol and 34.2 ± 6.9 µmol methane, respectively, until the end of the incubation period. FK and FA produced significantly more methane than their counterparts without additional electron acceptors MA and MK, which yielded only 24.4 ± 8.3 and 22.0 ± 8.7 µmol methane, respectively (Fig. ). SA samples also produced high amounts of methane (31.1 ± 3.3 µmol). Although statistical significance was not found between SA and MA, the SA samples show tendentially higher methane yield (Fig. ). The addition of alkanes showed only a slight increase in methane yields in the presence of sulphate compared to SK in the last 2 weeks. Carbon dioxide-containing samples (MCK and MCA) yielded less methane than their carbon dioxide-lacking counterparts (MK and MA). We also calculated the energy content of the produced methane. It was the highest in the FK samples 33.5 ± 3.7 J in total or 7.5 ± 0.8 J relative to 1 g of the original soil sample and the lowest in MCA 5.7 ± 2.3 J in total or 1.3 ± 0.5 J relative to the original soil (Table ). Hydrogen and oxygen concentrations were also followed but remained under the detection limit during the incubation period. Carbon dioxide evolution in the samples was followed to determine the metabolic activity of the microbial community in the non-CO 2 -amended samples. CO 2 -amended samples contained an initial concentration of 20% (v/v) CO 2 , which was far over the linear range of our TCD detector and made it impossible to measure any increase or decrease of the CO 2 concentration in the headspace. In KA, KAR and KK, an increase in carbon dioxide concentration was not detectable except for the first week when there was an initial increase from 0 to 0.1% (v/v). A slightly higher initial increase was observed in the aromatics mixture-amended samples (MAR, FAR, SAR), but the final concentration did not exceed 0.3% (v/v). Statistical analysis showed a significant difference between MAR, FAR, SAR and their corresponding abiotic control KAR on the last day of incubation. The other samples had a gradual increase in carbon dioxide, and the final concentration reached an average of 2.5% (v/v) except for the SK where a slight decrease was observable compared to the other samples with an average of 1.7% (v/v). This was in concordance with the methane concentrations of SK samples, where we observed a significant drop compared to that of SA. Bioconversion rate of hydrocarbons under different conditions The original soil contained weathered diesel contamination, which had been there for two decades. The concentration of the original contamination was 4500 mg/kg soil. We supplemented this indigenous carbon source with an aliphatic mixture in the case of MA, FA, SA and their abiotic control KA or aromatics mixture in the case of MAR, FAR, SAR and their abiotic control KAR. We measured the biodegradation rate on the last day of incubation for every component of the exogenous carbon source compared to their corresponding abiotic control (Fig. ). There was a significant elevation of the biodegradation rate in all aliphatics amended samples compared to KA. Generally, the biodegradation of alkanes was the slowest in the MA samples (14.2–19.2% was degraded after 8 weeks) and the fastest in MCA samples (26.7–31.8%). Carbon dioxide addition significantly increased the biodegradation rate of alkanes and cycloalkanes compared to MA. We also observed an elevated biodegradation rate in the presence of ferric ions or sulphate. However, n-hexadecane biodegradation showed a significant elevation in the FA samples compared to MA. The biodegradation of aromatics showed a different picture. Although neither carbon dioxide nor methane was generated in the aromatics mixture-amended sample, a substantial elevation in biodegradation of aromatics was observed in the MAR, MCAR, FAR and SAR samples compared to the KAR samples. The highest rate was shown in the SAR samples (29.5–36.4%), while the lowest was in MAR (19.7–21.1%). The biodegradation rate in FAR was only slightly lower than SAR, and both were significantly higher for all the components but methylnaphthalene compared to MAR and MCAR. The MAR and MCAR samples acted similarly on all components except for naphthalene and phenanthrene, which were degraded at a significantly higher rate in the presence of carbon dioxide. Shotgun metagenome sequencing DNA was extracted from the samples, and whole metagenome sequencing was performed in triplicates. In total, 38,909,341 paired-end reads were generated with an average length of 135 bases. After quality trimming, 84.5% of the reads were preserved with an average length of 129 bases. Microbial composition of the enrichment cultures The microbial composition of every sample was determined by assigning the reads to taxons with Kaiju. A bar chart shows the abundances of various prokaryotic classes (Fig. a), and a cluster map was generated from the data on genus level (Fig. b). The major classes of the original soil appeared in all the samples, but their ratios changed based on the treatment groups. In the original soil, multiple classes are dominant: Clostridia (14.79%), Anaerolineae (13.95%), Deltaproteobacteria (9.41%), Alphaproteobacteria (8.20%), Betaproteobacteria (8.01%) and Actinomycetia (6.69%). In the enrichment culture, Betaproteobacteria (13.71%) and Methanomicrobia (10.48%) became the most dominant, followed by Actinomycetia (8.62%). In contrast, Actinomycetes were predominant in the alkane-amended samples (27.94–64.75%) and in the biotic controls (23.67–30.75%). The abundance of Clostridia (3.03–13.52%, 8.17–16.05%, respectively) and Methanomicrobia (1.45–5.94%, 5.77–10.20%, respectively) is also remarkable in these samples. In the aromatics-amended samples, Clostridia (24.04–37.20%) became the most dominant class, and Bacteroidia (7.07–10.54%) also emerged. Actinomycetia (5.39–11.72%) and Methanomicrobia (1.64–8.45%) remained also dominant, but their relative abundances were much lower compared to the alkane-amended samples, and the biotic controls stayed in the range of the original soil and the enrichment culture. The samples are divided into two major clusters based on their microbial composition (Fig. B). The first cluster contains the aromatics-amended samples and the original soil. However, the original soil showed similarities in the microbial composition with the aromatics-amended samples. Its predominant genus, Smithella (formerly belonging to Deltaproteobacteria, reassigned to Desulfobacterota (Waite et al. )), which might be the main hydrocarbon degrader in the contaminated soil, was completely missing from the other samples. Some other genera, like Anaerolinea , Rhodoferax , Sphingopyxis , Rhizobium and Mesorhizobium , were missing or only sporadically present across the samples. The main cause of this radical change in the microbial composition of the samples was the temperature difference between the deep soil and the laboratory experiment. This is supported by the substantial changes in the community structure of the enrichment culture compared to the original soil. The enrichment culture had two dominant genera, Methanothrix (6.02%) and Thiobacillus (6.09%). Methanomicrobium (1.53%), Methanoregula (1.25%) and Cellulomonas (1.00%) were also present. Ruminococcus (5.89–9.16%) was predominant in the aromatics-amended cluster regardless of the type of TEA present (Fig. B). Bacteroides (3.03–4.78%) along with Faecalibacterium (1.31–2.23%) were also dominant genera in the aromatics-amended samples. Alistipes (0.91–1.78%), Clostridium (1.06–1.58%) and Cellulomonas (0.72–1.65%) are prevalent in the aromatics-amended samples. Even though active methanogenesis was not detected in the aromatics-amended samples, the genus Methanothrix (0.66–6.77%) was present in this cluster. The second cluster contained the alkane-amended samples and the biotic controls. The enrichment culture belonged to this cluster. The genus Cellulomonas was predominant among the alkane-amended samples (16.36–32.45%) and in the majority of the biotic controls (6.07–11.63%), although it had lower relative abundance in the latter samples. Streptomyces and Microbacterium had a higher abundance in MA and SA samples compared to the corresponding biotic controls (MK, SK). In FA and MCA samples, there was no significant difference in the abundance of the two genera compared to the corresponding controls (FK, MCK). In addition, another member of the Cellulomonadaceae, Actinotalea , was only present in MA, FA and SA samples. Geothrix (0.62–5.15%) was present among the alkane-amended samples and their corresponding controls with the highest abundance in MK and the lowest in MCA. Methanothrix was ubiquitous in this cluster of samples, but its abundance decreased in the presence of alkanes. Carbon dioxide also had a negative effect on the prevalence of the genus Methanothrix . Furthermore, in MA and MCA, as well as in MK and FK samples, Methanobacterium also appeared. This genus along with Methanoregula and Methanothrix composed the bulk of the methanogenic archaea in the enrichment culture. Genomes recovered from the microbial communities We have co-assembled the reads of the alkane- and the aromatics-amended samples. The assembly resulted in 155,159 contigs with a minimum length of 200 bp. The N50 value was 1755 with the largest contig of 869,339 bp. A total of 90% of the reads could be mapped back to the assembly. Twenty-five metagenome-assembled genomes (MAGs) were recovered; out of them, 18 were high quality, and 7 were medium quality (Table ) according to the MIMAG protocol (Bowers et al. ). Despite the high completeness and low contamination of the MAGs, only four of them could be identified on the species level and six others on the genus level. The other MAGs were identified on the family level or above. We have found 3 Euryarchaeota MAGs (bin 4, bin 11 and bin 18). All the 3 MAGs were methanogenic archaea, namely Methanothrix (bin 04), Methanobacterium (bin 11) and an unidentified genus (UBA288) of the family Methanosphaerulaceae (bin 18). This family was recently proposed, and Methanosphaerula was moved to it as a type genus from Methanoregulaceae (Rinke et al. ). The uncultivated Bacteria and Archaea (UBA) genera and other taxonomic groups were identified in a large genome recovery project (Parks et al. ). The Methanoregula sp. found in the read-based taxonomy belonged to this MAG since we used an older database with Kaiju created prior to the publication of the Rinke article. The other 22 MAGs belonged to the Eubacteria kingdom. Two Ruminococcus species Ruminococcus bromii (bin 15) and Ruminococcus bicirculans (bin 7) along with Faecalibacterium prausnitzii (bin 24) and Geothrix fermentans (bin 21) were identified on the species level. On genus level Thiobacillus (bin 1), Bellilinea (bin 12), and two Cellulomonas (bins 5 and 8), MAGs were found. Many MAGs were assigned to UBA or other uncultivated genera or higher taxonomic levels. The abundance of the MAGs in individual samples was calculated, and a cluster map was created from the data (Fig. ). The clustering of the samples showed a similar pattern as in the case of the read-based abundance (Fig. B). Two major clusters were formed. One cluster contained the alkane-amended samples and the biotic controls, while the other cluster consisted of the aromatics-amended samples. MA was an outgroup in this case mainly due to the high abundance (average GCPM = 66.48) of bin 23. This was an unidentified genus BRH-c25 belonging to the family Lutisporaceae (Clostridia) and has an average nucleotide identity (ANI) of 80.08% to its closest relative BRH-c25 sp001515955. This microbe is not present in other samples of our experiments except for FK and SK but in a low average copy number of 3.55 and 4.55, respectively. The members of this genus have been found in rock porewater metagenome (PRJNA257561) and anaerobic digesters (PRJNA602310, PRJNA797469). The only publication so far proposed that a member of the genus BRH-c25 was capable of reductive alkane dehalogenation (Ning et al. ). Bin 05 ( Cellulomonas sp.) has the highest abundance (2733.71 average GCPM) in the MA samples compared to other treatment groups (18.86–1224.38 average GCPM). Bin 5 had a high abundance among the alkane-amended samples and the biotic controls, while it decreased in the aromatic-amended samples. This was in concordance with the read-based results. The other Cellulomonas MAG (bin 08) had a low abundance across the samples. Therefore, bin 05 might be the main actor during alkane biodegradation. Our assumption was supported by the elevated abundance of bin 05 in the MA, FA and SA samples compared to their corresponding biotic controls MK, FK and SK. Another Actinomycetota MAG belonging to the UBA 6100 genus was prevalent in this sample cluster. The MAG shares 83.66% ANI with its closest relative UBA6100 sp002423585 which has been found in the metagenome of Syncrude tailings pond (Alberta, Canada), a heavily hydrocarbon-contaminated environment (Parks et al. ). The abundance of bin 20 was higher in the ferric ion and sulphur-amended samples compared to MA, MCA, MK and MCK. Also, this bacterium had a higher abundance in FA and SA compared to FK and SK. So, bin 20 was positively influenced by the presence of sulphate and ferric ions, which suggested it could utilise them as electron acceptors and/or nutrients. Five other MAGs (bins 01, 04, 09, 12, 21) had elevated abundance in the alkane-amended/biotic control cluster. Bin 01 is a Thiobacillus sp., and its closest relative is the Thiobacillus denitrificans with 88.72% ANI. This MAG was abundant in the MA, MCA and MK samples, and its incidences were decreased in the FA, SA, FK, SK and MCK samples. Bin 04 was identified as Methanothrix sp. with 93.73% ANI to its closest relative Methanothrix soehngenii . This archaea might be the main methane producer in the samples, except for MA and MCA where its numbers decreased and bin 11 ( Methanobacterium sp.) emerged. The abundance of Methanobacterium sp. and Methanothrix sp. was in an inverse proportionality. Bin 09 is another Actinomycetia bacterium belonging to the UBA2279 family, just as bin 20. Despite the high quality of this MAG (97% completeness and 0.8% contamination), there was no matched relative to it below the family level; therefore, this MAG represents a yet unknown genus. Bin 12 belongs to the genus Bellilinea (Chloroflexi) and is related to the Bellilinea sp004347695 with 92.51% ANI. This species was found in a 1-methylnaphthalene degrading consortium (Müller et al. ). Bin 21 is G. fermentans based on the 95.72% ANI. This bin shows a similar abundance pattern to the Geothrix in the read based results. In the aromatics-amended samples, bins 07 and 15 representing the two Ruminococcus spp. had high GCPM values compared to their corresponding biotic controls. Bin 20 was also abundant in this cluster of samples, but it had a lower average GCPM compared to the corresponding biotic controls except for MCAR where the abundance equalled to MCK. F. prausnitzii (bin 24) also had elevated GCPM values compared to the biotic controls and the alkane-amended samples and showed a similar pattern to the two Ruminococcus bins. There were bins with interesting abundance patterns (Fig. ). One of them was bin 16 which was only present in the ferric or sulphate ion-amended samples mainly in FAR, SAR, FA, SK and SA. This bin belongs to the Desulfobacterota phylum, and its closest relative is an uncultivated genus CG2-30–66-27 in the MBNT15 family. Bin 14 had an inverse pattern to this, and it was the most abundant in samples MA, MCA, MAR and MCAR. This bin belongs to the SC72 family of Dojkabacteria. Functional metagenomics of the hydrocarbon-degrading microbial communities Eighteen glycyl radical enzymes were found in 11 MAGs. These enzymes may catalyse the activation of hydrocarbon molecules by fumarate addition. Fumarate addition is a frequent way to initiate anaerobic biodegradation of hydrocarbons, and it is carried out by pyruvate-formate lyase (Pfl)-like glycyl radical enzymes (Laczi et al. ). Three types of such enzymes have been described so far. Benzyl-succinate synthase (BssA), alkyl-succinate synthase (AssA) and naphthyl-2-methyl succinate synthase (NmsA) catalyse the activation of alkanes, mono- and polyaromatic compounds respectively (Achong et al. ; Selesi et al. ; Zedelius et al. ). The Cellulomonas sp. (bin 05) harboured three glycyl radical enzymes belonging to the Pfl-like superfamily. The other Cellulomonas MAG (bin 08) harboured two. The two Ruminococcus MAGs as well as the Faecalibacterium prausnitzii contained one Pfl-like enzyme. Interestingly, bin 23 was only abundant in MA samples and harboured two Pfl-like enzymes. Besides bin 5, bin 23 also may contribute to the hydrocarbon biodegradation in MA samples. In the assembly, we found a gene not binned into the MAGs encoding an enzyme which was highly identical (> 60%) to a proteobacterial NmsA. The central metabolites of aromatics biodegradation are benzoyl-CoA and its derivatives. There are two benzoyl-CoA-reducing pathways known, ATP dependent and ATP independent (Fuchs et al. ; Boll et al. ). Bin 16 which was abundant in the aromatics-amended FAR and SAR samples contained a complete set of bcr genes encoding the class I type benzoyl-CoA reductase (BCR). This four-subunit enzyme belongs to the BCR/HAD family and is responsible for the ATP-dependent reduction of benzoyl-CoA. We found a similar enzyme set encoded in the two Ruminococcus MAGs (bins 07 and 15). In this case, a large enzyme, a fusion of two YjiL ATPase domains and a DUF2229 family domain belonging to the Hgd-D superfamily, were present. Next to this large enzyme coding gene, another Hgd-D-like protein was encoded. The BcrCBAD radical enzyme complex in T. aromatica consists of two ATP-binding subunits belonging to the YjiL ATPase family and two Hgd-D-like reductase subunits (Boll et al. ; Buckel et al. ). This suggests that the large enzyme of the Ruminococcus MAGs is an acyl-CoA reductase participating in aromatics biodegradation. The BCR/HAD acyl-CoA reductase family have numerous members with yet unknown functions (Buckel et al. ); therefore, the exact function of the ruminococcal acyl-CoA reductase should be investigated. The Faecalibacterium MAG also encoded a similar enzyme consisting of one ATP-binding and one CoA-ester binding domain. Class II type BCRs are responsible for the ATP-independent reduction of benzoyl-CoA. Two MAGs belonging to the UBA1429 order of the Chloroflexota (bins 19 and 22) harboured the full set of bam BCDEFGHI-like genes in two gene clusters. Other Chloroflexota MAGs lacked genes, e.g. in bin 13 bam D, while in bin 03, bam C was missing. Also, in bin 3, the bam B-like genes were not clustered with other bam- like genes, and in general, bam -like genes were scattered in the genome.
We have followed methane evolution during the entire length of the incubation period. The measurements were performed weekly. All samples produced methane during the 8 weeks except for the samples amended with the aromatics mixture (FAR, MAR, MCAR, SAR) and the abiotic controls (KA, KAR, KK) (Fig. ). In samples, where the methanogenesis was not inhibited, the methane concentration increased gradually as time passed. The fastest methane production was reached in the FK and FA samples. These samples produced 37.7 ± 4.1 µmol and 34.2 ± 6.9 µmol methane, respectively, until the end of the incubation period. FK and FA produced significantly more methane than their counterparts without additional electron acceptors MA and MK, which yielded only 24.4 ± 8.3 and 22.0 ± 8.7 µmol methane, respectively (Fig. ). SA samples also produced high amounts of methane (31.1 ± 3.3 µmol). Although statistical significance was not found between SA and MA, the SA samples show tendentially higher methane yield (Fig. ). The addition of alkanes showed only a slight increase in methane yields in the presence of sulphate compared to SK in the last 2 weeks. Carbon dioxide-containing samples (MCK and MCA) yielded less methane than their carbon dioxide-lacking counterparts (MK and MA). We also calculated the energy content of the produced methane. It was the highest in the FK samples 33.5 ± 3.7 J in total or 7.5 ± 0.8 J relative to 1 g of the original soil sample and the lowest in MCA 5.7 ± 2.3 J in total or 1.3 ± 0.5 J relative to the original soil (Table ). Hydrogen and oxygen concentrations were also followed but remained under the detection limit during the incubation period. Carbon dioxide evolution in the samples was followed to determine the metabolic activity of the microbial community in the non-CO 2 -amended samples. CO 2 -amended samples contained an initial concentration of 20% (v/v) CO 2 , which was far over the linear range of our TCD detector and made it impossible to measure any increase or decrease of the CO 2 concentration in the headspace. In KA, KAR and KK, an increase in carbon dioxide concentration was not detectable except for the first week when there was an initial increase from 0 to 0.1% (v/v). A slightly higher initial increase was observed in the aromatics mixture-amended samples (MAR, FAR, SAR), but the final concentration did not exceed 0.3% (v/v). Statistical analysis showed a significant difference between MAR, FAR, SAR and their corresponding abiotic control KAR on the last day of incubation. The other samples had a gradual increase in carbon dioxide, and the final concentration reached an average of 2.5% (v/v) except for the SK where a slight decrease was observable compared to the other samples with an average of 1.7% (v/v). This was in concordance with the methane concentrations of SK samples, where we observed a significant drop compared to that of SA.
The original soil contained weathered diesel contamination, which had been there for two decades. The concentration of the original contamination was 4500 mg/kg soil. We supplemented this indigenous carbon source with an aliphatic mixture in the case of MA, FA, SA and their abiotic control KA or aromatics mixture in the case of MAR, FAR, SAR and their abiotic control KAR. We measured the biodegradation rate on the last day of incubation for every component of the exogenous carbon source compared to their corresponding abiotic control (Fig. ). There was a significant elevation of the biodegradation rate in all aliphatics amended samples compared to KA. Generally, the biodegradation of alkanes was the slowest in the MA samples (14.2–19.2% was degraded after 8 weeks) and the fastest in MCA samples (26.7–31.8%). Carbon dioxide addition significantly increased the biodegradation rate of alkanes and cycloalkanes compared to MA. We also observed an elevated biodegradation rate in the presence of ferric ions or sulphate. However, n-hexadecane biodegradation showed a significant elevation in the FA samples compared to MA. The biodegradation of aromatics showed a different picture. Although neither carbon dioxide nor methane was generated in the aromatics mixture-amended sample, a substantial elevation in biodegradation of aromatics was observed in the MAR, MCAR, FAR and SAR samples compared to the KAR samples. The highest rate was shown in the SAR samples (29.5–36.4%), while the lowest was in MAR (19.7–21.1%). The biodegradation rate in FAR was only slightly lower than SAR, and both were significantly higher for all the components but methylnaphthalene compared to MAR and MCAR. The MAR and MCAR samples acted similarly on all components except for naphthalene and phenanthrene, which were degraded at a significantly higher rate in the presence of carbon dioxide.
DNA was extracted from the samples, and whole metagenome sequencing was performed in triplicates. In total, 38,909,341 paired-end reads were generated with an average length of 135 bases. After quality trimming, 84.5% of the reads were preserved with an average length of 129 bases.
The microbial composition of every sample was determined by assigning the reads to taxons with Kaiju. A bar chart shows the abundances of various prokaryotic classes (Fig. a), and a cluster map was generated from the data on genus level (Fig. b). The major classes of the original soil appeared in all the samples, but their ratios changed based on the treatment groups. In the original soil, multiple classes are dominant: Clostridia (14.79%), Anaerolineae (13.95%), Deltaproteobacteria (9.41%), Alphaproteobacteria (8.20%), Betaproteobacteria (8.01%) and Actinomycetia (6.69%). In the enrichment culture, Betaproteobacteria (13.71%) and Methanomicrobia (10.48%) became the most dominant, followed by Actinomycetia (8.62%). In contrast, Actinomycetes were predominant in the alkane-amended samples (27.94–64.75%) and in the biotic controls (23.67–30.75%). The abundance of Clostridia (3.03–13.52%, 8.17–16.05%, respectively) and Methanomicrobia (1.45–5.94%, 5.77–10.20%, respectively) is also remarkable in these samples. In the aromatics-amended samples, Clostridia (24.04–37.20%) became the most dominant class, and Bacteroidia (7.07–10.54%) also emerged. Actinomycetia (5.39–11.72%) and Methanomicrobia (1.64–8.45%) remained also dominant, but their relative abundances were much lower compared to the alkane-amended samples, and the biotic controls stayed in the range of the original soil and the enrichment culture. The samples are divided into two major clusters based on their microbial composition (Fig. B). The first cluster contains the aromatics-amended samples and the original soil. However, the original soil showed similarities in the microbial composition with the aromatics-amended samples. Its predominant genus, Smithella (formerly belonging to Deltaproteobacteria, reassigned to Desulfobacterota (Waite et al. )), which might be the main hydrocarbon degrader in the contaminated soil, was completely missing from the other samples. Some other genera, like Anaerolinea , Rhodoferax , Sphingopyxis , Rhizobium and Mesorhizobium , were missing or only sporadically present across the samples. The main cause of this radical change in the microbial composition of the samples was the temperature difference between the deep soil and the laboratory experiment. This is supported by the substantial changes in the community structure of the enrichment culture compared to the original soil. The enrichment culture had two dominant genera, Methanothrix (6.02%) and Thiobacillus (6.09%). Methanomicrobium (1.53%), Methanoregula (1.25%) and Cellulomonas (1.00%) were also present. Ruminococcus (5.89–9.16%) was predominant in the aromatics-amended cluster regardless of the type of TEA present (Fig. B). Bacteroides (3.03–4.78%) along with Faecalibacterium (1.31–2.23%) were also dominant genera in the aromatics-amended samples. Alistipes (0.91–1.78%), Clostridium (1.06–1.58%) and Cellulomonas (0.72–1.65%) are prevalent in the aromatics-amended samples. Even though active methanogenesis was not detected in the aromatics-amended samples, the genus Methanothrix (0.66–6.77%) was present in this cluster. The second cluster contained the alkane-amended samples and the biotic controls. The enrichment culture belonged to this cluster. The genus Cellulomonas was predominant among the alkane-amended samples (16.36–32.45%) and in the majority of the biotic controls (6.07–11.63%), although it had lower relative abundance in the latter samples. Streptomyces and Microbacterium had a higher abundance in MA and SA samples compared to the corresponding biotic controls (MK, SK). In FA and MCA samples, there was no significant difference in the abundance of the two genera compared to the corresponding controls (FK, MCK). In addition, another member of the Cellulomonadaceae, Actinotalea , was only present in MA, FA and SA samples. Geothrix (0.62–5.15%) was present among the alkane-amended samples and their corresponding controls with the highest abundance in MK and the lowest in MCA. Methanothrix was ubiquitous in this cluster of samples, but its abundance decreased in the presence of alkanes. Carbon dioxide also had a negative effect on the prevalence of the genus Methanothrix . Furthermore, in MA and MCA, as well as in MK and FK samples, Methanobacterium also appeared. This genus along with Methanoregula and Methanothrix composed the bulk of the methanogenic archaea in the enrichment culture.
We have co-assembled the reads of the alkane- and the aromatics-amended samples. The assembly resulted in 155,159 contigs with a minimum length of 200 bp. The N50 value was 1755 with the largest contig of 869,339 bp. A total of 90% of the reads could be mapped back to the assembly. Twenty-five metagenome-assembled genomes (MAGs) were recovered; out of them, 18 were high quality, and 7 were medium quality (Table ) according to the MIMAG protocol (Bowers et al. ). Despite the high completeness and low contamination of the MAGs, only four of them could be identified on the species level and six others on the genus level. The other MAGs were identified on the family level or above. We have found 3 Euryarchaeota MAGs (bin 4, bin 11 and bin 18). All the 3 MAGs were methanogenic archaea, namely Methanothrix (bin 04), Methanobacterium (bin 11) and an unidentified genus (UBA288) of the family Methanosphaerulaceae (bin 18). This family was recently proposed, and Methanosphaerula was moved to it as a type genus from Methanoregulaceae (Rinke et al. ). The uncultivated Bacteria and Archaea (UBA) genera and other taxonomic groups were identified in a large genome recovery project (Parks et al. ). The Methanoregula sp. found in the read-based taxonomy belonged to this MAG since we used an older database with Kaiju created prior to the publication of the Rinke article. The other 22 MAGs belonged to the Eubacteria kingdom. Two Ruminococcus species Ruminococcus bromii (bin 15) and Ruminococcus bicirculans (bin 7) along with Faecalibacterium prausnitzii (bin 24) and Geothrix fermentans (bin 21) were identified on the species level. On genus level Thiobacillus (bin 1), Bellilinea (bin 12), and two Cellulomonas (bins 5 and 8), MAGs were found. Many MAGs were assigned to UBA or other uncultivated genera or higher taxonomic levels. The abundance of the MAGs in individual samples was calculated, and a cluster map was created from the data (Fig. ). The clustering of the samples showed a similar pattern as in the case of the read-based abundance (Fig. B). Two major clusters were formed. One cluster contained the alkane-amended samples and the biotic controls, while the other cluster consisted of the aromatics-amended samples. MA was an outgroup in this case mainly due to the high abundance (average GCPM = 66.48) of bin 23. This was an unidentified genus BRH-c25 belonging to the family Lutisporaceae (Clostridia) and has an average nucleotide identity (ANI) of 80.08% to its closest relative BRH-c25 sp001515955. This microbe is not present in other samples of our experiments except for FK and SK but in a low average copy number of 3.55 and 4.55, respectively. The members of this genus have been found in rock porewater metagenome (PRJNA257561) and anaerobic digesters (PRJNA602310, PRJNA797469). The only publication so far proposed that a member of the genus BRH-c25 was capable of reductive alkane dehalogenation (Ning et al. ). Bin 05 ( Cellulomonas sp.) has the highest abundance (2733.71 average GCPM) in the MA samples compared to other treatment groups (18.86–1224.38 average GCPM). Bin 5 had a high abundance among the alkane-amended samples and the biotic controls, while it decreased in the aromatic-amended samples. This was in concordance with the read-based results. The other Cellulomonas MAG (bin 08) had a low abundance across the samples. Therefore, bin 05 might be the main actor during alkane biodegradation. Our assumption was supported by the elevated abundance of bin 05 in the MA, FA and SA samples compared to their corresponding biotic controls MK, FK and SK. Another Actinomycetota MAG belonging to the UBA 6100 genus was prevalent in this sample cluster. The MAG shares 83.66% ANI with its closest relative UBA6100 sp002423585 which has been found in the metagenome of Syncrude tailings pond (Alberta, Canada), a heavily hydrocarbon-contaminated environment (Parks et al. ). The abundance of bin 20 was higher in the ferric ion and sulphur-amended samples compared to MA, MCA, MK and MCK. Also, this bacterium had a higher abundance in FA and SA compared to FK and SK. So, bin 20 was positively influenced by the presence of sulphate and ferric ions, which suggested it could utilise them as electron acceptors and/or nutrients. Five other MAGs (bins 01, 04, 09, 12, 21) had elevated abundance in the alkane-amended/biotic control cluster. Bin 01 is a Thiobacillus sp., and its closest relative is the Thiobacillus denitrificans with 88.72% ANI. This MAG was abundant in the MA, MCA and MK samples, and its incidences were decreased in the FA, SA, FK, SK and MCK samples. Bin 04 was identified as Methanothrix sp. with 93.73% ANI to its closest relative Methanothrix soehngenii . This archaea might be the main methane producer in the samples, except for MA and MCA where its numbers decreased and bin 11 ( Methanobacterium sp.) emerged. The abundance of Methanobacterium sp. and Methanothrix sp. was in an inverse proportionality. Bin 09 is another Actinomycetia bacterium belonging to the UBA2279 family, just as bin 20. Despite the high quality of this MAG (97% completeness and 0.8% contamination), there was no matched relative to it below the family level; therefore, this MAG represents a yet unknown genus. Bin 12 belongs to the genus Bellilinea (Chloroflexi) and is related to the Bellilinea sp004347695 with 92.51% ANI. This species was found in a 1-methylnaphthalene degrading consortium (Müller et al. ). Bin 21 is G. fermentans based on the 95.72% ANI. This bin shows a similar abundance pattern to the Geothrix in the read based results. In the aromatics-amended samples, bins 07 and 15 representing the two Ruminococcus spp. had high GCPM values compared to their corresponding biotic controls. Bin 20 was also abundant in this cluster of samples, but it had a lower average GCPM compared to the corresponding biotic controls except for MCAR where the abundance equalled to MCK. F. prausnitzii (bin 24) also had elevated GCPM values compared to the biotic controls and the alkane-amended samples and showed a similar pattern to the two Ruminococcus bins. There were bins with interesting abundance patterns (Fig. ). One of them was bin 16 which was only present in the ferric or sulphate ion-amended samples mainly in FAR, SAR, FA, SK and SA. This bin belongs to the Desulfobacterota phylum, and its closest relative is an uncultivated genus CG2-30–66-27 in the MBNT15 family. Bin 14 had an inverse pattern to this, and it was the most abundant in samples MA, MCA, MAR and MCAR. This bin belongs to the SC72 family of Dojkabacteria.
Eighteen glycyl radical enzymes were found in 11 MAGs. These enzymes may catalyse the activation of hydrocarbon molecules by fumarate addition. Fumarate addition is a frequent way to initiate anaerobic biodegradation of hydrocarbons, and it is carried out by pyruvate-formate lyase (Pfl)-like glycyl radical enzymes (Laczi et al. ). Three types of such enzymes have been described so far. Benzyl-succinate synthase (BssA), alkyl-succinate synthase (AssA) and naphthyl-2-methyl succinate synthase (NmsA) catalyse the activation of alkanes, mono- and polyaromatic compounds respectively (Achong et al. ; Selesi et al. ; Zedelius et al. ). The Cellulomonas sp. (bin 05) harboured three glycyl radical enzymes belonging to the Pfl-like superfamily. The other Cellulomonas MAG (bin 08) harboured two. The two Ruminococcus MAGs as well as the Faecalibacterium prausnitzii contained one Pfl-like enzyme. Interestingly, bin 23 was only abundant in MA samples and harboured two Pfl-like enzymes. Besides bin 5, bin 23 also may contribute to the hydrocarbon biodegradation in MA samples. In the assembly, we found a gene not binned into the MAGs encoding an enzyme which was highly identical (> 60%) to a proteobacterial NmsA. The central metabolites of aromatics biodegradation are benzoyl-CoA and its derivatives. There are two benzoyl-CoA-reducing pathways known, ATP dependent and ATP independent (Fuchs et al. ; Boll et al. ). Bin 16 which was abundant in the aromatics-amended FAR and SAR samples contained a complete set of bcr genes encoding the class I type benzoyl-CoA reductase (BCR). This four-subunit enzyme belongs to the BCR/HAD family and is responsible for the ATP-dependent reduction of benzoyl-CoA. We found a similar enzyme set encoded in the two Ruminococcus MAGs (bins 07 and 15). In this case, a large enzyme, a fusion of two YjiL ATPase domains and a DUF2229 family domain belonging to the Hgd-D superfamily, were present. Next to this large enzyme coding gene, another Hgd-D-like protein was encoded. The BcrCBAD radical enzyme complex in T. aromatica consists of two ATP-binding subunits belonging to the YjiL ATPase family and two Hgd-D-like reductase subunits (Boll et al. ; Buckel et al. ). This suggests that the large enzyme of the Ruminococcus MAGs is an acyl-CoA reductase participating in aromatics biodegradation. The BCR/HAD acyl-CoA reductase family have numerous members with yet unknown functions (Buckel et al. ); therefore, the exact function of the ruminococcal acyl-CoA reductase should be investigated. The Faecalibacterium MAG also encoded a similar enzyme consisting of one ATP-binding and one CoA-ester binding domain. Class II type BCRs are responsible for the ATP-independent reduction of benzoyl-CoA. Two MAGs belonging to the UBA1429 order of the Chloroflexota (bins 19 and 22) harboured the full set of bam BCDEFGHI-like genes in two gene clusters. Other Chloroflexota MAGs lacked genes, e.g. in bin 13 bam D, while in bin 03, bam C was missing. Also, in bin 3, the bam B-like genes were not clustered with other bam- like genes, and in general, bam -like genes were scattered in the genome.
In this study, we have investigated the hydrocarbon biodegradation coupled with methanogenesis in consortia derived from chronically contaminated soil. Methane is a potent energy carrier with a calorific value of approximately 55.5 MJ/kg (Lee et al. ). Synthrophic microbes often engage in a mutualist relationship with methanogenic archaea channelling the products of their metabolism into methanogenic pathways (Morris et al. ). This phenomenon can be exploited not only in biogas plants but also during the remediation of contaminated sites that lack access to oxygen. Thus, a part of the energy can be recovered from the contaminants. Therefore, we monitored the evolution of methane in our samples on a weekly basis. Except for the abiotic control and the BTEX-containing samples, methane production was observed in all our samples. BTEX compounds in higher than 0.1% (w/v) concentration inhibit the methanogenesis (Siddique et al. ) which can explain the lack of methane in the aromatic mixture amended samples. A subset of samples were amended with sulphate or ferric ions to boost microbial activity. We applied them in sufficiently low concentrations to avoid disrupting methanogenesis. The presence of either ferric ions or sulphate increased the methane evolution rate. Methanogenesis and dissimilatory iron reduction are two competing pathways; the latter is more favourable thermodynamically. However, the presence of ferric chloride in low concentrations (less than 200 mg/L) may increase the efficiency of methanogenesis (Siegert et al. ; Zhan et al. ) instead of decreasing it. In the present experiment, the boosting effect of sulphate and ferric ions on methanogenesis is indirect and may derive from facilitating the metabolism of syntrophic partners. Furthermore, the sulphate reduction may co-exist with methanogenesis, and a low sulphate concentration can increase methane yield (Siegert et al. ; Sela-Adler et al. ). Carbon dioxide can be utilised in the hydrogenotrophic pathway of methanogenic archaea to yield methane. Certain hydrocarbon degraders like Smithella sp. or Desulfotomaculum sp. can produce hydrogen during hydrocarbon biodegradation (Abbasian et al. ). This hydrogen is used to reduce carbon dioxide to methane. Since Smithella sp. was the most abundant in the original soil samples (see the microbial composition and Fig. B), we added carbon dioxide to a subset of samples to induce the hydrogenotrophic pathway. However, the methane yield decreased in these samples. High carbon dioxide concentrations may inhibit methanogenesis, but our samples did not contain the high amounts reported previously (Hansson and Molin ). Carbon dioxide also can change the preferred methanogenic pathways towards hydrogenotrophy (Mayumi et al. ). The GC method for methane measurement separates hydrogen from methane and other gas components. Hence, the presence of hydrogen can be detected in the samples while measuring methane. During the 8 weeks of incubation, hydrogen was not detected in the headspace of the samples which may be explained by the disappearance of Smithella sp. The lack of hydrogen impedes the hydrogenotrophic pathway towards which the methanogenesis is driven by the carbon dioxide. Therefore, the methane yield in the carbon dioxide-amended samples decreased. It is worth mentioning that despite the dynamically increasing methane concentrations in the headspace, the final methane amount remains low, peaking at 37.7 µmol in FK samples at the 8th week of incubation. Relative to 1 g of original soil, this represents approximately 7.5 J of energy. This limits the amount of recoverable energy via methanogenesis-coupled hydrocarbon biodegradation. Therefore, the produced methane can only serve as a cost-reducing agent for bioremediation and is unlikely to generate profit. Collecting the methane is also a challenging and an important task, given methane’s status as a potent greenhouse gas. There are technologies for landfill gas collection that can be adapted; however, their feasibility highly depends on the amounts of methane generated. Some critical parameters like the radius of influence need to be considered while designing such systems (Parameswaran and Sivakumar Babu ) as well as the mathematical models for gas generation and migration (Oonk ; Majdinasab et al. ; Zeng ; Huang et al. ). The increase in carbon dioxide concentration indicates the metabolic activity of the microbes. In the abiotic controls, there was only a slight increase in CO 2 concentrations at the beginning without any further CO 2 evolution. We assume the initial CO 2 derives from the soil itself. In the aromatics-amended samples, there was a significantly higher CO 2 production compared to the abiotic controls. However, this increase was only initial and stopped after the first week of incubation. This suggests that the surplus of carbon dioxide is derived from the metabolic activity of the microbial community. Nevertheless, this is just a faint activity compared to the other biotic samples. This indicates that the microbial activity was severely impaired in the presence of toxic compounds, or the aromatic metabolism was stuck at a certain point. The substrates were not converted to CO 2 , or the conversion slowed down somewhere along the pathway. The biodegradation rate of the aliphatics was the largest in CO 2 -amended samples. A recent study found similar results with resins and asphaltenes when crude oil was degraded by Klebsiella michiganensis (Zhang et al. ). Carboxylases can be a key factor in the increased biodegradation of alkanes catalysing the conversion of aliphatic compounds into fatty acids (So et al. ; Erb ; Abbasian et al. ). However, some evidence shows this carboxylation is not direct but preceded by a hydroxylation reaction (Callaghan ). In the lack of sufficient carbon dioxide, carboxylation processes slow down or stop completely. Hence, the biodegradation rate decreases. In addition, sulphate and ferric ion-amended samples did not fall behind the CO 2 -amended ones, and they are a good alternative for enhancing both methanogenesis and alkane biodegradation via inducing the hydrocarbonoclastic syntrophic bacteria. In addition, the biodegradation of aromatics is also increased by sulphate and ferric ions. The most abundant genus in the aromatics-amended samples was the Ruminococcus . The hydrocarbon-degrading ability of ruminococci was not known so far, although they have been found several times as a dominant genus in hydrocarbon-contaminated samples. Astuti and coworkers reported the emergence of the genus Ruminococcus in an oil reservoir after the injection of nutrients, but their prevalence was linked to molasses degradation (Astuti et al. ). Ruminococcus was the most abundant genus of biofilters used in bioreactors for oil sands process water treatment (Arslan and Gamal El-Din ). Another study by Tang and colleagues showed an unidentified Ruminococcaceae bacterium with elevated abundance during PAH biodegradation when the digesters were replenished with straw (Tang et al. ). In this case, the strengthening of ruminococci was explained by their cellulolytic activity. In addition, they tolerated the presence of PAHs in the sludge, which is in concordance with our results. Furthermore, the increase in the abundance of the genus Ruminococcus along with other acetogenic bacteria was observed in the presence of phenanthrene in the waste-activated sludge (Luo et al. ). This suggests that ruminococci not only tolerate PAHs, but the presence of PAHs also positively affects them. However, it remains unclear whether they can utilise aromatics. We have reconstructed two ruminococcal genomes belonging to R. bicirculans and R. bromii . The presence of glycyl radical enzyme and benzoyl-CoA reductase-like genes in these MAGs further supports their involvement in aromatics biodegradation. Although, it should be mentioned that primary and secondary structure similarities are not sufficient to precisely predict the function of glycyl radical enzymes. This family encompasses a wide variety of enzymes with different activities (ribonucleotide reductases, decarboxylases, formate lyases, x-succinate synthases, and so on) with relatively high similarity to each other (Backman et al. ). Therefore, the role of ruminococci in aromatics biodegradation should be further investigated. The dominance of the genera Bacteroides and Faecalibacterium in aromatics-amended samples is in concordance with the study of Liu and colleagues in which they observed the Bacteroides and Faecalibacterium genera becoming dominant in compost treated with 5-ppm benzo[a]pyrene (Liu et al. ). The involvement of Faecalibacterium in aromatics biodegradation is further supported by the reconstructed MAG of F. prausnitzii . Similar glycidyl radical enzyme and benzoyl-CoA coding reductase genes are harboured in it like in the case of the two Ruminococcus MAGs. Another study concluded the involvement of the genus Bacteroides in toluene and MTBE removal, although they did not present genomic evidence (Hsia et al. ). Unfortunately, we could not recover any MAGs belonging to this genus either. So, the involvement of Bacteroides in aromatics biodegradation remains elusive. Other genera, namely Clostridium and Alistipes , were also present in aromatics amended samples. Clostridium is frequently found as a dominant genus during anaerobic aromatic hydrocarbon degradation (Zhou et al. ; Palanisamy et al. ; Lv et al. ). The genus Alistipes was recently shown as a biomarker of hydrocarbon pollution in the intestines of Atlantic cod (Walter et al. ), although its role in hydrocarbon biodegradation is unclear. We could not recover any genomes belonging to these genera. Cellulomonas may be involved in alkane biodegradation. Cellulomonas was previously reported as a hydrocarbon biodegrader and bioemulsifier producer (Cébron et al. ; Chunyan et al. ). Cellulomonas spp. are facultative anaerobes with the ability to a fermentative lifestyle (Batool et al. ) and dissimilatory metal reduction (Sani et al. ; Khanal et al. ). To our knowledge, anaerobic biodegradation of hydrocarbons by Cellulomonas spp. has not been reported yet. In addition, Cellulomonas spp. were not just abundant in the aliphatics-amended samples but were also present in the original soil and the enrichment culture as well. Genomic data showed that the two Cellulomonas MAGs harbour multiple glycyl radical enzyme genes which can be involved in alkane activation. Therefore, one or more Cellulomonas sp. can act as the main hydrocarbon degraders in the aliphatics-amended samples. However, like in the case of Ruminococcus and Faecalibacterium , their role should be further investigated. We observed the presence of Actinotalea in alkane-amended samples. Actinotalea has been previously found in various anaerobic hydrocarbon-degrading communities. Actinotalea has been suggested to act in the carbon turnover by fermenting carbohydrates and proteins of the dead biomass in the oil-degrading enrichment and providing acetate and carbon dioxide (Li et al. ; Semenova et al. ). In another study, Actinotalea was isolated from underground coal seams and described as a polysaccharide degrader microbe (Vick et al. ). Interestingly, another Actinomicota genus Streptomyces was found in the alkane-amended samples and their corresponding biotic controls. Streptomyces is a member of normal soil flora capable of hydrocarbon biodegradation, but it utilises the aerobic pathway (Baoune et al. ). Some Streptomyces spp. are also able to grow under anaerobic conditions (Sangeetha et al. ); however, to date, there is no evidence of their ability to utilise hydrocarbons in the absence of oxygen. In contrast, the members of the genus Microbacterium can utilise hydrocarbons, mainly PAHs and other aromatics, under anaerobic conditions (Qin et al. ; Dhar et al. ; Wartell et al. ). However, no MAGs were reconstructed from our samples belonging to these genera. In addition, we have observed the elevated abundance of another Actinobacteriota MAG in the presence of alkanes. This indicated the involvement of this bacterium in hydrocarbon biodegradation either as a primary degrader or a syntrophic partner. Although, we could not find in its genome any direct evidence of its involvement in hydrocarbon biodegradation. The genus Geothrix is present in the alkane-amended samples and the biotic controls. We also reconstructed a related MAG and identified as G. fermentans . This species was originally isolated from a hydrocarbon-contaminated aquifer (Coates et al. ), and their involvement in n-alkane biodegradation was proposed (Rizoulis et al. ). However, our genomic data could not support its involvement in anaerobic hydrocarbon biodegradation. On the other hand, G. fermentans is an electroactive bacterium which can facilitate interspecies electron transfer and therefor the metabolic activities of other microbes (Aulenta et al. ; Conners et al. ). Many Chloroflexota MAGs have been recovered showing different patterns of abundance among the samples. Members of Chloroflexi were proposed to be scavengers of necromass in hydrocarbon-degrading microbiomes (Tan et al. ). A recent study showed metatranscriptomic evidence of Bellilinea sp. utilising dead biomass in hydrocarbon-degrading anaerobic cultures (Liu et al. ). The recovered MAGs showed evidence of the capability of Chloroflexota to degrade benzoyl-CoA which is a central metabolite of aromatics biodegradation. Based on our results, bacteria belonging to the phylum Chloroflexota utilise the ATP-independent benzoyl-CoA reduction pathway. Therefore, they can be involved in aromatics biodegradation. We also found some MAGs with interesting abundance patterns. One of them belongs to the MBNT15 bacterial group. The members of MBNT15 were first observed in a boreal peatland microbiome. They were capable of dissimilatory iron reduction and were proposed to be scavengers (Begmatov et al. ). The other belongs to the Dojkabacteria which was first observed in hydrocarbon and chlorinated solvent-contaminated aquifers by Dojka and colleagues (Dojka et al. ). Then, it was named after their discoverer by Wrighton and colleagues (Wrighton et al. ). Members of this taxon are capable of CO 2 fixation via the pentose bisphosphate pathway. This could be an explanation for bin 14 having a high abundance in MCA and MCAR samples, but its numbers were low in MCK. We could observe a similar pattern in MA, MAR and MK. In MAR, we did not detect any CO 2 evolution during the incubation period, and MK had similar CO 2 levels as MA. This suggested that CO 2 fixation was not the main route for this bacterium in our samples, but it directly or indirectly participated in hydrocarbon biodegradation for its high abundance is linked to the additional carbon source amended samples. Methanogenic archaea are important in syntrophic relationships channelling the electrons derived from the metabolic pathways of syntrophic partners towards methane generation (Leahy et al. ). Thus, they are the key actors in methanogenesis-coupled hydrocarbon biodegradation. In the majority of our samples, Methanothrix sp. is the most abundant. The bulk of the members of this genus are strictly acetoclastic (Khanh Nguyen et al. ). In addition, Holmes and colleagues showed metatranscriptomic evidence that Methanothrix sp. can accept electrons via the conductive pili of Geobacter (Holmes et al. ; Zhou et al. ). Methanobacterium sp. is another methanogenic archaea found in our samples. The members of this genus utilise the hydrogenotrophic pathway to produce methane (Khanh Nguyen et al. ). In contrast to Methanothrix , Methanobacterium has a lower abundance suggesting that Methanothrix and the acetotrophic pathway are responsible for the majority of the methane produced. This is also supported by the reduced methane evolution rate in the presence of carbon dioxide. In addition, DIET between Methanothrix and the exoelectrogenic hydrocarbon degrader Geothrix is also possible. In conclusion, we have found that the addition of sulphate and ferric ions in low concentrations facilitate both the methanogenesis and the hydrocarbon biodegradation. The energy recovered in the form of methane is relatively low and only suitable for eventually decreasing rehabilitation costs; however, the contaminated site must be thoroughly investigated, and the gas collection system must be carefully designed prior to bioremediation. Whole metagenomic analysis pinpointed some new candidates for hydrocarbon biodegradation ( Cellulomonas , Ruminococcus , Faecalibacterium ), while the members of Chloroflexota can act as secondary degraders. Analysing their recovered genomes, we have identified genes coding for glycyl radical enzymes, which may function as alkyl- or benzylsuccinate synthases, and types I and II benzoyl-CoA reductases; however, their roles must be clarified in future studies.
Below is the link to the electronic supplementary material. Supplementary file1 (PDF 172 KB)
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Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation | 38524e1c-88d4-467c-b17c-3292c22016f7 | 11108179 | Microbiology[mh] | Phenazines are secreted secondary metabolites produced by diverse soil bacteria that microbes use in various ways: from quorum sensing to antimicrobial warfare to energy conservation under anoxia . Each of these biological roles is connected to the ability of phenazines to accept and donate electrons (i.e., their redox activity), a process that has been studied for over 120 years. Bacterial phenazine reduction was first proposed in the nineteenth century as an indicator for the presence of enterics in water supplies . Several decades later, pyocyanin, one of the phenazines produced by Pseudomonas aeruginosa , was described as an “accessory respiratory pigment” that increased the rate of oxygen consumption by Staphylococcus , Pneumococcus , and erythrocytes by shuttling electrons from the cells to oxygen . Once it became apparent that phenazines can have cytotoxic effects, they were characterized as antimicrobial compounds that destructively abstract electrons from the transport chain . It was then discovered that reducing phenazines can greatly benefit Pseudomonas aeruginosa by 1) regulating gene expression during quorum sensing by oxidizing a transcription factor; 2) acting as alternative terminal electron acceptors to promote anoxic survival; and 3) facilitating nutrient acquisition These reports paint a complex picture of the multifarious effects phenazines can have, but in each case, the conceptual model ends with the cell reducing the phenazine, which raises the question: how are phenazines recycled? The first answer is: abiotically. Phenazines are broadly reactive molecules and can be oxidized by a variety of oxidants, including molecular oxygen and manganese or iron minerals. When oxygen serves as the electron acceptor, superoxide is produced, harming both phenazine producers and other cell types . In contrast, when iron minerals, to which phosphate is adsorbed, serve as the electron acceptor, ferrous iron and phosphate can be released, alleviating nutrient limitation However, not all oxidants of higher redox potential (e.g., nitrate and nitrite) react quickly enough with phenazines to re-oxidize them abiotically on biologically relevant timescales. Nonetheless, bacteria with versatile respiratory electron transport chains, such as the soil isolate C . portucalensis MBL, can catalyze biological phenazine oxidation during anaerobic respiration . Moreover, the thermodynamics of biological phenazine oxidation with a variety of terminal electron acceptors are favorable (see our theoretical treatment of this subject in the supplement, ). These observations provide a second possible answer to the question of how phenazines are recycled: it stands to reason that in anoxic microenvironments where phenazine-reactive abiotic oxidants are limited, phenazine-reducing bacteria may benefit from the presence of phenazine-oxidizing bacteria. Furthermore, we hypothesize that phenazine oxidation itself might provide a survival advantage to phenazine-oxidizing cells under anaerobic conditions where electron donors are limited. To test these ideas, we used a systematic genetic approach to dissect how C . portucalensis oxidizes phenazine-1-carboxylic acid (PCA), which is naturally produced, widespread, and environmentally relevant . We focused on potential catalysts within the C . portucalensis electron transport chain (Figs and , and ). In addition to leading us to a generalizable mechanistic model for how biological PCA works, the development of a genetic system in this recently isolated soil organism exemplifies how the adaptation of existing tools can be used to rapidly gain new insights into microbial processes of environmental interest.
Inactivation of individual nitrate reductases yields mild PCA oxidation defects To investigate the mechanisms and dynamics of PCA oxidation by C . portucalensis MBL, we adapted Escherichia coli genetic engineering protocols . Given that C . portucalensis harbors three functionally redundant nitrate reductase complexes (whose catalytic subunits comprise NapA, NarG, or NarZ), we hypothesized that inactivating any one of these enzymes would be insufficient to abolish PCA-oxidation activity. To start, we compared the phenotypes of strains in which we perturbed nitrate reductases by two different methods: λRed recombination or oligo-mediated recombineering to make whole operon deletions or targeted translational knockouts of catalytic subunits, respectively . shows the three nitrate reductases we mutagenized, as well as their predicted orientations in the ETC. According to this model, deleting the entire operon or knocking out just the catalytic subunit should have the same effect: all three categories of PCA oxidation reactions would be abolished. Disrupting any single nitrate reductase by either genetic engineering method reduced but did not eliminate PCA oxidation. We observed no difference between the narZ translational knockout ( narZ-tlKO) and the operon deletion ( ΔnarZYWV ) in the rate and dynamics of PCA oxidation . Comparing narZ-tlKO and ΔnarUZYWV , we found that ΔnarUZYWV had a greater delay before PCA oxidation commenced, but the maximum rate of PCA oxidation was the same as for narZ-tlKO and ΔnarZYWV . All three mutants oxidized PCA later and more slowly than the wildtype . The lag in oxidation by the ΔnarUZYWV strain compared to the Δ narZYWV and narZ-tlKO strains may reflect the fact that narU is an inner membrane nitrate-nitrite antiporter : its absence may delay nitrate entrance to the cytosol where it can encounter NarZ or NarG . As observed for the narZ mutants, there was no difference between the narG-tlKO and ΔnarGHJI PCA oxidation phenotypes, and there was only mild loss of PCA reduction compared to the wildtype . The napA-tlKO strain appeared to have a more severe PCA oxidation defect than ΔnapFDAGHBC versus the wildtype, yet loss of PCA oxidation by these strains still only minimally slowed down PCA oxidation . For the rest of this study, we used oligo recombineering rather than λRed recombination to generate strains with multiple gene disruptions. PCA oxidation dynamics with nitrate depend on which nitrate reductases are present Given the presence of three nitrate reductases in the genome, we speculated that our ability to alter PCA oxidation dynamics via their deletion, alone or in combination, would depend on their expression under different growth conditions. Accordingly, we compared nitrate-driven PCA oxidation dynamics after two different pregrowth conditions in lysogeny broth (LB): slanted shaking overnight tubes and standing parafilm-sealed overnight tubes. These two conditions permitted fully aerated (oxic) and hypoxic growth, respectively. Depending on which cultivation condition was used, distinct nitrate reductases dominantly contributed to PCA oxidation . After oxic pregrowth , the narZ knockout had the most severe phenotype of the single mutants, and eliminating both narG and narZ was sufficient to abolish PCA oxidation, implying that under this condition napA is irrelevant . The time it took for the different strains to oxidize 50% of the provided reduced PCA was correlated with the average maximum PCA oxidation rate, and the narZ single knockout was again the slowest among the other single mutants . The abiotic, narGnarZ double knockout, and napAnarZnarG triple knockout conditions never reached this 50% threshold . Relative to the wildtype, only the narZ single knockout strain had a significantly different time to 50% oxidation (Figs , and ). Cells pre-grown in stationary cultures provided a subtle but important contrast to the shaking pregrowth results . Here, each of the double knockouts had a detectable PCA oxidation rate, indicating that any of the three nitrate reductases can drive the oxidation . Moreover, rather than narZ , the greatest loss of oxidation in a single mutant background came from the narG . Once again, the time to 50% oxidation was correlated to the maximum oxidation rate , but in this condition, the narZ single mutant did not delay reaching this threshold relative to the wildtype activity . These results imply that the pregrowth condition determines the relative presence and activity of the different nitrate reductases prior to the oxidation assay, leading to different phenotypes for a given strain. Because pre-growth in stationary cultures allowed us to observe the contributions of each of the three nitrate reductases, we employed this condition for the rest of the genetic experiments, including complementation assays . When the nitrate reductases were individually overexpressed in the triple knockout background during overnight stationary pre-growth , only the narZ overexpression strain had a statistically significant rescue of PCA oxidation . The rescue effect was small, and the napA and narG overexpression strains did shift toward higher oxidation rates, so this is likely due to the overexpression system being unoptimized for C . portucalensis MBL. Summarily, PCA oxidation was abolished only when all three terminal nitrate reductases were knocked out. The PCA oxidation dynamics and phenotypes can generally be summarized by the time it takes to reach a threshold concentration of reduced PCA (e.g., ) and the maximal PCA oxidation rate (e.g., ), but this can obscure some more nuanced phenotypes, such as the biphasic nature of many of the PCA oxidation curves (e.g., , narZ-tlKO ). While the fit and oxidation metrics depend on the parametrization of the model, we were able to identify a conservative parametrization that robustly represented all tested strains . For the remaining experiments, we report only the maximum PCA oxidation rates as the phenotypes of interest. Loss of quinones in C. portucalensis MBL significantly disrupts PCA oxidation , yet mild oxidation is still achieved by nitrate reductases in their absence Nitrate-driven PCA oxidation by C . portucalensis is fully abolished only when all three terminal nitrate reductases are knocked out; any one remaining reductase enables PCA oxidation . This observation raises the question whether the nitrate reductases oxidize PCA directly ( and , Category 1 reactions) or if PCA contributes electrons to an upstream pool, such as the quinols ( and , Category 2 reactions). PCA oxidation was largely lost when the biosynthesis of all three quinones (ubiquinone, menaquinone, and demethylmenaquinone) was disrupted in the menAubiC -tlKO strain, indicating that PCA oxidation by quinones is the main component of the PCA oxidation rate . However, the PCA oxidation rate in this genetic background did not drop to abiotic levels, indicating that cellular nitrate reduction can drive PCA oxidation at a low rate without quinones as intermediaries . In other words, PCA oxidation by terminal reductases also can occur. The loss of ubiquinones alone in the ubiC-tlKO background did not affect PCA oxidation, but the loss of (demethyl)menaquinones in the menA - tlKO strain did have an effect . In the quinone-null background, PCA oxidation persists with any one of the nitrate reductases , as evidenced by the positive oxidation rate in the menAubiCnapAnarZ- , menAubiCnarGnapA- , and menAubiCnarGnarZ-tlKO strains. Interestingly, the strains with no quinones and only intact NarG or NapA had faster PCA oxidation rates than the menAubiC strain on its own . The co-occurrence of PCA transferring electrons to both terminal reductases and quinones (Category 1 and 2 reactions) implies that PCA oxidation may be plastic: it does not necessarily depend on a specifically evolved enzyme or pathway to proceed. We were unable to genetically complement the loss of quinones during nitrate-driven PCA oxidation . Curiously, overexpressing menA exacerbated the menAubiC-tlKO phenotype . Because each nitrate reductase retains some PCA oxidation activity even in the absence of quinones , it may be the case that exogenously expressing a low level of quinones is not enough to give a signal over the independent nitrate reductase activity. However, our greater degree of success with complementing menA for the other terminal electron acceptors indicates that the system works in principle (see the alternative TEA section, below). Pseudomonas aeruginosa PA14 replicates nitrate reductase knockout phenotypes from C. portucalensis MBL Given the apparent generalizability of PCA oxidation to the three nitrate reductases in C . portucalensis MBL and the observation that other bacteria perform the same metabolism (15,29), we assessed whether P . aeruginosa PA14 nitrate reductase mutants conform to the same mechanistic model. P . aeruginosa is particularly relevant as a point of comparison because it biosynthesizes PCA, has been studied extensively as a PCA reducer, and, like C . portucalensis MBL, also has both respiratory and periplasmic nitrate reductases (albeit only one homolog of the respiratory nitrate reductase complex, NarGHI) . We compared wildtype, ΔnarG , and ΔnapAB strains of P . aeruginosa PA14 after pregrowth in shaking and standing conditions, as we had done for C . portucalensis MBL. We observed that in the shaking pregrowth condition, the ΔnarG strain had no phenotype versus the wildtype, while the ΔnapAB strain had a severe, though incomplete, PCA oxidation defect ( , left). In contrast, after either of the standing pregrowth treatments, the ΔnapAB strain did not substantially differ from the wildtype, while the Δ narG strain had severe, though incomplete, loss of PCA oxidation ( , right). This pattern of distinct nitrate reductases dominating PCA oxidation activity depending on the pre-growth condition corresponds to the fact that napA and narG are regulated by distinct systems: RpoS and Anr, respectively , meaning that the P . aeruginosa napA gene is regulated like the C . portucalensis narZ gene. The P . aeruginosa phenotypes thus correspond to the difference we observed between C . portucalensis MBL narG and narZ translational knockouts depending on pre-growth conditions , which implies that the PCA oxidation mechanism is not specific to a given organism or enzyme but rather the architecture of the electron transport chain. Alternative TEAs drive PCA oxidation analogously to nitrate Having found a generalizable mechanism for nitrate-driven PCA oxidation in C . portucalensis MBL, we tested whether it extended to the other anaerobic respiratory metabolisms. We found that fumarate (terminal reductase catalytic subunit FrdA), DMSO (terminal reductase catalytic subunit DmsA), and TMAO (terminal reductase catalytic subunit TorA) can all drive PCA oxidation by C . portucalensis MBL . Analogously to nitrate-driven PCA oxidation, there was no abiotic reaction between reduced PCA and these alternative terminal electron acceptors. Knocking out the terminal reductase was sufficient to abolish fumarate-driven PCA oxidation . In fact, the frdA knockout strain quenched residual oxygen, thus lowering the apparent oxidation rate beyond that of the abiotic control (Figs and ). The menA knockout strain, lacking (demethyl-)menaquinones lost some PCA oxidation activity, but the ubiC knockout strain was indistinguishable from the wildtype . Unlike in the case of nitrate-driven PCA oxidation , the menAubiC knockout strain did not have a more severe phenotype than the menA knockout alone, which corresponds to the standard model that the fumarate reductase does not engage ubiquinone . It is also consistent that MenA complementation strain rescues some activity, and the UbiC complementation strain does not. In the complementation assays, the frdA overexpression vector was able to rescue roughly half of the wildtype activity over the frdA knockout background ( , frdA-tlKO vs. frdA pFE21-FrdA), and the menA overexpression vector rescued PCA oxidation rates entirely (Figs and . menAubiC-tlKO vs. menAubiC pFE21-MenA). The ubiC overexpression vector gave no effect (Figs and , menAubiC-tlKO vs. menAubiC pFE21-UbiC). Overall, the results here are analogous to what we observed while exploring nitrate-driven PCA oxidation: the abiotic control rules out Category 3 (electron transfer to terminal electron acceptor) PCA oxidation, the frdA knockout abolishes PCA oxidation entirely (just as the napAnarZnarG triple knockout did for nitrate), and the full loss of quinones retains partial PCA oxidation activity, which indicates that both Category 1 and 2 PCA oxidation reactions play a role. DMSO-driven PCA oxidation followed largely the same patterns as for nitrate and fumarate, with the surprising exception of ubiquinone phenotypes. There was no abiotic activity with DMSO, ruling out Category 3 PCA oxidation . The dmsA knockout lost all activity, which was partially complemented by the DmsA overexpression strain (Figs and ). The menA knockout did not have a strong phenotype, but the ubiC knockout had a substantially faster PCA oxidation than the wildtype (Figs and ). However, the menAubiC double knockout had a severe loss of PCA oxidation, though still measurable above the abiotic control (Figs and ). The MenA complementation strain slightly rescued PCA oxidation relative to the menAubiC background, but the UbiC complementation strain had no rescue. These results again support the roles of both Category 1 and 2 PCA oxidation reactions, but our model does not explain why the loss of ubiquinones alone would increase PCA oxidation, while loss of ubiquinones on top of (demethyl-)menaquinones would decrease it. Our exploration of TMAO-driven PCA oxidation is less complete because we were not able to fully abolish PCA oxidation by knocking out the catalytic subunits of terminal reductases. Knocking out torA increased the rate of PCA oxidation relative to the wildtype (Figs and ). We found that the dmsA knockout strain had a PCA oxidation deficit during TMAO-driven PCA oxidation, which is consistent with the Dms complex promiscuously reducing many N-oxides . The dmsAtorA double knockout strain was faster at oxidizing PCA than the dmsA single knockout strain, implying that C . portucalensis MBL possesses other TMAO reductases. We did not pursue a full study of the role of quinones during TMAO-driven PCA oxidation because we could not constrain the full activity. Nitrate-driven PCA oxidation provides a survival benefit for C. portucalensis MBL in a bioelectrochemical reactor With a new understanding of the mechanism of biological PCA oxidation, we can assess its impact on bacterial fitness over longer time scales. After having grown to stationary phase in LB, C . portucalensis MBL continuously oxidizes PCA, so long as nitrate is available in a bioelectrochemical chamber with a working electrode poised to a reducing voltage . Upon spiking fresh nitrate into the reactor after PCA oxidation ceases, the oxidation immediately resumes, indicating that nitrate availability is the limiting factor for these cultures . Adding more nitrate while PCA oxidation was active does not affect the current, which implies that the minimum threshold for the permissible nitrate concentration is relatively low ( , green trace). After several days of oxidizing PCA with nitrate, the apparent rate of nitrate consumption goes down, as evidenced by a persistent current in the 24-hour spiking condition after the third day ( , yellow trace). In the cultures that received additional nitrate, there was a slight but steady decrease in total current over time ( , yellow and green traces). We did not assess whether there are analogous dynamics for PCA oxidation in the bioelectrochemical chamber when provided any of the other terminal electron acceptors that we compared above. Having observed PCA oxidizing activity over multiple days, we compared three potential metrics of C . portucalensis MBL fitness (colony forming units (CFUs), cellular ATP content, and nitrite production) in two chamber bioelectrochemical reactor conditions, with and without PCA, both providing a reducing working electrode and an initial concentration of 10 mM nitrate as the terminal electron acceptor . In the chamber with PCA, the cells converted nitrate to nitrite faster than in the chamber without PCA, which coincided with the loss of current as nitrite concentrations reached their asymptote ( , third and fourth time points). Despite the PCA oxidation current ceasing between days one and three, the effect on survival was not apparent until day four, on which the cultures with PCA began to die . Throughout the time course, there were no samples that indicated a difference in cellular ATP content between the two conditions . Although this experiment cannot speak to the underlying reason for differential cell survival, it is apparent that extended PCA oxidation was beneficial under these conditions, in which PCA was the sole electron donor provided.
To investigate the mechanisms and dynamics of PCA oxidation by C . portucalensis MBL, we adapted Escherichia coli genetic engineering protocols . Given that C . portucalensis harbors three functionally redundant nitrate reductase complexes (whose catalytic subunits comprise NapA, NarG, or NarZ), we hypothesized that inactivating any one of these enzymes would be insufficient to abolish PCA-oxidation activity. To start, we compared the phenotypes of strains in which we perturbed nitrate reductases by two different methods: λRed recombination or oligo-mediated recombineering to make whole operon deletions or targeted translational knockouts of catalytic subunits, respectively . shows the three nitrate reductases we mutagenized, as well as their predicted orientations in the ETC. According to this model, deleting the entire operon or knocking out just the catalytic subunit should have the same effect: all three categories of PCA oxidation reactions would be abolished. Disrupting any single nitrate reductase by either genetic engineering method reduced but did not eliminate PCA oxidation. We observed no difference between the narZ translational knockout ( narZ-tlKO) and the operon deletion ( ΔnarZYWV ) in the rate and dynamics of PCA oxidation . Comparing narZ-tlKO and ΔnarUZYWV , we found that ΔnarUZYWV had a greater delay before PCA oxidation commenced, but the maximum rate of PCA oxidation was the same as for narZ-tlKO and ΔnarZYWV . All three mutants oxidized PCA later and more slowly than the wildtype . The lag in oxidation by the ΔnarUZYWV strain compared to the Δ narZYWV and narZ-tlKO strains may reflect the fact that narU is an inner membrane nitrate-nitrite antiporter : its absence may delay nitrate entrance to the cytosol where it can encounter NarZ or NarG . As observed for the narZ mutants, there was no difference between the narG-tlKO and ΔnarGHJI PCA oxidation phenotypes, and there was only mild loss of PCA reduction compared to the wildtype . The napA-tlKO strain appeared to have a more severe PCA oxidation defect than ΔnapFDAGHBC versus the wildtype, yet loss of PCA oxidation by these strains still only minimally slowed down PCA oxidation . For the rest of this study, we used oligo recombineering rather than λRed recombination to generate strains with multiple gene disruptions.
Given the presence of three nitrate reductases in the genome, we speculated that our ability to alter PCA oxidation dynamics via their deletion, alone or in combination, would depend on their expression under different growth conditions. Accordingly, we compared nitrate-driven PCA oxidation dynamics after two different pregrowth conditions in lysogeny broth (LB): slanted shaking overnight tubes and standing parafilm-sealed overnight tubes. These two conditions permitted fully aerated (oxic) and hypoxic growth, respectively. Depending on which cultivation condition was used, distinct nitrate reductases dominantly contributed to PCA oxidation . After oxic pregrowth , the narZ knockout had the most severe phenotype of the single mutants, and eliminating both narG and narZ was sufficient to abolish PCA oxidation, implying that under this condition napA is irrelevant . The time it took for the different strains to oxidize 50% of the provided reduced PCA was correlated with the average maximum PCA oxidation rate, and the narZ single knockout was again the slowest among the other single mutants . The abiotic, narGnarZ double knockout, and napAnarZnarG triple knockout conditions never reached this 50% threshold . Relative to the wildtype, only the narZ single knockout strain had a significantly different time to 50% oxidation (Figs , and ). Cells pre-grown in stationary cultures provided a subtle but important contrast to the shaking pregrowth results . Here, each of the double knockouts had a detectable PCA oxidation rate, indicating that any of the three nitrate reductases can drive the oxidation . Moreover, rather than narZ , the greatest loss of oxidation in a single mutant background came from the narG . Once again, the time to 50% oxidation was correlated to the maximum oxidation rate , but in this condition, the narZ single mutant did not delay reaching this threshold relative to the wildtype activity . These results imply that the pregrowth condition determines the relative presence and activity of the different nitrate reductases prior to the oxidation assay, leading to different phenotypes for a given strain. Because pre-growth in stationary cultures allowed us to observe the contributions of each of the three nitrate reductases, we employed this condition for the rest of the genetic experiments, including complementation assays . When the nitrate reductases were individually overexpressed in the triple knockout background during overnight stationary pre-growth , only the narZ overexpression strain had a statistically significant rescue of PCA oxidation . The rescue effect was small, and the napA and narG overexpression strains did shift toward higher oxidation rates, so this is likely due to the overexpression system being unoptimized for C . portucalensis MBL. Summarily, PCA oxidation was abolished only when all three terminal nitrate reductases were knocked out. The PCA oxidation dynamics and phenotypes can generally be summarized by the time it takes to reach a threshold concentration of reduced PCA (e.g., ) and the maximal PCA oxidation rate (e.g., ), but this can obscure some more nuanced phenotypes, such as the biphasic nature of many of the PCA oxidation curves (e.g., , narZ-tlKO ). While the fit and oxidation metrics depend on the parametrization of the model, we were able to identify a conservative parametrization that robustly represented all tested strains . For the remaining experiments, we report only the maximum PCA oxidation rates as the phenotypes of interest.
C. portucalensis MBL significantly disrupts PCA oxidation , yet mild oxidation is still achieved by nitrate reductases in their absence Nitrate-driven PCA oxidation by C . portucalensis is fully abolished only when all three terminal nitrate reductases are knocked out; any one remaining reductase enables PCA oxidation . This observation raises the question whether the nitrate reductases oxidize PCA directly ( and , Category 1 reactions) or if PCA contributes electrons to an upstream pool, such as the quinols ( and , Category 2 reactions). PCA oxidation was largely lost when the biosynthesis of all three quinones (ubiquinone, menaquinone, and demethylmenaquinone) was disrupted in the menAubiC -tlKO strain, indicating that PCA oxidation by quinones is the main component of the PCA oxidation rate . However, the PCA oxidation rate in this genetic background did not drop to abiotic levels, indicating that cellular nitrate reduction can drive PCA oxidation at a low rate without quinones as intermediaries . In other words, PCA oxidation by terminal reductases also can occur. The loss of ubiquinones alone in the ubiC-tlKO background did not affect PCA oxidation, but the loss of (demethyl)menaquinones in the menA - tlKO strain did have an effect . In the quinone-null background, PCA oxidation persists with any one of the nitrate reductases , as evidenced by the positive oxidation rate in the menAubiCnapAnarZ- , menAubiCnarGnapA- , and menAubiCnarGnarZ-tlKO strains. Interestingly, the strains with no quinones and only intact NarG or NapA had faster PCA oxidation rates than the menAubiC strain on its own . The co-occurrence of PCA transferring electrons to both terminal reductases and quinones (Category 1 and 2 reactions) implies that PCA oxidation may be plastic: it does not necessarily depend on a specifically evolved enzyme or pathway to proceed. We were unable to genetically complement the loss of quinones during nitrate-driven PCA oxidation . Curiously, overexpressing menA exacerbated the menAubiC-tlKO phenotype . Because each nitrate reductase retains some PCA oxidation activity even in the absence of quinones , it may be the case that exogenously expressing a low level of quinones is not enough to give a signal over the independent nitrate reductase activity. However, our greater degree of success with complementing menA for the other terminal electron acceptors indicates that the system works in principle (see the alternative TEA section, below).
PA14 replicates nitrate reductase knockout phenotypes from C. portucalensis MBL Given the apparent generalizability of PCA oxidation to the three nitrate reductases in C . portucalensis MBL and the observation that other bacteria perform the same metabolism (15,29), we assessed whether P . aeruginosa PA14 nitrate reductase mutants conform to the same mechanistic model. P . aeruginosa is particularly relevant as a point of comparison because it biosynthesizes PCA, has been studied extensively as a PCA reducer, and, like C . portucalensis MBL, also has both respiratory and periplasmic nitrate reductases (albeit only one homolog of the respiratory nitrate reductase complex, NarGHI) . We compared wildtype, ΔnarG , and ΔnapAB strains of P . aeruginosa PA14 after pregrowth in shaking and standing conditions, as we had done for C . portucalensis MBL. We observed that in the shaking pregrowth condition, the ΔnarG strain had no phenotype versus the wildtype, while the ΔnapAB strain had a severe, though incomplete, PCA oxidation defect ( , left). In contrast, after either of the standing pregrowth treatments, the ΔnapAB strain did not substantially differ from the wildtype, while the Δ narG strain had severe, though incomplete, loss of PCA oxidation ( , right). This pattern of distinct nitrate reductases dominating PCA oxidation activity depending on the pre-growth condition corresponds to the fact that napA and narG are regulated by distinct systems: RpoS and Anr, respectively , meaning that the P . aeruginosa napA gene is regulated like the C . portucalensis narZ gene. The P . aeruginosa phenotypes thus correspond to the difference we observed between C . portucalensis MBL narG and narZ translational knockouts depending on pre-growth conditions , which implies that the PCA oxidation mechanism is not specific to a given organism or enzyme but rather the architecture of the electron transport chain.
Having found a generalizable mechanism for nitrate-driven PCA oxidation in C . portucalensis MBL, we tested whether it extended to the other anaerobic respiratory metabolisms. We found that fumarate (terminal reductase catalytic subunit FrdA), DMSO (terminal reductase catalytic subunit DmsA), and TMAO (terminal reductase catalytic subunit TorA) can all drive PCA oxidation by C . portucalensis MBL . Analogously to nitrate-driven PCA oxidation, there was no abiotic reaction between reduced PCA and these alternative terminal electron acceptors. Knocking out the terminal reductase was sufficient to abolish fumarate-driven PCA oxidation . In fact, the frdA knockout strain quenched residual oxygen, thus lowering the apparent oxidation rate beyond that of the abiotic control (Figs and ). The menA knockout strain, lacking (demethyl-)menaquinones lost some PCA oxidation activity, but the ubiC knockout strain was indistinguishable from the wildtype . Unlike in the case of nitrate-driven PCA oxidation , the menAubiC knockout strain did not have a more severe phenotype than the menA knockout alone, which corresponds to the standard model that the fumarate reductase does not engage ubiquinone . It is also consistent that MenA complementation strain rescues some activity, and the UbiC complementation strain does not. In the complementation assays, the frdA overexpression vector was able to rescue roughly half of the wildtype activity over the frdA knockout background ( , frdA-tlKO vs. frdA pFE21-FrdA), and the menA overexpression vector rescued PCA oxidation rates entirely (Figs and . menAubiC-tlKO vs. menAubiC pFE21-MenA). The ubiC overexpression vector gave no effect (Figs and , menAubiC-tlKO vs. menAubiC pFE21-UbiC). Overall, the results here are analogous to what we observed while exploring nitrate-driven PCA oxidation: the abiotic control rules out Category 3 (electron transfer to terminal electron acceptor) PCA oxidation, the frdA knockout abolishes PCA oxidation entirely (just as the napAnarZnarG triple knockout did for nitrate), and the full loss of quinones retains partial PCA oxidation activity, which indicates that both Category 1 and 2 PCA oxidation reactions play a role. DMSO-driven PCA oxidation followed largely the same patterns as for nitrate and fumarate, with the surprising exception of ubiquinone phenotypes. There was no abiotic activity with DMSO, ruling out Category 3 PCA oxidation . The dmsA knockout lost all activity, which was partially complemented by the DmsA overexpression strain (Figs and ). The menA knockout did not have a strong phenotype, but the ubiC knockout had a substantially faster PCA oxidation than the wildtype (Figs and ). However, the menAubiC double knockout had a severe loss of PCA oxidation, though still measurable above the abiotic control (Figs and ). The MenA complementation strain slightly rescued PCA oxidation relative to the menAubiC background, but the UbiC complementation strain had no rescue. These results again support the roles of both Category 1 and 2 PCA oxidation reactions, but our model does not explain why the loss of ubiquinones alone would increase PCA oxidation, while loss of ubiquinones on top of (demethyl-)menaquinones would decrease it. Our exploration of TMAO-driven PCA oxidation is less complete because we were not able to fully abolish PCA oxidation by knocking out the catalytic subunits of terminal reductases. Knocking out torA increased the rate of PCA oxidation relative to the wildtype (Figs and ). We found that the dmsA knockout strain had a PCA oxidation deficit during TMAO-driven PCA oxidation, which is consistent with the Dms complex promiscuously reducing many N-oxides . The dmsAtorA double knockout strain was faster at oxidizing PCA than the dmsA single knockout strain, implying that C . portucalensis MBL possesses other TMAO reductases. We did not pursue a full study of the role of quinones during TMAO-driven PCA oxidation because we could not constrain the full activity.
C. portucalensis MBL in a bioelectrochemical reactor With a new understanding of the mechanism of biological PCA oxidation, we can assess its impact on bacterial fitness over longer time scales. After having grown to stationary phase in LB, C . portucalensis MBL continuously oxidizes PCA, so long as nitrate is available in a bioelectrochemical chamber with a working electrode poised to a reducing voltage . Upon spiking fresh nitrate into the reactor after PCA oxidation ceases, the oxidation immediately resumes, indicating that nitrate availability is the limiting factor for these cultures . Adding more nitrate while PCA oxidation was active does not affect the current, which implies that the minimum threshold for the permissible nitrate concentration is relatively low ( , green trace). After several days of oxidizing PCA with nitrate, the apparent rate of nitrate consumption goes down, as evidenced by a persistent current in the 24-hour spiking condition after the third day ( , yellow trace). In the cultures that received additional nitrate, there was a slight but steady decrease in total current over time ( , yellow and green traces). We did not assess whether there are analogous dynamics for PCA oxidation in the bioelectrochemical chamber when provided any of the other terminal electron acceptors that we compared above. Having observed PCA oxidizing activity over multiple days, we compared three potential metrics of C . portucalensis MBL fitness (colony forming units (CFUs), cellular ATP content, and nitrite production) in two chamber bioelectrochemical reactor conditions, with and without PCA, both providing a reducing working electrode and an initial concentration of 10 mM nitrate as the terminal electron acceptor . In the chamber with PCA, the cells converted nitrate to nitrite faster than in the chamber without PCA, which coincided with the loss of current as nitrite concentrations reached their asymptote ( , third and fourth time points). Despite the PCA oxidation current ceasing between days one and three, the effect on survival was not apparent until day four, on which the cultures with PCA began to die . Throughout the time course, there were no samples that indicated a difference in cellular ATP content between the two conditions . Although this experiment cannot speak to the underlying reason for differential cell survival, it is apparent that extended PCA oxidation was beneficial under these conditions, in which PCA was the sole electron donor provided.
Our observations that (i) any respirable terminal electron acceptor stimulates PCA oxidation and (ii) that the presence of quinones is required for a large fraction of the oxidation rate imply a generalizable mechanism: any cell that can perform anaerobic respiration and has electron flux through the quinol pool should oxidize PCA. This is reflected in our inability to find a bacterium that does not oxidize PCA under suitable conditions . Notably, had we not compared PCA oxidation by C . portucalensis MBL under differing pre-growth conditions, we may have had arrived at an errant model—linking PCA oxidation to particular terminal reductases. For example, had we relied on oxic-pregrowth cultures, we would have concluded that the periplasmic nitrate reductase is not involved in PCA oxidation , yet it is now clear that multiple reductases can promote PCA oxidation by driving flux through the ETC when conditions are right for them to be active. When comparing how different organisms and different terminal electron acceptors induce PCA oxidation, it is important to keep in mind the biosynthetic capacity of the organism (both the presence of the pathway and its regulation) to make the various quinones , as well as the ability of a given terminal reductase to use each of the quinones. For instance, C . portucalensis MBL synthesizes ubiquinone, menaquinone, and demethylmenaquinone, but the production of those quinones is differentially regulated depending on aerobic or anaerobic growth, and the periplasmic nitrate reductase only makes use of ubiquinone and menaquinone (not demethylmenaquinone) (Figs and ) . Meanwhile, Pseudomonas aeruginosa PA14 can produce only ubiquinone, has a very limited capacity for fermentative growth and can only use nitrate for anaerobic respiration . These features of P . aeruginosa PA14 made it unfeasible to test quinone-null or double nitrate reductase mutants , but they illuminated the generalizable principles of anaerobic PCA oxidation. Species-specific quirks accounted for, the theoretical and experimental framework we describe in this report leads to several testable predictions (see the supplement, ). First, it forecasts the genetic and chemical conditions under which other phenazines such as pyocyanin (PYO, E°’ 1/2 = -0.040 V) would be oxidized . PYO’s redox midpoint potential, being more positive than that of PCA, indicates that its oxidation could be mediated by ubiquinone or demethylmenaquinone but not menaquinone . For the same reason, fumarate should not be a suitable terminal electron acceptor to drive PYO oxidation. A knockout of the gene ubiE , which disrupts ubiquinone and menaquinone synthesis but keeps demethylmenaquinone synthesis intact , would enable a more detailed exploration of the interactions of phenazines, quinones, and the anaerobically respiring electron transport chain . While the ubiCmenA double knockout had a more severe PCA oxidation phenotype than the menA knockout alone, the ubiC single knockout phenotypes and the complementation results suggest that (demethyl-)menaquinones play a more significant role than ubiquinones (e.g., ). Recent work from our lab demonstrated that ubiquinone spontaneously and instantaneously oxidizes PCA in aqueous solution ; the results presented here imply that the plasma membrane environment changes the dynamics of how phenazines and quinones interact. This motivates further dissection of the role of specific quinones during in vivo PCA oxidation in future studies. We observed that, in the absence of quinones, PCA oxidation can still be driven by terminal reductases in the case of nitrate, fumarate, and DMSO. In other words, PCA electron transfer to terminal reductases may play a subtle, but significant role during anaerobic respiration. This may mean that either PCA is functionally substituting for a quinone, that there is an unknown other intermediate between PCA and the terminal reductase, or that there is an unappreciated abiotic oxidation of PCA in these contexts. The last case is unlikely: while nitrite that is produced during nitrate respiration can oxidize PCA at a low rate , its production requires that electrons be donated to the terminal nitrate reductase; without quinones, the next logical source of electrons would be the PCA, meaning that the PCA is ultimately oxidized by the terminal reductase and not nitrite. Furthermore, the fumarate and DMSO observations do not provide the same abiotic recourse as nitrite. There may well be an unknown factor that mediates electron transfer between PCA and the terminal nitrate and fumarate reductases. However, the existence of archaeal methanophenazine suggests that such a factor is not necessary. Methanophenazine is effectively the core phenazine molecule with a long aliphatic tail and is the membrane electron carrier in Methanosarcina mazei , which lacks quinones . In other words, M . mazei provides an example of canonical electron shuttles being completely replaced by a phenazine in the membrane. Given this example and the fact that five different terminal reductase complexes catalyze PCA oxidation in the absence of quinones, the most parsimonious explanation is that reduced PCA replaces quinols as an electron donor to these complexes at an appreciable rate. The efficacy with which different phenazines may substitute for quinones likely depends on their hydrophobicity and diffusion within the plasma membrane. The flexibility of conditions under which C . portucalensis MBL oxidizes PCA is underscored by the functional redundancy of the various quinones and of certain terminal reductases. This is most evident in the terminal nitrate reductases, where NarG, NarZ, and NapA each contribute different fractions of PCA oxidation depending on how the cultures were pre-grown. Assuming that the regulation of these genes is analogous to their orthologs’ regulation in E . coli , NarG and NapA are regulated primarily by Fnr in response to hypoxia and nitrate availability. In E . coli , NarZ is primarily regulated by RpoS in response to growth arrest. This regulatory schema corresponds to our observation that the narZ single knockout has the most severe phenotype in the slanted shaking pre-growth treatment (in which cultures reach stationary phase) and that the narG single knockout has the most severe phenotypes in the standing pre-growth treatment . This is further supported by our comparison of the nap and nar knockouts in Pseudomonas aeruginosa . Thus, the ability of a cell to oxidize PCA is defined by both its genetic content and regulatory state. The redundant pathways for PCA oxidation enable organisms like C . portucalensis MBL to perform the process under many different environmental conditions, all of which have common electron flux through the electron transport chain and the quinone pool. In the case of TMAO-driven PCA oxidation, we were unable to fully abolish the activity by knocking out terminal reductases. We found that the DMSO reductase, as represented by the catalytic subunit DmsA, contributed to PCA oxidation by TMAO . This corresponds to the promiscuity of the DMSO reductase that has been reported in other organisms . However, the torA knockout had no phenotype on its own, and the dmsAtorA double knockout oxidized PCA faster than the dmsA knockout alone . This implies that there is another TMAO reductase(s) in C . portucalensis MBL that also participates in TMAO-driven PCA oxidation. Indeed, C . portucalensis MBL has two homologs to the catalytic subunit of the E . coli TorYZ reductase (NCBI accession IDs NUH53377.1 and NUH54782.1), which may be the culprits . If knocking out these two genes in addition to dmsA and torA is not sufficient to abolish PCA oxidation activity with TMAO, the causative enzyme could potentially be identified via a mutant screen in the quadruple knockout genetic background, looking for loss of PCA oxidation activity during TMAO respiration. In addition to illuminating the molecular factors responsible for PCA oxidation, our work suggests that PCA oxidation may provide a fitness benefit for C . portucalensis MBL under some conditions . We presume that such a benefit would most likely arise when cells are starved for electron donors; in future follow-up studies, depletion of cellular internal prior to a survival assay would be expected to magnify a survival benefit. From our results, it is not clear how PCA oxidation promotes survival, as there was no effect on cellular ATP content over our experimental time period , yet given its interactions with ETC components, it seems plausible that it could beneficially modulate membrane properties. Whether this is in fact the case, and whether PCA oxidation can support the growth of autotrophic organisms that rely on exogenous electron donors to fix inorganic carbon, are interesting physiological puzzles for future research. Regardless, given that PCA reduction can provide a fitness benefit for Pseudomonas aeruginosa , it may be possible to pair phenazine oxidizing and reducing bacteria, such that the complete PCA redox cycle supports the survival of one or both . Beyond employing PCA oxidation to conserve energy, it is also possible for a bacterium to use PCA oxidation to compete against its neighbors . Given that some bacteria appear to reduce PCA to release bioavailable iron and phosphorus , if an organism like C . portucalensis MBL were to intercept and oxidize the PCA before it reached the target mineral, the PCA reducer would remain starved for iron or phosphorus . Thus, depending on the context, PCA oxidation may serve either mutualistic or competitive interactions between bacteria. Identifying these partnerships may enable new model systems for studying bacterial interactions beyond metabolite exchange or antibiotic secretion. Given the redox nature of these interactions, they are worthwhile to explore in bioelectrochemical reactors, where bacterial phenazine oxidation/reduction processes may compete with or supplement electrode activity. A clear direction for future studies is to assess the relative survival or fitness of different C . portucalensis MBL genotypes in electrode chambers. For example, a quinone null strain with only one of the three nitrate reductases subjected to a reducing electrode under anoxia would be forced to rely on electrons from PCA to drive nitrate reduction: will this culture survive better than one provided no PCA? No electrode? How might its PCA oxidation activity impact another organism grown in co-culture? The methods and results of this report open the field to ask detailed questions about the role of electron donors, separate from carbon sources, in bacterial metabolism. We end by noting that this work was made possible by recent advances in genetic engineering methods, allowing us to take an organism out of soil and develop a full genetic toolkit to test hypotheses in the span of a few years . Notably, this work underscores the importance of testing phenotypes across multiple physiological conditions, as some gene activities may be cryptic under a given gene regulatory mode. There are likely many other similar redox metabolisms in the environment that await discovery, and our report provides an example of how they may be pursued.
Reagents All chemical compounds used were purchased from Sigma-Aldrich. We want to note for future researchers that we encountered batch effects with different lots of TMAO: some lots (particularly the hydrated TMAO stocks) abiotically oxidize PCA while others do not. With TMAO, it may be important to try a couple of batches of the compound. Strains and culturing We employed wildtype and mutant C . portucalensis MBL and P . aeruginosa UCBPP-PA14 in this study . How the mutants were generated is described in the following section and in the supplement . In brief, all strains were cultured in lysogeny broth (LB) prior to PCA oxidation assays. Each pre-growth in 5 mL LB was for 17 hours at 30°C in borosilicate glass culture tubes. In , we compare pre-growth in slanted shaking tubes (250 rpm) and standing tubes sealed with parafilm to reduce oxygen permeance. In the bioelectrochemical studies (Figs and ), wildtype C . portucalensis MBL was first pre-grown overnight in shaking slanted tubes and then outgrown to a larger volume before concentrating the cells and inoculating them into the reactors (see the supplement for a detailed protocol, ). For all other experiments, C . portucalensis MBL strains were pre-grown in standing, sealed tubes without supplemented nitrate. PCA oxidation assays The basal PCA oxidation assay medium was composed of 20 mM potassium phosphate buffer (pH 7–7.1); 1 mM sodium sulfate; 10 mM ammonium chloride; and 1× freshwater salt solution (17.1 mM sodium chloride, 1.97 mM magnesium chloride, 0.68 mM calcium chloride, and 6.71 mM potassium chloride). Reduced PCA was prepared in this basal medium at a concentration of 1.2 mM by electrolysis (working electrode poised to -500 mV). Reduced PCA was the only electron donor added to the assay medium. We performed PCA oxidation assays either by tracking the fluorescence of reduced PCA over time in a plate reader (BioTek Synergy 4 or HTX) housed in a Coy-brand anoxic chamber or by tracking the current generated by a culture when provided PCA and an electrode poised to continuously reduce any PCA that the cells oxidized. Depending on the experiment, 10 mM of the terminal electron acceptor (nitrate, fumarate, DMSO, or TMAO) was added to the assay medium. The current traces were measured using a Gamry potentiostat, and these experiments were conducted in an mBraun-brand anoxic chamber, which scrubs hydrogen from the headspace. A detailed protocol for the plate reader assay is available via protocols.io ( dx.doi.org/10.17504/protocols.io.bp2l6xm6dlqe/v1 ), and an explanation of how our electrode chamber assay differed from a previously published protocol is available in the supplement . Genetic engineering of C. portucalensis MBL Detailed protocols for genetically engineering C . portucalensis MBL are available in the supplement . In brief, we employed both λRed-mediated homologous recombination to delete endogenous operons and oligo-mediated recombineering to cause translational knockouts of genes of interest . In the λRed approach, the operons were replaced by a kanamycin resistance cassette by homologous recombination using the λRed recombinase expressed on a transient plasmid . The oligonucleotides and primers we used to accomplish this are listed in Tables and . For generating the deletion strains, we used the helper plasmids pKD46 (for the λRed machinery), pKD4 (for the kanamycin resistance cassette flanked by FRT sites), and pCP20 (for the FLPase). In the oligo-mediated recombineering approach, we replaced three subsequent codons with the three different stop codons (TAA, TGA, and TAG, not necessarily in that order) in the first half of the coding sequence of the catalytic subunit of the given gene(s). We used the helper plasmid pHelper_Ec1_V1_gentR, which was derived from pORTMAGE-Ec1 by adding a SacB gene for sucrose counterselection to cure the plasmid. These helper plasmids are listed in . We transformed C . portucalensis MBL by electroporation: 200 Ω, 25 μF, 2.5 kV in 2 mm gap cuvettes. Our protocols for preparing electrocompetent C . portucalensis MBL, the Datsenko-Wanner knockouts, and the oligo-mediated recombineering are available via protocols.io: Electrocompetent cells: dx.doi.org/10.17504/protocols.io.kqdg3x7r7g25/v1 Datsenko-Wanner knockouts: dx.doi.org/10.17504/protocols.io.ewov1q5e7gr2/v1 Oligo-mediated recombineering: dx.doi.org/10.17504/protocols.io.eq2lyj1rwlx9/v1 Cloning and induction of complementation vectors We cloned the complementation vectors using the plasmid pFE21 as a backbone for inducible expression of wildtype C . portucalensis MBL genes . We used Gibson assembly to construct the new vectors from the pFE21 backbone and gene amplicons, and the primers we used are listed in . We induced the vector by incubating the cultures with 50 nM anhydrotetracycline during the pre-growth phase of the assay.
All chemical compounds used were purchased from Sigma-Aldrich. We want to note for future researchers that we encountered batch effects with different lots of TMAO: some lots (particularly the hydrated TMAO stocks) abiotically oxidize PCA while others do not. With TMAO, it may be important to try a couple of batches of the compound.
We employed wildtype and mutant C . portucalensis MBL and P . aeruginosa UCBPP-PA14 in this study . How the mutants were generated is described in the following section and in the supplement . In brief, all strains were cultured in lysogeny broth (LB) prior to PCA oxidation assays. Each pre-growth in 5 mL LB was for 17 hours at 30°C in borosilicate glass culture tubes. In , we compare pre-growth in slanted shaking tubes (250 rpm) and standing tubes sealed with parafilm to reduce oxygen permeance. In the bioelectrochemical studies (Figs and ), wildtype C . portucalensis MBL was first pre-grown overnight in shaking slanted tubes and then outgrown to a larger volume before concentrating the cells and inoculating them into the reactors (see the supplement for a detailed protocol, ). For all other experiments, C . portucalensis MBL strains were pre-grown in standing, sealed tubes without supplemented nitrate.
The basal PCA oxidation assay medium was composed of 20 mM potassium phosphate buffer (pH 7–7.1); 1 mM sodium sulfate; 10 mM ammonium chloride; and 1× freshwater salt solution (17.1 mM sodium chloride, 1.97 mM magnesium chloride, 0.68 mM calcium chloride, and 6.71 mM potassium chloride). Reduced PCA was prepared in this basal medium at a concentration of 1.2 mM by electrolysis (working electrode poised to -500 mV). Reduced PCA was the only electron donor added to the assay medium. We performed PCA oxidation assays either by tracking the fluorescence of reduced PCA over time in a plate reader (BioTek Synergy 4 or HTX) housed in a Coy-brand anoxic chamber or by tracking the current generated by a culture when provided PCA and an electrode poised to continuously reduce any PCA that the cells oxidized. Depending on the experiment, 10 mM of the terminal electron acceptor (nitrate, fumarate, DMSO, or TMAO) was added to the assay medium. The current traces were measured using a Gamry potentiostat, and these experiments were conducted in an mBraun-brand anoxic chamber, which scrubs hydrogen from the headspace. A detailed protocol for the plate reader assay is available via protocols.io ( dx.doi.org/10.17504/protocols.io.bp2l6xm6dlqe/v1 ), and an explanation of how our electrode chamber assay differed from a previously published protocol is available in the supplement .
C. portucalensis MBL Detailed protocols for genetically engineering C . portucalensis MBL are available in the supplement . In brief, we employed both λRed-mediated homologous recombination to delete endogenous operons and oligo-mediated recombineering to cause translational knockouts of genes of interest . In the λRed approach, the operons were replaced by a kanamycin resistance cassette by homologous recombination using the λRed recombinase expressed on a transient plasmid . The oligonucleotides and primers we used to accomplish this are listed in Tables and . For generating the deletion strains, we used the helper plasmids pKD46 (for the λRed machinery), pKD4 (for the kanamycin resistance cassette flanked by FRT sites), and pCP20 (for the FLPase). In the oligo-mediated recombineering approach, we replaced three subsequent codons with the three different stop codons (TAA, TGA, and TAG, not necessarily in that order) in the first half of the coding sequence of the catalytic subunit of the given gene(s). We used the helper plasmid pHelper_Ec1_V1_gentR, which was derived from pORTMAGE-Ec1 by adding a SacB gene for sucrose counterselection to cure the plasmid. These helper plasmids are listed in . We transformed C . portucalensis MBL by electroporation: 200 Ω, 25 μF, 2.5 kV in 2 mm gap cuvettes. Our protocols for preparing electrocompetent C . portucalensis MBL, the Datsenko-Wanner knockouts, and the oligo-mediated recombineering are available via protocols.io: Electrocompetent cells: dx.doi.org/10.17504/protocols.io.kqdg3x7r7g25/v1 Datsenko-Wanner knockouts: dx.doi.org/10.17504/protocols.io.ewov1q5e7gr2/v1 Oligo-mediated recombineering: dx.doi.org/10.17504/protocols.io.eq2lyj1rwlx9/v1
We cloned the complementation vectors using the plasmid pFE21 as a backbone for inducible expression of wildtype C . portucalensis MBL genes . We used Gibson assembly to construct the new vectors from the pFE21 backbone and gene amplicons, and the primers we used are listed in . We induced the vector by incubating the cultures with 50 nM anhydrotetracycline during the pre-growth phase of the assay.
S1 Fig KEGG reference pathway for quinone biosynthesis (map00130). The relevant genes are indicated by red boxes and the relevant quinones by red ellipses. Note: MenG is a homolog to UbiE from photosynthetic organisms and is not present in γ-Proteobacteria like C . portucalensis MBL; the alternative pathway for menaquinone biosynthesis via futalosine (the Mqn genes) is also absent in C . portucalensis MBL . Loss of UbiC results in the loss of ubiquinones. Loss of MenA results in the loss of menaquinones and demethylmenaquinones. Loss of UbiE results in the loss of ubiquinones and demethylmenaquinones. Note: Pseudomonas aeruginosa only has ubiquinones in its ETC under both aerobic and anaerobic growth conditions . Pathway diagram used with permission from Kanehisa Laboratories (permission received November 6, 2023). (TIF) S2 Fig All pairwise comparisons of PCA oxidation dynamics of nitrate reductase knockouts, including shaking (oxic) versus standing (hypoxic) overnight pregrowth. (A) Pairwise comparisons of the maximum PCA oxidation rate by all nitrate reductase genotypes after oxic pregrowth. This corresponds to . (B) Pairwise comparisons of the maximum PCA oxidation rate by all nitrate reductase genotypes after hypoxic pregrowth, corresponding to . (C) All pairwise comparisons for the time to oxidize half of the provided PCA, corresponding to . (D) Finally, all the pairwise comparisons for the half-max oxidation time for . (TIF) S3 Fig Quality of LOWESS fit depends on the parametrization of its scanning window. (A-B) A demonstration of the analysis pipeline. (A) We used a locally weighted scatterplot smoothing (LOWESS) algorithm to fit a curve to our empirical data, here showing an example of one of the biological replicates for wildtype C . portucalensis MBL oxidizing PCA with nitrate after hypoxic overnight pregrowth. The data are in semitransparent circles and the LOWESS fit is the red line. The fitting was parametrized to use 5% of the data in its sliding window. This fit allowed us to determine the time it took the cultures to oxidize half of the provided PCA (T half max , or any arbitrary threshold) and the derivative, or rate, of PCA oxidation. (B) This derivative allowed us to estimate the maximum rate of PCA oxidation and the time at which it occurred. The output of the LOWESS algorithm depends on one key parameter: the fraction of data that it considers for each smoothing window. (C) Examples of different fits to the same data as in , scanning the fraction parameter from 0 to 1. The value that was used for the analysis, 0.05, is in red. (D) How the estimated maximum PCA oxidation rate depends on the scanning window. The red square indicates the value at 0.05. (E) How the time to half of PCA being oxidized depends on the scanning window. The red square indicates the value at 0.05. (F) A 2D representation of both the maximum oxidation rate and the time at which it occurs. The red square indicates the value at 0.05. 0.05 was chosen as the window for all the analyses in this report as it appeared to give stable outputs while using a minimal window for fitting. (TIF) S4 Fig Complementation of nitrate reductase knockouts after overnight standing pre-growth. (A) The maximum PCA oxidation rate for the triple knockout strain and overexpressed individual nitrate reductases in that genetic background. Squares represent the means of technical triplicates and circles represent independent biological replicates. The negative value in the triple knockout background indicates that the cells were further reducing the provided stock of PCA, rather than oxidizing it. (B) Pairwise statistical tests against the null hypothesis that there is no difference between the mean maximum oxidation rates of two given genotypes. Given six comparisons, the Bonferroni-corrected p-value threshold for significance is p < 0.00833. (TIF) S5 Fig Overexpression of MenA and UbiC does not complement the PCA oxidation phenotype of the menAubiC double mutant. (A) Maximum PCA oxidation rates in complemented quinone knockout backgrounds. (B) Pairwise comparisons of the mean maximum PCA oxidation rates in (C). Given six comparisons, the Bonferroni-corrected threshold for significance is p < 0.00833. (TIF) S6 Fig All pairwise comparisons for the strains in . (A) Statistical significance matrix for the maximum PCA oxidation rate in the presence of fumarate. (B) Statistical significance matrix for the maximum PCA oxidation rate in the presence of DMSO. (C) Statistical significance matrix for the maximum PCA oxidation rate in the presence of TMAO. (TIF) S7 Fig PCA oxidation depends on nitrate availability in a bioelectrochemical reactor. Each chart presents a time course of current (μA on a linear scale) during incubation of C . portucalensis MBL in a bioelectrochemical reactor with a working electrode that continuously reduces PCA. Current indicates that the culture is oxidizing PCA. Vertical black bars represent the timing of nitrate spiking, when appropriate. Each chart is titled according to the initial concentration of nitrate in the medium and the spiking schedule. (TIF) S1 Text Supplementary results. (DOCX)
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CSCO guidelines for metastatic colorectal cancer: personalized medicine in clinical practice | 11c17cb7-3637-4ce6-af23-ed98c3bf69a4 | 10546093 | Internal Medicine[mh] | Approximately 10%–15% of CRC patients present with MSI-H/dMMR and have a better prognosis compared to microsatellite stable (MSS)/mismatch repair proficient (pMMR) CRC patients. Thus, the proportion of MSI-H/dMMR patients among all mCRC patients is lower (4%–5%). KEYNOTE-177 was a landmark study involving immune checkpoint inhibitor (ICI) use in the treatment of mCRC (NCT02563002). A total of 307 patients with MSI-H/dMMR mCRC who had not previously received treatment were randomly assigned to receive either pembrolizumab or chemotherapy (mFOLFOX6 or FOLFIRI with or without bevacizumab or cetuximab). Pembrolizumab treatment resulted in a significantly longer progression-free survival (PFS) than chemotherapy (16.5 vs . 8.2 months; P = 0.0002), with a considerably lower occurrence of treatment-related grade ≥ 3 adverse events (22% vs . 66%). Therefore, pembrolizumab was added as a class I recommendation (Level 1A evidence) in the first-line treatment for MSI-H/dMMR mCRC patients to the 2021 CSCO guidelines . CheckMate-142 was another important study involving ICIs in the treatment of MSI-H/dMMR mCRC. As early as 2018, the significance of a double-immune treatment strategy (nivolumab + ipilimumab) was affirmed in the late-line treatment of mCRC patients with MSI-H/dMMR (NCT02060188); however, due to the lack of availability of ipilimumab, CSCO guidelines have not recommended this regimen for the palliative treatment of MSI-H/dMMR mCRC patients. The 5-year follow-up results of nivolumab plus low-dose ipilimumab in the first-line therapy cohort from the phase II CheckMate-142 study was presented at the American Society of Clinical Oncology (ASCO) in 2022. Nivolumab + ipilimumab achieved a sustained overall survival (OS) and PFS benefits (48-month OS rate = 72%; 48-month PFS rate = 51%), as well as an encouraging objective response rate (ORR = 71%). Because ipilimumab had been approved in China, this double-ICI treatment regimen was added as a class III recommendation for palliative first-line treatment of metastatic dMMR/MSI-H CRC patients (Level 3 evidence) in the 2023 CSCO guidelines . PD-1 inhibitors were added as a class II recommendation (Level 2A evidence) for the late-line treatment of MSI-H/dMMR mCRC patients; however, the detailed types of PD-1 inhibitors were not defined in the 2021 CSCO guidelines. Domestic drugs [envafolimab (NCT03667170), tislelizumab (NCT03736889) and serplulimab (NCT03941574)] have shown good efficacy in the treatment of MSI-H/dMMR mCRC patients who had previously failed standard therapy with ORRs of 43.1%, 39.1%, and 46.7%, respectively. Pembrolizumab, envafolimab, serplulimab, tislelizumab, and nivolumab + ipilimumab were recommended as a priority in the late-line treatment of MSI-H/dMMR mCRC patients in the 2023 CSCO guidelines . Insights: Researchers continue to try improving the cure rate in mCRC patients on the basis of first-line immunotherapy. COMMIT was a phase III clinical study that evaluated the efficacy and safety of atezolizumab monotherapy versus mFOLFOX6 + bevacizumab + atezolizumab as first-line therapy in MSI-H/dMMR mCRC patients (NCT02997228). The COMMIT trial may determine whether the addition of chemotherapy and an anti-angiogenic agent to an ICI further improved the efficacy in MSI-H/dMMR mCRC patients. CheckMate 8HW was another phase III clinical trial that compared the efficacy of three regimens in the first-line treatment of MSI-H/dMMR mCRC [nivolumab monotherapy, nivolumab + ipilimumab, and chemotherapy (NCT04008030)]. Additionally, researchers are currently exploring whether ICI can change the adjuvant treatment of MSI-H/dMMR patients. ATOMIC is an ongoing phase III randomized trial to determine whether the addition of atezolizumab to adjuvant FOLFOX improves disease-free survival (DFS) versus FOLFOX alone in patients with stage III CRC with dMMR (NCT02912559).
BRAF V600E mutation Approximately 10% of CRC patients carry a BRAF gene mutation. Among these patients, 90% carry the BRAF V600E mutation and 21% of patients with the BRAF V600E mutation also have tumors with dMMR/MSI-H. The 5-year survival rate of patients carrying the BRAF V600E mutation is significantly less than wild-type patients. The BRAF inhibitor vemurafenib was approved by the U.S. food & drug administration (FDA) for the treatment of BRAF -mutated melanoma in 2011; however, vemurafenib monotherapy did not provide benefits in patients with BRAF -mutated mCRC . Yang et al. constructed CRC cell lines and corresponding transplanted tumor mouse models carrying the BRAF V600E mutation and reported that dual inhibition of BRAF and EGFR by vemurafenib and cetuximab combined with irinotecan (VIC) yielded a significant anti-tumor effect. Furthermore, a phase II clinical trial (SWOG1406) revealed that the VIC regimen resulted in a prolongation of PFS (4.4 vs . 2.0 months) and a higher disease control rate (67% vs . 22%) than irinotecan plus cetuximab (NCT02164916). Therefore, the VIC regimen was added as a class III recommendation (Level 2B evidence) in the late-line palliative treatment of mCRC patients with the BRAF V600E mutation in the 2019 CSCO guidelines . The mechanism underlying the lower effectiveness of BRAF inhibitor monotherapy treatment in BRAF -mutant mCRC compared to melanoma likely involves reactivation of the MAPK pathway downstream of BRAF, which may be overcome by blocking BRAF, EGFR, and MEK together. BEACON CRC was a phase III clinical trial designed to evaluate the efficacy and safety of cetuximab and encorafenib (a selective BRAF kinase inhibitor) with or without binimetinib (a MEK inhibitor) compared to chemotherapy for previously treated mCRC patients with BRAF V600E (NCT02928224). The enrolled participants were divided into three groups: triplet-combination (cetuximab + encorafenib + binimetinib); doublet-combination (cetuximab + encorafenib); and the chemotherapy control. The median OS of patients receiving 3-drug treatment was nearly 2-fold compared to the control group and the risk of disease death was reduced by 48%. In addition, data from several other phase I/II clinical trials confirmed the efficacy of dabrafenib + trametinib and dabrafenib + trametinib + panitumumab (NCT01750918). Therefore, considering drug availability, dabrafenib + trametinib + cetuximab was added as a class III recommendation (Level 2B evidence) in the late-line treatment for patients with BRAF V600E in the 2020 CSCO guidelines . The updated results of the BEACON CRC study at the 2020 ASCO annual meeting showed that the triplet- and doublet-combination groups both achieved a significantly longer OS (9.3 vs . 9.3 vs . 5.9 months), a longer PFS (4.5 vs . 4.3 vs . 1.5 months), and a higher ORR (27% vs . 20% vs . 2%) compared with the control group. Because there was no difference in OS and PFS between the triplet- and doublet-combination groups, the 2021 version of the CSCO guidelines revised the original dabrafenib + trametinib + cetuximab regimen to a BRAF inhibitor + cetuximab ± MEK inhibitor regimen as a class III recommendation (Level 2B evidence) . In addition, a new note was added in the 2021 CSCO guidelines, as follows: “BRAF inhibitor + cetuximab + MEK inhibitor can be considered for patients with extensive metastatic sites, high tumor burden or obvious tumor-related symptoms” . Insights: The doublet-combination group has also been tested in the first-line setting. BREAKWATER was a phase III clinical trial involving a BRAF inhibitor + cetuximab versus a BRAF inhibitor + cetuximab ± chemotherapy (FOLFOX6 or FOLFIRI) versus two- or three-drug chemotherapy ± bevacizumab in the first-line treatment of mCRC with BRAF V600E and MSS. The efficacy data of the safety lead-in period was reported (NCT04607421) at the 2023 ASCO-GI annual meeting. First-line treatment with encorafenib + cetuximab combined with FOLFOX6 or FOLFIRI achieved an encouraging ORR (68.4% and 75.0%, respectively), as well as a promising mPFS (11.1 months and not estimable, respectively). Due to the better efficacy data of FOLFIRI + encorafenib + cetuximab, the study supplemented cohort 3 to compare the efficacy of encorafenib + cetuximab + FOLFIRI (arm D) and FOLFIRI ± bevacizumab (arm E) based on the previous 3 arms (A: encorafenib + cetuximab, B: encorafenib + cetuximab + FOLFOX, and C: FOLFOX/FOLFIRINOX/XELOX ± bevacizumab). At the same time, investigators have begun to consider whether BRAF inhibitors further improve the DFS after adjuvant therapy in patients with BRAF -mutant CRC. The study protocol of a randomized trial (NCT05710406) was recently presented at the 2023 ASCO annual meeting. The protocol was designed to determine if encorafenib + cetuximab improves the DFS in patients with resected BRAF V600E and MSS/pMMR high-risk stage II (pT4) or stage III colon cancer after standard adjuvant therapy.
HER-2 amplification HER-2 , also known as ERBB2 , is a member of the ERBB family. An abstract presented at the 2017 ASCO annual meeting reported HER-2 alterations in mCRC patients. Comprehensive genomic sequencing in 8874 mCRC patients was analyzed . A total of 433 (4.9%) HER-2 alterations were detected, of which 265 (3.0%) were HER-2 amplifications, 164 (1.9%) were HER-2 mutations, and 4 (0.5%) were HER-2 fusions. In the Molecular Pathological Section of the 2020 CSCO guidelines “detection of HER-2 status” was added as a class III recommendation for surgery/biopsy specimens of mCRC after standard treatment failure. The HER-2 status detection method was similar to breast and gastric cancers, including immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Currently, the criteria for judging HER-2 positivity in CRC are only derived from clinical research. The definition of HER-2 - positive by IHC was as follows in the HERACLES study: ≥ 50% of the tumor tissue was 3+ positive; and for an HER-2 score of 2+, the HER-2 status should be further clarified by FISH (≥ 50% of tumor cells with a HER-2/CEP17 ratio ≥ 2.0). The HERACLES trial showed that KRAS codon 12/13 wild-type, HER-2-positive mCRC patients receiving trastuzumab + lapatinib had a 30% ORR and the PFS reached 21 weeks . Moreover, 57 HER-2 -amplified mCRC patients enrolled in the MyPathway basket study were treated with pertuzumab + trastuzumab as third-line therapy (NCT02091141). Eighteen patients (32%) achieved an objective response, including one complete response. The median PFS and OS were 2.9 and 11.5 months, respectively. Although there is still a lack of data on anti-HER-2 targeted therapy for HER-2 -amplified mCRC in the Chinese population, referring to the latest version of the NCCN guidelines, trastuzumab + pertuzumab or trastuzumab + lapatinib is recommended as third-line treatment for HER2 -amplified mCRC as a class III recommendation (Level 2B evidence) per the 2020 CSCO guidelines . Insights: To date, the efficacy and survival data of a number of phase I/II clinical trials on the combination of anti-HER-2 monoclonal antibodies and small molecule tyrosine kinase inhibitors in the treatment of HER-2 -amplified mCRC have been published. MOUTAINEER reported that among HER-2-positive patients, the response rate of tucatinib monotherapy was only approximately 3%, while the response rate of tucatinib + trastuzumab reached 38.1% (NCT03043313). The ongoing MOUNTAINEER-03 study aims to compare the efficacy of tucatinib + trastuzumab + mFOLFOX6 versus mFOLFOX6 + bevacizumab/cetuximab in the first-line treatment of HER-2-positive and RAS wild-type mCRC patients (NCT05253651). The antibody-drug conjugate, trastuzumab deruxtecan (DS-8201), is another attractive HER2-targeted drug. DESTINY-CRC01 demonstrated the efficacy and safety of DS-8201 (6.4 mg/kg) in 53 patients with HER2-positive mCRC (IHC3+ or IHC 2+/FISH+) who failed multiple lines of therapy. The ORR reached 45.3%, but none of the patients responded in the low-level HER2 expression (IHC2+/FISH− or IHC1+) mCRC group (NCT03384940). Interestingly, DESTINY-CRC02 demonstrated that the efficacy-to-risk ratio for DS-8201 at 5.4 mg/kg was better than 6.4 mg/kg, with a higher ORR (37.8% vs . 27.5%) and a lower incidence of grade ≥ 3 adverse events (49.4% vs . 59.9%) in the treatment of HER-2-positive mCRC patients (IHC3+ or IHC 2+/FISH+) (NCT04744831).
KRAS G12C mutation Although KRAS was described in CRC 30–40 years ago, KRAS was considered to be an undruggable target until the advent of sotorasib (AMG510), which is active against solid tumors with KRAS G12C . However, among CRC patients, those patients with KRAS G12C account for only 3%–4% . Detection of KRAS mutations is a routine clinical practice before treating mCRC. With respect to KRAS G12C, the 2023 CSCO guidelines specifically emphasize that in addition to exons 2, 3, and 4, attention should also be paid to whether the detection method covers some important gene mutation sites and mutation forms, such as G12C and G12D. Insights: The currently available KRAS G12C inhibitors include sotorasib (AMG510) and adagrasib (MRTX 849). When using a single drug, the effective rate of the two drugs is < 20%. However, if KRAS G12C inhibitors are combined with anti-EGFR therapy, the curative effect is doubled , . Therefore, the current focus is mainly on the treatment of small-molecule drugs targeting KRAS G12C combined with anti-EGFR therapy. CodeBreak 101 showed that the sotorasib + panitumumab + FOLFIRI regimen was associated with good clinical benefits in the late-line treatment of KRAS G12C-mutant mCRC patients, with an ORR of 55% and a disease control rate (DCR) of 93%. The efficacy was not related to the number of previous treatment lines and whether or not irinotecan treatment failed (NCT04185883). Some domestic small-molecule KRAS G12C inhibitors are also worthy of our attention. Both IBI351 [GFH925 (600 mg BID, NCT05005234 and NCT05497336)] and D-1553 (NCT04585035) monotherapy in the treatment of KRAS G12C-mutated mCRC have shown great efficacy, with an ORR reaching 42.9% and 20.8%, and a DCR reaching 88.1% and 95.8%, respectively. CodeBreak 300 is an ongoing study comparing sotorasib + panitumumab with standard third-line therapy (NCT05198934). The KRYSTAL-10 study, which compares adagrasib + cetuximab head-to-head with standard second-line therapy (NCT04793958), is similar and also ongoing.
NTRK gene fusion NTRK gene fusion is very rare in CRC, with an incidence of approximately 0.35%. In the basket study of NTRK inhibitors treating NTRK gene fusion tumors, mCRC with NTRK gene fusions showed a gratifying response rate to NTRK inhibitors; 4 patients with mCRC who failed multiple lines of therapy received larotrectinib (NCT02637687 and NCT02576431), with an ORR of 75%, and 4 patients received entrectinib with (NCT02097810, NCT02568267 and EudraCT 2012-000148-88), an ORR of 50%. Like HER-2 status detection, the 2020 CSCO guidelines also added “detection of NTRK gene fusion” as a class III recommendation for surgery/biopsy specimens of mCRC after standard treatment failure . There are several methods for detecting NTRK gene fusion, including IHC, FISH, DNA-based next-generation sequencing (NGS), and RNA-based NGS . IHC is a fast and economical method for primary screening with a sensitivity of 87.9% and a specificity of 81.1%, but NTRK gene fusion still needs to be verified by DNA-based NGS. Moreover, RNA-based NGS is considered to be the best detection method for NTRK gene fusion.
This editorial mainly focused on the research pertaining to the treatment of mCRC with relatively rare molecular features, such as dMMR/MSI-H, BRAF V600E, HER-2 amplification, KRAS G12C, and NTRK gene fusion. In addition to these alterations, some molecular characteristics are also worthy of our attention, such as the PIK3CA mutation, MET amplification, POLE/POLD1 mutation, ALK gene fusion, and CLDN18.2 expression. Compared to the international guidelines, the CSCO guidelines focus more on the results of clinical trials based on the Chinese population and recommend appropriate treatment options considering the availability of drugs in China. It is believed that with the increasingly intense international information exchange, the CSCO guidelines will continue to approach the latest anti-tumor drugs, while maintaining unique Chinese characteristics.
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The effect of the Covid-19 Pandemic on pediatric urology | a5bc812f-915f-441a-ade2-869e091291aa | 7719986 | Pediatrics[mh] | Since the COVID-19 epidemic was first declared in China in December 2019 , the virus has spread rapidly around the World owing to its characteristics: rapid spread, high contagiousness, and mortality from viral pneumonia. Critically, hospitals in many countries have had to transform. In Europe as of April 28, there have been 880,000 cases of COVID-19, and specifically in Spain 213,000 cases have been confirmed by Polymerase Chain Reaction (PCR) . We have had to convert the departments of our hospitals in an attempt to ensure that human resources and medical infrastructure were adequate to treat patients affected by COVID-19, and a key element of these efforts has been an increase in staff levels through the involvement of doctors from different specialties in the care of these patients. As our healthcare system has become increasingly saturated, most nurses have been moved to COVID-19 areas and the majority of the OR personnel have been moved to the ICU owing to the rise in the need for ventilated beds; these changes have entailed the added difficulty of obtaining adequate personal protective equipment (PPE). Other specialized hospitals have been declared COVID-19 free in order to allow treatment of all patients considered non-infected. Pregnant COVID-19 patients have been transferred to these centers, and all cases of pediatric disease are being treated exclusively in maternal and pediatric hospitals. The COVID-19 crisis has forced health care providers to establish priorities for the treatment of pathologies and to suspend elective surgeries, all with the aim of increasing the number of personnel, and this, too, has meant an involuntary change in our health care systems . The decision on which type of care should be postponed and which should continue will need to be reviewed as the pandemic situation changes. Medical and surgical societies around the World have provided rapid guidance, underpinned by the best knowledge available, on the adaptation of their guidelines recommendations to the current situation . But we must also ask ourselves what strategy to follow for those COVID-19 patients who require surgical interventions, bearing in mind the very limited scientific evidence currently available, especially in our subspecialty of pediatric urology. Here, we carry out a review of the scant scientific evidence based mainly on the few publications available to date and on the recommendations of the main scientific societies regarding which patients should undergo surgery, when surgery should be performed, how patient visits should be organized, which risks need to be addressed, which surgical techniques are safer in this pandemic, how we should protect ourselves, and what risks a child faces when undergoing an operation affected by COVID-19.
The rapid spread of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic, with infection of individuals of all ages residing in almost every country in the World . The pediatric population appears to be affected in much smaller proportions than adults, with only 2% of cases described in patients under age 20. According to data published by the Chinese Center for Disease Control and Prevention, only 1% of cases occur in those aged between 10 and 19 years and l% in children under 10 years old . An epidemiologic report described 731 confirmed COVID-19 cases in the pediatric population, with over 90% of patients characterized as asymptomatic or as having mild or moderate symptoms . In more severe cases, symptoms can include gastrointestinal symptoms and patients can progress to respiratory failure, shock, coagulation dysfunction, renal damage, septic shock, and multiorgan failure. A case of Kawasaki disease with concurrent COVID-19 infection has recently been published in the literature , and cutaneous manifestations of COVID-19 have also been reported in children . As we know, at the moment there is no specific treatment. Symptomatic treatment is administered in mild and moderate cases, with supportive measures and/or treatment of complications in severe cases. Numerous controlled clinical trials with newly developed molecules and drugs already authorized for other indications have been launched for complicated cases, primarily within hospitals . In Spain, the above-described trend continued in April: as of 3 April there were 111 confirmed cases in children under 2 years of age (0.2%), 39 in children aged between 2 and 4 years (0.1%), and 193 in children aged between 5 and 14 years (0.3%). Data were extracted from 54% of the reported cases (63,002 cases) as of that date (117,710 cases) . Based on the currently available data, children with COVID-19 have a better prognosis than adults, with few reported severe cases, and in mild cases recovery occurs within 1-2 weeks of disease onset. Most of the confirmed cases were secondary to exposure to family contacts. However, transmission from children to adults and other children can occur, as documented in a number of pediatric cases in China. On the other hand, it has been reported that the elimination of the virus in respiratory secretions and feces occurs over a longer period in children with mild symptoms than in adults, a fact that represents a great challenge for infection control . Transmission of the virus from asymptomatic children and a carrier period of up to 21 days have also been demonstrated. These data may explain a greater number of infections. Therefore, children should participate in the usual preventive actions to contain spread of the infection, and the protection of health professionals during evaluation and examination of children with respiratory infections is crucial . While most cases of COVID-19 in children are not severe, there is a population with higher risk factors for poor disease course . One of the questions frequently asked by our urological pediatric patients or parents is whether they are at higher risk of suffering from COVID-19 due to their congenital urological diseases the fact that up to now there is no scientific evidence that congenital uropathy is a risk factor for poor evolution for the development of complications in patients with SARS-COVID-2. When a patient is admitted to our pediatric urology unit and we suspect COVID-19 due to fever or suggestive symptoms, we carry out the PCR test with a pharyngeal sample to rule it out, as well as blood tests (hemogram, coagulation, venous blood gas, biochemistry with LDH, PCR, and PCT) and chest radiography (ideally portable). The use of chest ultrasound should be considered if it is available and if trained personnel are available to perform it. A single family member or other companion authorized by the parents must remain with the patient at all times, complying with the recommended isolation measures (surgical mask, gown, and frequent hand washing). It is recommended that the companion always be the same. In patients with severe disease, measurement of CPK, troponin, BNP, fibrinogen, D-dimer, and ferritin levels is recommended, as well as the acquisition of other data on hemophagocytic lympho- -histiocytosis. Need for lumbar puncture will be assessed if neurological symptoms arise. Indication for other complementary tests will be evaluated according to the circumstances in each case.
Scientific societies for urology, such as the European Association of Urology (EAU) and the American Urological Association (AUA) , have created their own information centers for COVID-19 where they can be consulted. The American College of Surgeons has established basic principles for clinical practice during this period. They recommend minimization of exposure to the hospital environment, with the following guiding principles: The goal is to provide timely surgical care to children with emergent and urgent pediatric surgical issues while optimizing patient care resources (e.g., hospital and intensive care unit beds, personal protective equipment, ventilators) and preserving the health of caregivers. There is no substitute for sound surgical judgment. Surgery should be performed only if delaying the procedure is likely to prolong hospital stay, increase the likelihood of later hospital admission, or cause harm to the patient. Children who have failed attempts at medical management of a surgical condition should be considered for surgery to decrease the future use of resources (e.g., recurrent infections in a branchial cleft cyst following a course of antibiotics). Shared multidisciplinary decisions regarding surgical scheduling should be made in the context of available institutional resources that will be variable and rapidly evolving. Telemedicine and teleconsultation services should be used for patient and physician interaction when available. For this, the creation of local review committees for decision-making related to COVID-19 surgical triage is very important .
When the term “urgent surgery” is applied in the specialty of urology, and specifically in adult patients, one most commonly thinks of surgeries for oncological conditions or obstructive urolithiasis with risk of sepsis, which are much less frequent in children. On the other hand, within pediatric urology, one might think of testicular torsions as requiring urgent surgery, or of Wilms tumors, but these are much less frequent than the indications in adults. Most of our patients have congenital pathologies, and in our day-to-day practice we perform mostly reconstructive surgeries, although we can also treat obstructive lithiasis or pathologies involving risk of loss of kidney function. Prioritizing what is urgent or “elective” in this context may be more difficult than in adults. Due to these problems, European societies such as British Association of Pediatrics Urologist (BAPU) and the EAU/ESPU have published recommendations for pediatric urological procedures . EAU/ESPU Recommendations Panels were asked to provide tables with recommendations based on the level of priority, including those that the panels felt were critical drivers of outcome and would especially be impacted by the current crisis, and always based on the highest level of evidence that was possible. LOW PRIORITY : Clinical harm (very unlikely if postponed for 6 months). INTERMEDIATE PRIORITY : Cancel procedure but reconsider if there is an increase in capacity (postponement for more than 3 months not recommended: clinical harm possible but unlikely if the procedure is postponed for more than 3 months). HIGH PRIORITY : The last procedure to be cancelled; prevent delay of >6 weeks. Clinical harm (e.g., loss of organ function) very likely if the procedure is postponed for >6 weeks). EMERGENCY : Cannot be postponed more than 24 hours. Life-threatening/organ function-threatening condition. The BAPU also recommends that routine surgery be discontinued. Emergency surgery should be limited to category 4 or 5, unless local capacity is good enough to allow category 3 to be considered .
Panels were asked to provide tables with recommendations based on the level of priority, including those that the panels felt were critical drivers of outcome and would especially be impacted by the current crisis, and always based on the highest level of evidence that was possible. LOW PRIORITY : Clinical harm (very unlikely if postponed for 6 months). INTERMEDIATE PRIORITY : Cancel procedure but reconsider if there is an increase in capacity (postponement for more than 3 months not recommended: clinical harm possible but unlikely if the procedure is postponed for more than 3 months). HIGH PRIORITY : The last procedure to be cancelled; prevent delay of >6 weeks. Clinical harm (e.g., loss of organ function) very likely if the procedure is postponed for >6 weeks). EMERGENCY : Cannot be postponed more than 24 hours. Life-threatening/organ function-threatening condition. The BAPU also recommends that routine surgery be discontinued. Emergency surgery should be limited to category 4 or 5, unless local capacity is good enough to allow category 3 to be considered .
Is PCR recommended in all children before any surgery? The EAU/ESPU guidelines recommend performing PCR for the COVID-19 test prior to any surgical intervention whenever possible . Nasopharyngeal swab with RT-PCR performed within 48 h preoperatively for the detection of COVID-19 unfortunately shows a false negative rate of 30%-40% ; however, it is always useful . If it cannot be performed or the test result is unknown, the patient is to be treated as positive and the number of personnel present in the operating room limited in order to reduce risks. Unfortunately, the literature regarding the effect of surgery on susceptibility to COVID-19 is very limited and relates only to adults. In one study, the mean age of 34 patients who underwent elective surgeries (levels 3 and 4) during the incubation period of COVID-19 was 55 years. All patients developed COVID-19 pneumonia shortly after surgery; 44.1% of the patients required admission to ICU during disease progression and 20.5% died after admission to ICU . Regional or local anesthesia should be considered whenever possible to prevent the need for mechanical ventilation, although local anesthesia is very rare in children compared with adults . Risk of contagion in operating room situations We know that certain procedures in the operating room generate aerosols (aerosol-generating procedures, AGP) and thereby increase the risk for surgical personnel if the patient is infected or in the incubation period . These include intubation, extubation, bronchoscopy, the introduction of chest tubes, electrocautery, and the use of ultrasonic devices. AGP should only be performed with full PPE, including an N95 mask or a powered air-purifying respirator (PAPR) designed for the operating room. It is advisable to use suction devices as much as possible. Laparoscopic/robotic/open surgical techniques The European Association for Endoscopic Surgery reports that there is very little scientific evidence on the relative risks of minimally invasive surgery versus conventional open surgery in the context of COVID-19. However, it recommends that the risk of viral contamination of personnel during surgery, whether open, laparoscopic, or robotic, be considered and that protective measures be used strictly to ensure the safety of operating room personnel and to maintain a functioning workforce. For minimally invasive procedures, the use of devices to filter released CO2 for aerosol particles should be considered . While insufficient data are available to recommend for or against an open approach versus a laparoscopic/robotic approach, the surgical team must choose an approach that minimizes operating time and maximizes safety for both patients and staff . In the Chinese experience, 3,387 healthcare workers were infected with COVID-19 with a mortality of 0.6%. In this setting, special caution is mandatory to reduce the infection among healthcare workers caring for COVID-19 patients. The EAU Robotic Urology Section (ERUS) has developed guidelines for robotic surgery during the COVID-19 emergency. In the case of nondeferrable surgery, the release of surgical smoke during laparoscopic procedures may carry small viral particles. As a consequence, any laparoscopic or robotic surgery should be performed only when necessary. It may be of particular importance to perform robotic surgery at the lowest permissible intraabdominal pressure . As reported by Zheng et al. , ultrasonic scalpels or the electrical equipment commonly used in minimally invasive surgery can easily produce large amounts of surgical smoke, and in particular, the low-temperature aerosol from ultrasonic scalpels or scissors cannot effectively deactivate the cellular components of the virus in patients. These authors concluded that the particle concentration of the smoke in laparoscopic surgery is significantly higher than that in traditional open surgery . Thus, it is recommended that lower electrocautery power settings be used as much as possible. It is mandatory to confirm the complete and correct deflation of the pneumoperitoneum at the end of the procedure. In fact, due to the low gas mobility in the pneumoperitoneum, the aerosol formed during the operation tends to concentrate in the abdominal cavity. Sudden release of trocar valves, non-airtight exchange of instruments, or even small abdominal extraction incisions can potentially expose the health care team to the pneumoperitoneum aerosol. Therefore, CO2 should be aspirated as much as possible before removing trocars. In order to minimize the use of the operating room and optimize the use of surgical resources, procedures must be performed by experienced surgeons . Endoscopic procedures Only one report in the literature has demonstrated the presence of SARS-COV-2 in urine specimens, in 6.9% of patients, and there is no available evidence on urine transmission . It is recommended that endoscopic procedures and urethral catheterization be performed with caution and that surgeons should be completely protected against infection if the patient has suspected or confirmed COVID-19.
The EAU/ESPU guidelines recommend performing PCR for the COVID-19 test prior to any surgical intervention whenever possible . Nasopharyngeal swab with RT-PCR performed within 48 h preoperatively for the detection of COVID-19 unfortunately shows a false negative rate of 30%-40% ; however, it is always useful . If it cannot be performed or the test result is unknown, the patient is to be treated as positive and the number of personnel present in the operating room limited in order to reduce risks. Unfortunately, the literature regarding the effect of surgery on susceptibility to COVID-19 is very limited and relates only to adults. In one study, the mean age of 34 patients who underwent elective surgeries (levels 3 and 4) during the incubation period of COVID-19 was 55 years. All patients developed COVID-19 pneumonia shortly after surgery; 44.1% of the patients required admission to ICU during disease progression and 20.5% died after admission to ICU . Regional or local anesthesia should be considered whenever possible to prevent the need for mechanical ventilation, although local anesthesia is very rare in children compared with adults .
We know that certain procedures in the operating room generate aerosols (aerosol-generating procedures, AGP) and thereby increase the risk for surgical personnel if the patient is infected or in the incubation period . These include intubation, extubation, bronchoscopy, the introduction of chest tubes, electrocautery, and the use of ultrasonic devices. AGP should only be performed with full PPE, including an N95 mask or a powered air-purifying respirator (PAPR) designed for the operating room. It is advisable to use suction devices as much as possible.
The European Association for Endoscopic Surgery reports that there is very little scientific evidence on the relative risks of minimally invasive surgery versus conventional open surgery in the context of COVID-19. However, it recommends that the risk of viral contamination of personnel during surgery, whether open, laparoscopic, or robotic, be considered and that protective measures be used strictly to ensure the safety of operating room personnel and to maintain a functioning workforce. For minimally invasive procedures, the use of devices to filter released CO2 for aerosol particles should be considered . While insufficient data are available to recommend for or against an open approach versus a laparoscopic/robotic approach, the surgical team must choose an approach that minimizes operating time and maximizes safety for both patients and staff . In the Chinese experience, 3,387 healthcare workers were infected with COVID-19 with a mortality of 0.6%. In this setting, special caution is mandatory to reduce the infection among healthcare workers caring for COVID-19 patients. The EAU Robotic Urology Section (ERUS) has developed guidelines for robotic surgery during the COVID-19 emergency. In the case of nondeferrable surgery, the release of surgical smoke during laparoscopic procedures may carry small viral particles. As a consequence, any laparoscopic or robotic surgery should be performed only when necessary. It may be of particular importance to perform robotic surgery at the lowest permissible intraabdominal pressure . As reported by Zheng et al. , ultrasonic scalpels or the electrical equipment commonly used in minimally invasive surgery can easily produce large amounts of surgical smoke, and in particular, the low-temperature aerosol from ultrasonic scalpels or scissors cannot effectively deactivate the cellular components of the virus in patients. These authors concluded that the particle concentration of the smoke in laparoscopic surgery is significantly higher than that in traditional open surgery . Thus, it is recommended that lower electrocautery power settings be used as much as possible. It is mandatory to confirm the complete and correct deflation of the pneumoperitoneum at the end of the procedure. In fact, due to the low gas mobility in the pneumoperitoneum, the aerosol formed during the operation tends to concentrate in the abdominal cavity. Sudden release of trocar valves, non-airtight exchange of instruments, or even small abdominal extraction incisions can potentially expose the health care team to the pneumoperitoneum aerosol. Therefore, CO2 should be aspirated as much as possible before removing trocars. In order to minimize the use of the operating room and optimize the use of surgical resources, procedures must be performed by experienced surgeons .
Only one report in the literature has demonstrated the presence of SARS-COV-2 in urine specimens, in 6.9% of patients, and there is no available evidence on urine transmission . It is recommended that endoscopic procedures and urethral catheterization be performed with caution and that surgeons should be completely protected against infection if the patient has suspected or confirmed COVID-19.
To date, no specific treatment is available for COVID-19 infection and it is generally accepted that social distancing is the main and perhaps the only measure to prevent or contain the spread of infection so that the number of critical cases does not dramatically exceed the resources of a health system at risk of collapse. Reduction in outpatient clinic visits during various stages of severity of the COVID-19 pandemic is recommended. Pediatric urology telemedicine can lead to fewer patient contacts, lower infection rates among staff, and continuation of pediatric urological care by quarantined urologists. However, the proportion of patients eligible for telemedicine, their wish to use telemedicine, and their demographic risk profile for acquiring a severe pandemic infection are unknown. The ESPU has provided guidance on the reduction of outpatient clinic visits during the various stages of severity of the COVID-19 pandemic: Stage 1: Start to reduce outpatient cases such as benign scrotal and penile pathology as well as incontinence. Stage 2 : See only cases that are at least semi urgent, such as those requiring initial postoperative ultrasound after upper tract reconstruction. Consider postponing prolonged (postoperative) follow-up in stable patients. Stage 3 : Continue care for urgent cases in which delay may cause irreversible progression of disease or organ damage. This includes ultrasound and voiding cystography in suspected severely obstructive uropathy in which surgery is still considered. Stage 4 : Continue all care for cases in which a delay of care is potentially organ-threatening or life-threatening. In the case of postoperative follow-up of patients with genitourinary pathologies, it is advisable to carry out the follow-up by sending photographic documentation in compliance with the General Data Protection Regulation (GDPR). If the visit has to be in person, the patient should be accompanied by a single caregiver . A distance of 2 m should be maintained between patients. Every child with suspected respiratory infection should wear a mask. Children under one year of age must be kept in their strollers and in baby seats or restraint systems and away from other patients. In pediatric waiting rooms, there will be no materials such as toys, books, or other objects that children can share and that cannot guarantee that recommended material hygiene and cleanliness standards are met, in addition to evidence of transmission before the manifestation of symptoms. If there are COVID-19 symptoms, the child or caregiver has tested positive for COVID-19, or they are in quarantine, they should be seen in a COVID-dedicated area of the hospital without interaction with other patients .
All interhospital staff movements with residents training in other hospitals and all undergraduate clinical rounds have been canceled. All training programs for residents as well as fellowship programs in pediatric urology in Spain have been affected. Many residents have had to become so-called front-line doctors caring for patients affected by COVID-19. It is recommended that all procedures are performed by experienced urologists confident in the procedure. Procedures should be performed with the minimum number of staff members, who should also be fully trained and experienced. Furthermore, no external observers (i.e., fellows or students) should be present during procedures until the pandemic has been controlled, which we hope will be in the approaching period . Currently, training meetings held between companies or for the same department are scheduled via telematics.
There is no existing knowledge on the adverse impacts of loss of surgical capacity on patients' surgical condition and associated health or on prognosis. A new model will have to be established after the pandemic based on the length of the surgical waiting list.
Countries in Latin America are following the programs applied in Europe because the European countries have more experience with COVID-19. In preparation for potential surges in cases of COVID-19, most governments have chosen to create new healthcare facilities and have emphasized the need for careful planning around elective procedures, taking into account multiple considerations such as adequacy of supplies of PPE and other essential equipment, testing capacity, sanitation protocols, and workforce availability. Hospitals need to maintain adequate staffing levels to cover a potential surge in COVID-19 cases and should have enough beds, PPE, ventilators, and trained staff to allow these surgeries to take place without resorting to a crisis standard of care. Elective surgeries were initially suspended to preserve hospital bed capacity and PPE. When the data indicate a better position regarding hospital capacity, and provided individual institutions can accommodate their internal demand for PPE, it may be time to start performing some of these procedures again. As in many countries, training programs have continued through societies, webinars, and virtual masterclasses. Across Latin America, and indeed in all developing countries facing the COVID-19 pandemic, there are many unanswered key questions relating to impacts on the economy, levels of poverty, social and psychological problems, crime post quarantine, etc. No nation is prepared to face this crisis, but in developing countries the problem is even worse because they are all constantly in a state of crisis. In this context the post-COVID-19 era represents a huge challenge.
The COVID-19 virus has been impacting dramatically on the normal life of the departments. Because of the necessity to adopt strategies to contain the diffusion, all surgical departments have to be restricted. Perform surgery only in cases of organ-threatening or life-threatening disease. Suggested reduction in outpatient clinic visits during various stages of severity of the COVID-19 pandemic.
Consider treating only high-priority and emergency cases surgically during the COVID pandemic. Consider treating intermediate-priority patients if capacity is available but not during the COVID surge. Non-surgical management should be considered, to begin with, including medical treatment (e.g. antibiotics for vesico-ureteral reflux associated urinary tract infections), endovascular embolization (e.g. for bleeding renal traumas), or urinary tract diversion. Perform PCR for the COVID-19 test prior to any surgical intervention whenever possible. Follow the local recommendations for personal protective equipment (PPE). Avoid or reduce the use of monopolar electrosurgery, ultrasonic dissectors, and advanced bipolar devices, as these can lead to particle aerosolization. All minimally invasive procedures should preferably be performed by experienced surgeons. It is recommended that electrocautery power setting be lowered as much as possible in order to reduce the surgical smoke production, especially in laparoscopic surgery. During access, electrocautery should be provided with automatic suction system. Reduction in outpatient clinic visits during various stages of severity of the COVID-19 pandemic is recommended Multidisciplinary team meetings are recommended to offer the optimum therapeutics. Regional or local anesthesia should be considered whenever possible to prevent the need for mechanical ventilation.
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Energy management education for persons living with long COVID-related fatigue (EMERGE): protocol of a two-parallel arms target trial emulation study in a multicentre outpatient intervention setting with an online control group register | 896a1272-a8e2-4804-b45a-cfb36ee611e0 | 11808863 | Patient Education as Topic[mh] | This study will use a target trial emulation approach, which presents an alternative to a randomised controlled trial design when it is unethical or impractical. This study will deliver data on the effectiveness of an intervention recommended by guidelines and its cost-effectiveness. The problem of confounders in observational studies will be counteracted by using a propensity score matching technique, but not completely solved. Post-COVID-19 condition, also known as long COVID, occurs in individuals with a history of probable or confirmed SARS-CoV-2 infection, usually 3 months from the onset of COVID-19, with symptoms that last for at least 2 months, and cannot be explained by an alternative diagnosis. Prevalence estimates a range from 2.3% to 53% in adults. Long COVID may be more common in persons with a more severe acute illness or preexisting conditions. According to systematic reviews, fatigue, post-exertional malaise and dyspnoea are the most reported symptoms. Fatigue is experienced mainly as severe, impacting cognition, extended activities of daily living and social activities, which causes significant distress and disability to individuals, preventing their return to previous life routines, work and social life. Research on primarily mild-to-moderate and non-hospitalised SARS-CoV-2 cases reported that approximately 12%–23% of affected persons remained absent from work (or had long absence periods) at 3–7 months after acute disease. Currently, national and international guidelines, still mainly based on expert opinion, indicate a multidisciplinary approach for managing long COVID-related fatigue. This approach includes in addition to occupational therapy (OT), physiotherapy or psychological interventions, self-management education, peer support and symptom management strategies. The body of evidence supporting rehabilitation programmes for people with long COVID is gradually increasing, though it remains limited. According to a recent systematic review, which included studies up to May 2022, the evidence supporting physical training and breathing exercises to reduce fatigue and improve physical capacity and quality of life is limited. Another systematic review of registered interventional clinical trials for post-COVID syndrome, with a search date of September 2022, found that 168 out of 388 trials focused on physical rehabilitation, while only four trials focused on educational interventions. The most recent review and meta-analysis, which searched for randomised controlled trials (RCTs) until December 2023, included 24 trials with totally 3695 individuals. It identified “moderate certainty evidence that an online CBT programme probably improves fatigue and concentration, and a combined programme of physical and mental health rehabilitation probably increases the proportion of patients who experience recovery or important improvements” (p.9). Moderate certainty evidence was found for intermittent aerobic compared with continuous exercise, probably improving physical function. Despite OT, and particularly the use of energy management strategies, being one of the recommended interventions for persons with long COVID-related fatigue, data are missing on its effectiveness in managing the impact of fatigue on daily life and their costs. Since 2021, many healthcare providers in Switzerland have started providing interprofessional in- and outpatient rehabilitation programmes for persons with long COVID. In a citizen science project in Switzerland in 2021, persons with long COVID discussed and identified the needs of affected persons. From a patient-centred perspective, rehabilitation and chronic care management, availability of interfaces for treatment continuity, availability of specific healthcare structures and knowledge among professionals were being identified as priorities for a research agenda and allocation of funding resources. Switzerland has not implemented a long COVID register yet. Self-management education is the primary OT approach for persons with chronic fatigue syndrome and populations with chronic disease-related fatigue. Self-management refers to an individual’s ability to manage the symptoms, treatment, physical and psychosocial consequences and lifestyle changes inherent in living with a chronic condition. In 2017, group-based energy management education (EME) was developed for persons with multiple sclerosis-related fatigue. A detailed treatment manual and workbook for participants were written based on evidence-based intervention protocols for energy conservation and cognitive–behavioural therapy approaches, enriched with new elements from behavioural theories, principles for adult education and the inclusion of opinions from users and OT experts. Since the beginning of the COVID-19 pandemic, occupational therapists (OTs) played a role in treating people with acute and post-acute COVID-19 and post-COVID-19 conditions in different settings (acute care, inpatient rehabilitation and outpatient reintegration). High-impact symptoms such as fatigue or post-exertional malaise affect the performance in daily activities, social participation and well-being. Some EME-trained OTs have started to include persons with post-COVID-19 condition-related fatigue in their EME groups or used the EME materials as part of individual therapy. In May 2021, nine OTs reported that the EME protocol was feasible and appropriate, although some optimisations for persons with long COVID-related fatigue were necessary. Since March 2021, the Clinic for Neurorehabilitation Basel (REHAB) has provided interprofessional consultation and day-hospital treatment programmes for persons with long COVID. EME is the standard OT intervention for persons with fatigue. In this clinical context, we performed a feasibility study in 2022 using a mixed-method design to improve the treatment materials and to prepare this study with promising results. The purpose of this project is threefold. The primary aim is to evaluate if adding EME to the standard care improves outcomes for persons with long COVID-related fatigue. The secondary aim is to explore the energy management behavioural strategies applied in daily routines after participating in EME and investigate the influencing factors of implementing behavioural changes. The third aim is to perform a cost-effectiveness analysis of EME. Project status The recruitment at the three intervention group sites and for the control group (CG) register started in July 2024. The time between registration in the study and matching for the CG depends on the recruitment rate of the EME participants. The first matching of 15 participants of the experimental group with 15 participants out of the CG register was performed in December 2024. Study participation lasts for each study participant approximately 24 weeks; therefore, data collection was not finished with any participant before submission of this article. The recruitment will be finished during late summer/early autumn 2025, and data collection will be finished at approximately the end of 2025. Data analysis will begin at the end of 2025 after the last participant has completed T3. The total duration of the research project is 42 months. Study design The gold standard for investigating the efficacy of a treatment is an RCT, which guarantees high internal validity by minimising clinician and patient biases. In contrast, pragmatic trials prioritise external validity by studying interventions in the context of broad-based and normal clinical practice conditions. In rehabilitation research, pragmatic or real-world trials are increasingly being conducted. They aim to identify the most effective intervention for a patient-centred primary endpoint. Considering the above elements of the current context, we will emulate a prospective interventional study that draws causal inferences on the effectiveness of adding EME to standard care using a target trial emulation approach. Ethical approval has been obtained from the competent Swiss ethics commission (2024–00938; Rif. CE 4596). Interventions Standard care: any interventions tailored to the specific needs and goals that aim to support individuals with long COVID on their way back to normality, such as breathing exercises, individual OT, relaxation techniques, mindfulness, cognitive–behavioural therapy, physical exercise training and consultations by different health professionals (eg, physicians, cardiologists, pulmonologists, psychologists, dietitians and social workers). All participants will follow the standard care personalised to their needs and preferences. The CG will receive the standard care only. Energy management education (EME), revised outpatient version 2023: this updated version integrates the findings of the feasibility study performed in 2022. EME aims to increase self-management skills for managing the available amount of energy and achieving a satisfactory and meaningful daily routine despite fatigue. During EME, participants learn about the factors influencing their energy level and experiment with skills to manage their energy using behavioural strategies (eg, pace, plan and prioritise activities and optimise communication, environment and ergonomic behaviour). Subsequently, they identify and implement tailored behavioural modifications. To support the reflection on and use of energy-managing strategies and new behaviours, the leading OTs use behavioural change techniques. The outpatient EME protocol consists of seven group sessions (90 min) once a week plus an eighth session approximately 8 weeks after the seventh one. The first, seventh and eighth sessions can be done in individual sessions (45 min) alternatively. Sessions are led by EME-trained OTs. The experimental group will receive personalised standard care plus EME. The overview of the topics and further details are presented in . At the University Hospital Bern (INSEL) and Clinic for Neurorehabilitation Basel (REHAB), the intervention starts with an individual lesson (45 min), whereas at University Hospital Zürich (USZ), the first lesson is already delivered in groups (90 min). With a frequency of one lesson per week, lessons 2–7 are provided (duration 90 min, appr. five participants). EME finishes with a group lesson, approximately 8 weeks after lesson 7. REHAB provides lessons 7 and 8 as individual lessons of 45 min. The EME-leading OTs use the treatment manual, which describes in detail the tasks of each lesson. All EME participants receive the workbook, which contains relevant information on the topics covered in each session, including worksheets and space for personal notes plus self-training tasks. Setting and recruiting Experimental group (EME, outpatient version 2023, plus standard care): persons with long COVID-related fatigue following EME (revised edition 2023) in an outpatient setting at USZ, INSEL or REHAB, as part of their clinical routine and standard care, will be informed about this ongoing research and will be kindly asked if they are willing to participate in this project. If individuals agree, have signed the consent form and meet the inclusion/exclusion criteria checked by the study therapist on site, they will be included in the experimental group (EG). In these three public institutions, a group of trained OTs leads continuously EME groups and has received up-to-date training for the EME version 2023 in Spring 2024. CG (standard care ): a register of potential CG participants will be created through a contact with the Altea Network for Long COVID Association (ALTEA) and the patient organisation Long Covid Switzerland (LCS), both of which are partners in the study project and have been involved since the design phase. Using the contacts of ALTEA, LCS and specialised healthcare providers, information about the study will be disseminated, allowing interested persons to register themselves through a public link or QR code and to participate in a first online survey. Registration for the study has been open since 15 July 2024 and will remain open until summer 2025. Thereafter, based on the sociodemographic characteristics and clinical history of COVID-19, the CG participants will be selected from the register by their propensity score (PS) and matched with EG participants. Subsequently, the selected persons from the CG will be screened for eligibility, asked for their informed consent and included in the study. Eligibility criteria The target study population consists of individuals aged 18 years or older, living in Switzerland, with a post-COVID-19 condition and a confirmed experience of fatigue. Eligible participants must not have major depression and/or significant cognitive impairments, must have sufficient ability to understand and speak one of the national languages and must provide signed informed consent. Persons are not eligible if they have previously participated in an EME group, have insufficient comprehension of one of the national languages or have a history of severe neurological diseases (eg, stroke, multiple sclerosis, Parkinson’s disease) or mental health disorders (eg, schizophrenia, major depression, or dementia). Objectives The primary aim is to compare the effectiveness of the standard care for persons with long COVID-related fatigue plus participating in an EME group (revised edition 2023), with the standard care without EME at post-intervention (T2; week 14). The secondary aim is to assess the energy management behavioural strategies applied in daily routines in persons living with long COVID, their feasibility and the factors influencing behavioural changes (T3, week 24). The third aim is to evaluate the cost-effectiveness profile of the intervention by performing an economic evaluation alongside the trial. Outcomes Primary outcomes The impact of fatigue on physical, mental and social activities of daily routines will be measured with the Modified Fatigue Impact Scale. It contains 21 items, on a scale from 0 (never) to 4 (almost always), thus total ranging from 0 to 84. Self-efficacy in performing energy management strategies is the most proximal outcome of EME. Increased perceived self-efficacy in performing energy management behaviours and strategies correlates to the impact of fatigue in daily life and predicts behavioural changes after patient education. This will be measured using the ‘Self-Efficacy for Performing Energy Conservation Strategies Assessment’. This self-report questionnaire reports the mean scores of perceived confidence in performing 14 different energy management strategies (1=low confidence, 10=high confidence). It is a valid and reliable instrument with a minimal clinically important difference of 0.92 in people with chronic conditions and fatigue. Secondary outcomes The Occupational Self-Assessment (OSA 2.2 ; focuses on perceived competency to perform 21 daily tasks, which scores from 1 (I have many problems) to 4 (I do extremely well). The raw data are converted to interval levels (0–100). OSA has been validated in German and has shown the ability to detect changes in self-reported competence for daily activities over time. Health-related quality of life will be measured by using the Short-Form 12 Item Health-Related Quality of Life Survey. This survey is a practical, reliable, valid and often used measure of physical and mental health. It assesses the same eight health domains as the SF-36, but with only one or two questions per domain: Physical Functioning, Role-Physical, Bodily Pain, General Health, Vitality, Social Functioning, Role-Emotional and Mental Health. Each health domain score contributes to the Physical Component Summary and Mental Component Summary scores. The Behavioural Changes in Energy Management Survey will be administered to describe the stable use of energy management strategies in daily life. This survey consists of eight questions and documents changes in using energy management strategies in daily routines, the effectiveness of stable strategy use (VAS 0–10), the simplicity of implementing them (VAS 0–10) and the actual stage of change. This survey has been used in two previous studies and was feasible (complete filled-out questionnaire response rate: 70%, average response duration: 10 min). For the cost-effectiveness profile of the EME intervention assessed vs the standard care comparator, a self-reported questionnaire will be administered at several measure points to collect disease-related and productivity cost data allowing a more comprehensive assessment of the cost dimension. The dimensions for which data are collected include not only the ability to work and its remuneration and medical costs but also changes in costs, for example, mobility, leisure time and support at home. Patient and public involvement With Altea Network for Long COVID Association (ALTEA) and the patient organisation Long Covid Switzerland (LCS) as project partners, the two relevant patient associations in Switzerland are part of this research project during all steps from the very beginning until the end. They participated during the protocol development like giving feedback about targeted research questions and outcome measures (eg, adequacy and burden), co-developed most patient information (informed consents, flyers, posts for social media, etc), are actively involved in the recruitment for the CG register and are giving feedback and meanings from the patient community about our study. These two associations will be participating in the interpretation of the results of this study and their dissemination by co-authorship at scientific publications and congress participation and by having the lead in the dissemination to the patient community as to the public and politics. Voluntary study participants’ feedback was and will be taken seriously and has resulted in minor changes. Study participants and participants in the CG register will be informed about the results of the study and their dissemination. Data collection All questionnaires and surveys will be available in the three national languages (German, French and Italian) for both groups and at all time points online via Redcap ( http://www.project-redcap.org/ ). If preferred, they will also be available on paper, either delivered personally by the local investigator or sent by post. Self-reported questionnaires documenting the outcomes of interest will be collected at different time points. In , all procedures and data collection time points involving the CG and the EG are listed, respectively. Reasons for discontinuation are the following: (1) withdrawal of informed consent, (2) participants from the CG (without EME) starting in an OT-EME group during the data collection phase and (3) participants of the EG (with EME) missing more than three of the eight EME lessons. All data collected until withdrawal will be used in the analysis. If a participant decides to withdraw from the follow-up, the reasons for the withdrawal will be recorded for the subsequent analysis respectively the interpretation of the results. We will make all attempts to closely follow up participants and therefore minimise missingness throughout data collection. At each time point, participants of both groups will be invited to fill in the questionnaires via Redcap by presenting or e-mailing the correct web link and, if necessary, up to two reminders after 4–5 days each. No reaction will lead to a reminder for contacting the principal investigator, if they need any support to continue with the study, with a clear statement, that they are not obliged to answer this reminder or justify their drop-out if they do not want to. Data management The local investigators at USZ, REHAB, INSEL and EME-OTs will be carefully trained by the principal investigator in their research-relevant tasks. All data collected on paper are entered into Redcap by the local investigator and regularly checked for accuracy by the principal investigator. Project data will be handled with the uttermost discretion and is only accessible to authorised personnel who require the data to fulfil their duties within the scope of the research project. On the case report forms and other project-specific documents, participants are only identified by a unique participant number. At the end of this project, we will remove any direct identifiers in the data before depositing the anonymised data on Zenodo Repository assigned with a persistent identifier (DOI). Statistical methods and analysis Sample size The target study population are people with post-COVID-19 condition living in Switzerland. To calculate the sample size of this study, we used data from two previous studies. In the feasibility study with persons with long COVID-related fatigue, the mean difference of the primary outcome (self-efficacy in performing energy conservation strategies) at T2 (post-intervention) was 1.9 (95% CI: 2.89 to 0.90). We hypothesised, therefore, a difference of 1.5 points at T2 and conservative assumption of a SD of 2.2 points. Including a follow-up rate of 24% at T2, the estimated sample size to detect a difference of 1.5 points with 90% power (0.05 alpha) is 122 persons. Therefore, we aim at a total of 122 persons included in the study (eg, 61 and CG: 61). Statistical analysis We will emulate a prospective interventional study that makes causal inferences on the effectiveness of adding EME to the standard care using a target trial emulation approach (see ). To emulate random assignment, each participant in the EG will be prospectively matched to a participant in the register according to their baseline sociodemographic characteristics (sex and age) and clinical history of long-COVID-related fatigue. We will choose 1:1 optimal matching using PS as a distance measure. We will employ a prospective matched design to compare outcomes between the experimental group and CGs. This ensures that CG participants are carefully selected in real time, concurrently with the enrolment of EG participants, to maximise group comparability and reduce confounding. For every 15 participants who are enrolled in the EG, we will prospectively match one CG participant to each EG participant. Optimal matching minimises the overall sum of pair-wise distances between treatment subjects and matched control subjects and is recommended when matching for the purpose of selecting well-matched controls for follow-up (eg, when the outcome values are not yet available). Descriptive statistics will be used for all outcome measures at each measurement time to summarise results. To ensure that PS matching adequately balances measured confounders, we will carefully assess and report standardised differences between treatment groups before and after matching. Standardised differences are widely used because they are unaffected by sample size and provide a consistent measure of imbalance. A standardised difference of less than 10% (0.1 in absolute value) will be considered indicative of a clinically relevant balance between groups. Variables with standardised differences of greater than 10% will be flagged for potential residual confounding and addressed in sensitivity analyses. Our causal contrast of interest will be the per-protocol effect, so to analyse the effect of adding EME to the standard care on the dependent continuous variables for both primary and secondary outcomes, an adjusted repeated measures mixed-effects linear regression model will be used. In addition to the covariates already included in the matching phase, we are aiming to include additional potential covariates related to the clinical history of long-COVID (eg, fatigue intensity, fatigue impact) and socio-demographic data (eg, education, occupation status). Using PS matching combined with regression methods is a robust approach to account for measured confounding. However, this approach does not address unmeasured confounding, which could bias the results. To acknowledge this limitation and mitigate its impact, we will perform a sensitivity analysis to evaluate the robustness of your findings to potential unmeasured confounding. We will perform a simulated bias analysis to estimate the potential impact of unmeasured confounding. We will specify plausible distributions for an unmeasured confounder and assess its potential effect on the treatment-outcome relationship. In addition, we plan to conduct an E-value analysis for assessing whether our results are likely robust even in the presence of unmeasured confounders. The E-value quantifies the minimum strength of association that an unmeasured confounder would need to have with both the treatment and the outcome (beyond measured confounders) to explain away the observed treatment effect. Cost-effectiveness of EME intervention against the standard care only will be evaluated by performing an economic evaluation, using the direct costs of delivering the intervention, disease-related and productivity as the cost input and various outcomes as health effects. A probabilistic sensitivity analysis will be performed to consider the uncertainty in the various input parameters and their effects on the decision-making process. After the maximal efforts have been employed to minimise the presence missing data, we will employ, beyond the default complete case analysis, a multiple imputation approach to assess the robustness of results. Statistical analysis will be performed using Stata (Stata, College Station, Texas, USA) and R (R Core Team (2023) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria; < https://www.R-project.org/ >). Missing data After maximal efforts have been employed to minimise the presence of missing data, we will employ, beyond the default complete case analysis, a multiple imputation approach to assess the robustness of results. Volunteer bias The fact that only persons who have actively registered themselves in the register can potentially get part of the CG will be carefully considered when interpreting and discussing the results of this study. Selection bias This common flaw of observational studies, we address by not using any information collected in the EG after the baseline period to determine which individuals from the register should be enrolled in the CG. By defining the recruitment of controls in two stages and having clear eligibility criteria, we create a structured approach to estimate causal effects even in the absence of true randomisation, based on an unbiased sample. Ethics and dissemination Fatigue is a common and disabling condition for people with long COVID. The self-management education of persons enables them to implement new behavioural habits and engage more successfully in daily routines and social participation by managing their available energy. In the field of rehabilitation, this research will deliver urgently needed data on the effectiveness of an intervention recommended by guidelines for persons with long COVID and give a perspective on the potential and meaning of self-management education concerning health and well-being. Furthermore, the register of persons with a clinical history of long COVID can be used for further observational and evaluation studies, especially considering a long-term perspective. In the actual healthcare context in Switzerland, several factors prevent conducting an RCT or quasi-RCT aiming at investigating the effectiveness of EME in persons with long COVID-related fatigue: (1) in those institutions where the EME protocol with peer groups has been implemented as part of the standard care, it would not be feasible and/or ethical to introduce an alternative OT intervention acting as a CG intervention. (2) Enough patients must enter an OT service to continuously form and maintain EME groups. Randomisation into two groups would reduce the possibility of forming groups and prevent EME from being performed. (3) Furthermore, randomisation into a waiting list is not feasible because, in the case of EME, this would require at least 15 weeks, which would be an unnecessary additional patient burden. Regarding OT, this research project will support the further development of evidence-based practice and use of standardised treatment protocols, which are until to date lowly applied in the daily practice of OTs in Switzerland. Data from the survey on the implemented energy management strategies will inform OTs about the most effective as well as the most difficult strategies to implement and provide insights into predicting factors and successful behavioural changes. This kind of knowledge in OT was, until now, missing or is, at present, only subjectively and empirically. Furthermore, OT researchers are asked to contribute their perspective to the development and discussion of the science of habit because a better understanding of the factors that support changes in habits and routines is also relevant in other health areas (eg, pain management, ergonomic behavior, stress management and lifestyle behaviour). The findings of this study will support the training of OTs in general and especially those delivering self-management education and support their skills in coaching and empowering patients and offering a more informed perspective for persons with long COVID. This study will answer whether a higher effect justifies the effort of organising groups or if individual OTs are sufficiently empowering. At the same time, data from the cost-effective analysis will support rehabilitation managers, assurances and policymakers with data for informed decisions about group-based rehabilitation interventions. The results of this study will be published as articles in scientific journals from various fields and presented at national and international congresses. In addition, we will disseminate the results through the social media channels of the partner organisations (Altea Network and Long-COVID-Switzerland), media dedicated to practitioners (OT and medical staff) and general media. In the training courses for EME-leading OTs and workshops for other healthcare professionals (eg, nurses, physicians), which AW and RH are organising in all parts of Switzerland and the neighboring countries, the results of this study will be integrated, and participants will function as multiplicators of the new knowledge. The recruitment at the three intervention group sites and for the control group (CG) register started in July 2024. The time between registration in the study and matching for the CG depends on the recruitment rate of the EME participants. The first matching of 15 participants of the experimental group with 15 participants out of the CG register was performed in December 2024. Study participation lasts for each study participant approximately 24 weeks; therefore, data collection was not finished with any participant before submission of this article. The recruitment will be finished during late summer/early autumn 2025, and data collection will be finished at approximately the end of 2025. Data analysis will begin at the end of 2025 after the last participant has completed T3. The total duration of the research project is 42 months. The gold standard for investigating the efficacy of a treatment is an RCT, which guarantees high internal validity by minimising clinician and patient biases. In contrast, pragmatic trials prioritise external validity by studying interventions in the context of broad-based and normal clinical practice conditions. In rehabilitation research, pragmatic or real-world trials are increasingly being conducted. They aim to identify the most effective intervention for a patient-centred primary endpoint. Considering the above elements of the current context, we will emulate a prospective interventional study that draws causal inferences on the effectiveness of adding EME to standard care using a target trial emulation approach. Ethical approval has been obtained from the competent Swiss ethics commission (2024–00938; Rif. CE 4596). Standard care: any interventions tailored to the specific needs and goals that aim to support individuals with long COVID on their way back to normality, such as breathing exercises, individual OT, relaxation techniques, mindfulness, cognitive–behavioural therapy, physical exercise training and consultations by different health professionals (eg, physicians, cardiologists, pulmonologists, psychologists, dietitians and social workers). All participants will follow the standard care personalised to their needs and preferences. The CG will receive the standard care only. Energy management education (EME), revised outpatient version 2023: this updated version integrates the findings of the feasibility study performed in 2022. EME aims to increase self-management skills for managing the available amount of energy and achieving a satisfactory and meaningful daily routine despite fatigue. During EME, participants learn about the factors influencing their energy level and experiment with skills to manage their energy using behavioural strategies (eg, pace, plan and prioritise activities and optimise communication, environment and ergonomic behaviour). Subsequently, they identify and implement tailored behavioural modifications. To support the reflection on and use of energy-managing strategies and new behaviours, the leading OTs use behavioural change techniques. The outpatient EME protocol consists of seven group sessions (90 min) once a week plus an eighth session approximately 8 weeks after the seventh one. The first, seventh and eighth sessions can be done in individual sessions (45 min) alternatively. Sessions are led by EME-trained OTs. The experimental group will receive personalised standard care plus EME. The overview of the topics and further details are presented in . At the University Hospital Bern (INSEL) and Clinic for Neurorehabilitation Basel (REHAB), the intervention starts with an individual lesson (45 min), whereas at University Hospital Zürich (USZ), the first lesson is already delivered in groups (90 min). With a frequency of one lesson per week, lessons 2–7 are provided (duration 90 min, appr. five participants). EME finishes with a group lesson, approximately 8 weeks after lesson 7. REHAB provides lessons 7 and 8 as individual lessons of 45 min. The EME-leading OTs use the treatment manual, which describes in detail the tasks of each lesson. All EME participants receive the workbook, which contains relevant information on the topics covered in each session, including worksheets and space for personal notes plus self-training tasks. Experimental group (EME, outpatient version 2023, plus standard care): persons with long COVID-related fatigue following EME (revised edition 2023) in an outpatient setting at USZ, INSEL or REHAB, as part of their clinical routine and standard care, will be informed about this ongoing research and will be kindly asked if they are willing to participate in this project. If individuals agree, have signed the consent form and meet the inclusion/exclusion criteria checked by the study therapist on site, they will be included in the experimental group (EG). In these three public institutions, a group of trained OTs leads continuously EME groups and has received up-to-date training for the EME version 2023 in Spring 2024. CG (standard care ): a register of potential CG participants will be created through a contact with the Altea Network for Long COVID Association (ALTEA) and the patient organisation Long Covid Switzerland (LCS), both of which are partners in the study project and have been involved since the design phase. Using the contacts of ALTEA, LCS and specialised healthcare providers, information about the study will be disseminated, allowing interested persons to register themselves through a public link or QR code and to participate in a first online survey. Registration for the study has been open since 15 July 2024 and will remain open until summer 2025. Thereafter, based on the sociodemographic characteristics and clinical history of COVID-19, the CG participants will be selected from the register by their propensity score (PS) and matched with EG participants. Subsequently, the selected persons from the CG will be screened for eligibility, asked for their informed consent and included in the study. The target study population consists of individuals aged 18 years or older, living in Switzerland, with a post-COVID-19 condition and a confirmed experience of fatigue. Eligible participants must not have major depression and/or significant cognitive impairments, must have sufficient ability to understand and speak one of the national languages and must provide signed informed consent. Persons are not eligible if they have previously participated in an EME group, have insufficient comprehension of one of the national languages or have a history of severe neurological diseases (eg, stroke, multiple sclerosis, Parkinson’s disease) or mental health disorders (eg, schizophrenia, major depression, or dementia). The primary aim is to compare the effectiveness of the standard care for persons with long COVID-related fatigue plus participating in an EME group (revised edition 2023), with the standard care without EME at post-intervention (T2; week 14). The secondary aim is to assess the energy management behavioural strategies applied in daily routines in persons living with long COVID, their feasibility and the factors influencing behavioural changes (T3, week 24). The third aim is to evaluate the cost-effectiveness profile of the intervention by performing an economic evaluation alongside the trial. Primary outcomes The impact of fatigue on physical, mental and social activities of daily routines will be measured with the Modified Fatigue Impact Scale. It contains 21 items, on a scale from 0 (never) to 4 (almost always), thus total ranging from 0 to 84. Self-efficacy in performing energy management strategies is the most proximal outcome of EME. Increased perceived self-efficacy in performing energy management behaviours and strategies correlates to the impact of fatigue in daily life and predicts behavioural changes after patient education. This will be measured using the ‘Self-Efficacy for Performing Energy Conservation Strategies Assessment’. This self-report questionnaire reports the mean scores of perceived confidence in performing 14 different energy management strategies (1=low confidence, 10=high confidence). It is a valid and reliable instrument with a minimal clinically important difference of 0.92 in people with chronic conditions and fatigue. Secondary outcomes The Occupational Self-Assessment (OSA 2.2 ; focuses on perceived competency to perform 21 daily tasks, which scores from 1 (I have many problems) to 4 (I do extremely well). The raw data are converted to interval levels (0–100). OSA has been validated in German and has shown the ability to detect changes in self-reported competence for daily activities over time. Health-related quality of life will be measured by using the Short-Form 12 Item Health-Related Quality of Life Survey. This survey is a practical, reliable, valid and often used measure of physical and mental health. It assesses the same eight health domains as the SF-36, but with only one or two questions per domain: Physical Functioning, Role-Physical, Bodily Pain, General Health, Vitality, Social Functioning, Role-Emotional and Mental Health. Each health domain score contributes to the Physical Component Summary and Mental Component Summary scores. The Behavioural Changes in Energy Management Survey will be administered to describe the stable use of energy management strategies in daily life. This survey consists of eight questions and documents changes in using energy management strategies in daily routines, the effectiveness of stable strategy use (VAS 0–10), the simplicity of implementing them (VAS 0–10) and the actual stage of change. This survey has been used in two previous studies and was feasible (complete filled-out questionnaire response rate: 70%, average response duration: 10 min). For the cost-effectiveness profile of the EME intervention assessed vs the standard care comparator, a self-reported questionnaire will be administered at several measure points to collect disease-related and productivity cost data allowing a more comprehensive assessment of the cost dimension. The dimensions for which data are collected include not only the ability to work and its remuneration and medical costs but also changes in costs, for example, mobility, leisure time and support at home. The impact of fatigue on physical, mental and social activities of daily routines will be measured with the Modified Fatigue Impact Scale. It contains 21 items, on a scale from 0 (never) to 4 (almost always), thus total ranging from 0 to 84. Self-efficacy in performing energy management strategies is the most proximal outcome of EME. Increased perceived self-efficacy in performing energy management behaviours and strategies correlates to the impact of fatigue in daily life and predicts behavioural changes after patient education. This will be measured using the ‘Self-Efficacy for Performing Energy Conservation Strategies Assessment’. This self-report questionnaire reports the mean scores of perceived confidence in performing 14 different energy management strategies (1=low confidence, 10=high confidence). It is a valid and reliable instrument with a minimal clinically important difference of 0.92 in people with chronic conditions and fatigue. The Occupational Self-Assessment (OSA 2.2 ; focuses on perceived competency to perform 21 daily tasks, which scores from 1 (I have many problems) to 4 (I do extremely well). The raw data are converted to interval levels (0–100). OSA has been validated in German and has shown the ability to detect changes in self-reported competence for daily activities over time. Health-related quality of life will be measured by using the Short-Form 12 Item Health-Related Quality of Life Survey. This survey is a practical, reliable, valid and often used measure of physical and mental health. It assesses the same eight health domains as the SF-36, but with only one or two questions per domain: Physical Functioning, Role-Physical, Bodily Pain, General Health, Vitality, Social Functioning, Role-Emotional and Mental Health. Each health domain score contributes to the Physical Component Summary and Mental Component Summary scores. The Behavioural Changes in Energy Management Survey will be administered to describe the stable use of energy management strategies in daily life. This survey consists of eight questions and documents changes in using energy management strategies in daily routines, the effectiveness of stable strategy use (VAS 0–10), the simplicity of implementing them (VAS 0–10) and the actual stage of change. This survey has been used in two previous studies and was feasible (complete filled-out questionnaire response rate: 70%, average response duration: 10 min). For the cost-effectiveness profile of the EME intervention assessed vs the standard care comparator, a self-reported questionnaire will be administered at several measure points to collect disease-related and productivity cost data allowing a more comprehensive assessment of the cost dimension. The dimensions for which data are collected include not only the ability to work and its remuneration and medical costs but also changes in costs, for example, mobility, leisure time and support at home. With Altea Network for Long COVID Association (ALTEA) and the patient organisation Long Covid Switzerland (LCS) as project partners, the two relevant patient associations in Switzerland are part of this research project during all steps from the very beginning until the end. They participated during the protocol development like giving feedback about targeted research questions and outcome measures (eg, adequacy and burden), co-developed most patient information (informed consents, flyers, posts for social media, etc), are actively involved in the recruitment for the CG register and are giving feedback and meanings from the patient community about our study. These two associations will be participating in the interpretation of the results of this study and their dissemination by co-authorship at scientific publications and congress participation and by having the lead in the dissemination to the patient community as to the public and politics. Voluntary study participants’ feedback was and will be taken seriously and has resulted in minor changes. Study participants and participants in the CG register will be informed about the results of the study and their dissemination. All questionnaires and surveys will be available in the three national languages (German, French and Italian) for both groups and at all time points online via Redcap ( http://www.project-redcap.org/ ). If preferred, they will also be available on paper, either delivered personally by the local investigator or sent by post. Self-reported questionnaires documenting the outcomes of interest will be collected at different time points. In , all procedures and data collection time points involving the CG and the EG are listed, respectively. Reasons for discontinuation are the following: (1) withdrawal of informed consent, (2) participants from the CG (without EME) starting in an OT-EME group during the data collection phase and (3) participants of the EG (with EME) missing more than three of the eight EME lessons. All data collected until withdrawal will be used in the analysis. If a participant decides to withdraw from the follow-up, the reasons for the withdrawal will be recorded for the subsequent analysis respectively the interpretation of the results. We will make all attempts to closely follow up participants and therefore minimise missingness throughout data collection. At each time point, participants of both groups will be invited to fill in the questionnaires via Redcap by presenting or e-mailing the correct web link and, if necessary, up to two reminders after 4–5 days each. No reaction will lead to a reminder for contacting the principal investigator, if they need any support to continue with the study, with a clear statement, that they are not obliged to answer this reminder or justify their drop-out if they do not want to. The local investigators at USZ, REHAB, INSEL and EME-OTs will be carefully trained by the principal investigator in their research-relevant tasks. All data collected on paper are entered into Redcap by the local investigator and regularly checked for accuracy by the principal investigator. Project data will be handled with the uttermost discretion and is only accessible to authorised personnel who require the data to fulfil their duties within the scope of the research project. On the case report forms and other project-specific documents, participants are only identified by a unique participant number. At the end of this project, we will remove any direct identifiers in the data before depositing the anonymised data on Zenodo Repository assigned with a persistent identifier (DOI). Sample size The target study population are people with post-COVID-19 condition living in Switzerland. To calculate the sample size of this study, we used data from two previous studies. In the feasibility study with persons with long COVID-related fatigue, the mean difference of the primary outcome (self-efficacy in performing energy conservation strategies) at T2 (post-intervention) was 1.9 (95% CI: 2.89 to 0.90). We hypothesised, therefore, a difference of 1.5 points at T2 and conservative assumption of a SD of 2.2 points. Including a follow-up rate of 24% at T2, the estimated sample size to detect a difference of 1.5 points with 90% power (0.05 alpha) is 122 persons. Therefore, we aim at a total of 122 persons included in the study (eg, 61 and CG: 61). Statistical analysis We will emulate a prospective interventional study that makes causal inferences on the effectiveness of adding EME to the standard care using a target trial emulation approach (see ). To emulate random assignment, each participant in the EG will be prospectively matched to a participant in the register according to their baseline sociodemographic characteristics (sex and age) and clinical history of long-COVID-related fatigue. We will choose 1:1 optimal matching using PS as a distance measure. We will employ a prospective matched design to compare outcomes between the experimental group and CGs. This ensures that CG participants are carefully selected in real time, concurrently with the enrolment of EG participants, to maximise group comparability and reduce confounding. For every 15 participants who are enrolled in the EG, we will prospectively match one CG participant to each EG participant. Optimal matching minimises the overall sum of pair-wise distances between treatment subjects and matched control subjects and is recommended when matching for the purpose of selecting well-matched controls for follow-up (eg, when the outcome values are not yet available). Descriptive statistics will be used for all outcome measures at each measurement time to summarise results. To ensure that PS matching adequately balances measured confounders, we will carefully assess and report standardised differences between treatment groups before and after matching. Standardised differences are widely used because they are unaffected by sample size and provide a consistent measure of imbalance. A standardised difference of less than 10% (0.1 in absolute value) will be considered indicative of a clinically relevant balance between groups. Variables with standardised differences of greater than 10% will be flagged for potential residual confounding and addressed in sensitivity analyses. Our causal contrast of interest will be the per-protocol effect, so to analyse the effect of adding EME to the standard care on the dependent continuous variables for both primary and secondary outcomes, an adjusted repeated measures mixed-effects linear regression model will be used. In addition to the covariates already included in the matching phase, we are aiming to include additional potential covariates related to the clinical history of long-COVID (eg, fatigue intensity, fatigue impact) and socio-demographic data (eg, education, occupation status). Using PS matching combined with regression methods is a robust approach to account for measured confounding. However, this approach does not address unmeasured confounding, which could bias the results. To acknowledge this limitation and mitigate its impact, we will perform a sensitivity analysis to evaluate the robustness of your findings to potential unmeasured confounding. We will perform a simulated bias analysis to estimate the potential impact of unmeasured confounding. We will specify plausible distributions for an unmeasured confounder and assess its potential effect on the treatment-outcome relationship. In addition, we plan to conduct an E-value analysis for assessing whether our results are likely robust even in the presence of unmeasured confounders. The E-value quantifies the minimum strength of association that an unmeasured confounder would need to have with both the treatment and the outcome (beyond measured confounders) to explain away the observed treatment effect. Cost-effectiveness of EME intervention against the standard care only will be evaluated by performing an economic evaluation, using the direct costs of delivering the intervention, disease-related and productivity as the cost input and various outcomes as health effects. A probabilistic sensitivity analysis will be performed to consider the uncertainty in the various input parameters and their effects on the decision-making process. After the maximal efforts have been employed to minimise the presence missing data, we will employ, beyond the default complete case analysis, a multiple imputation approach to assess the robustness of results. Statistical analysis will be performed using Stata (Stata, College Station, Texas, USA) and R (R Core Team (2023) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria; < https://www.R-project.org/ >). Missing data After maximal efforts have been employed to minimise the presence of missing data, we will employ, beyond the default complete case analysis, a multiple imputation approach to assess the robustness of results. Volunteer bias The fact that only persons who have actively registered themselves in the register can potentially get part of the CG will be carefully considered when interpreting and discussing the results of this study. Selection bias This common flaw of observational studies, we address by not using any information collected in the EG after the baseline period to determine which individuals from the register should be enrolled in the CG. By defining the recruitment of controls in two stages and having clear eligibility criteria, we create a structured approach to estimate causal effects even in the absence of true randomisation, based on an unbiased sample. The target study population are people with post-COVID-19 condition living in Switzerland. To calculate the sample size of this study, we used data from two previous studies. In the feasibility study with persons with long COVID-related fatigue, the mean difference of the primary outcome (self-efficacy in performing energy conservation strategies) at T2 (post-intervention) was 1.9 (95% CI: 2.89 to 0.90). We hypothesised, therefore, a difference of 1.5 points at T2 and conservative assumption of a SD of 2.2 points. Including a follow-up rate of 24% at T2, the estimated sample size to detect a difference of 1.5 points with 90% power (0.05 alpha) is 122 persons. Therefore, we aim at a total of 122 persons included in the study (eg, 61 and CG: 61). We will emulate a prospective interventional study that makes causal inferences on the effectiveness of adding EME to the standard care using a target trial emulation approach (see ). To emulate random assignment, each participant in the EG will be prospectively matched to a participant in the register according to their baseline sociodemographic characteristics (sex and age) and clinical history of long-COVID-related fatigue. We will choose 1:1 optimal matching using PS as a distance measure. We will employ a prospective matched design to compare outcomes between the experimental group and CGs. This ensures that CG participants are carefully selected in real time, concurrently with the enrolment of EG participants, to maximise group comparability and reduce confounding. For every 15 participants who are enrolled in the EG, we will prospectively match one CG participant to each EG participant. Optimal matching minimises the overall sum of pair-wise distances between treatment subjects and matched control subjects and is recommended when matching for the purpose of selecting well-matched controls for follow-up (eg, when the outcome values are not yet available). Descriptive statistics will be used for all outcome measures at each measurement time to summarise results. To ensure that PS matching adequately balances measured confounders, we will carefully assess and report standardised differences between treatment groups before and after matching. Standardised differences are widely used because they are unaffected by sample size and provide a consistent measure of imbalance. A standardised difference of less than 10% (0.1 in absolute value) will be considered indicative of a clinically relevant balance between groups. Variables with standardised differences of greater than 10% will be flagged for potential residual confounding and addressed in sensitivity analyses. Our causal contrast of interest will be the per-protocol effect, so to analyse the effect of adding EME to the standard care on the dependent continuous variables for both primary and secondary outcomes, an adjusted repeated measures mixed-effects linear regression model will be used. In addition to the covariates already included in the matching phase, we are aiming to include additional potential covariates related to the clinical history of long-COVID (eg, fatigue intensity, fatigue impact) and socio-demographic data (eg, education, occupation status). Using PS matching combined with regression methods is a robust approach to account for measured confounding. However, this approach does not address unmeasured confounding, which could bias the results. To acknowledge this limitation and mitigate its impact, we will perform a sensitivity analysis to evaluate the robustness of your findings to potential unmeasured confounding. We will perform a simulated bias analysis to estimate the potential impact of unmeasured confounding. We will specify plausible distributions for an unmeasured confounder and assess its potential effect on the treatment-outcome relationship. In addition, we plan to conduct an E-value analysis for assessing whether our results are likely robust even in the presence of unmeasured confounders. The E-value quantifies the minimum strength of association that an unmeasured confounder would need to have with both the treatment and the outcome (beyond measured confounders) to explain away the observed treatment effect. Cost-effectiveness of EME intervention against the standard care only will be evaluated by performing an economic evaluation, using the direct costs of delivering the intervention, disease-related and productivity as the cost input and various outcomes as health effects. A probabilistic sensitivity analysis will be performed to consider the uncertainty in the various input parameters and their effects on the decision-making process. After the maximal efforts have been employed to minimise the presence missing data, we will employ, beyond the default complete case analysis, a multiple imputation approach to assess the robustness of results. Statistical analysis will be performed using Stata (Stata, College Station, Texas, USA) and R (R Core Team (2023) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria; < https://www.R-project.org/ >). After maximal efforts have been employed to minimise the presence of missing data, we will employ, beyond the default complete case analysis, a multiple imputation approach to assess the robustness of results. The fact that only persons who have actively registered themselves in the register can potentially get part of the CG will be carefully considered when interpreting and discussing the results of this study. This common flaw of observational studies, we address by not using any information collected in the EG after the baseline period to determine which individuals from the register should be enrolled in the CG. By defining the recruitment of controls in two stages and having clear eligibility criteria, we create a structured approach to estimate causal effects even in the absence of true randomisation, based on an unbiased sample. Fatigue is a common and disabling condition for people with long COVID. The self-management education of persons enables them to implement new behavioural habits and engage more successfully in daily routines and social participation by managing their available energy. In the field of rehabilitation, this research will deliver urgently needed data on the effectiveness of an intervention recommended by guidelines for persons with long COVID and give a perspective on the potential and meaning of self-management education concerning health and well-being. Furthermore, the register of persons with a clinical history of long COVID can be used for further observational and evaluation studies, especially considering a long-term perspective. In the actual healthcare context in Switzerland, several factors prevent conducting an RCT or quasi-RCT aiming at investigating the effectiveness of EME in persons with long COVID-related fatigue: (1) in those institutions where the EME protocol with peer groups has been implemented as part of the standard care, it would not be feasible and/or ethical to introduce an alternative OT intervention acting as a CG intervention. (2) Enough patients must enter an OT service to continuously form and maintain EME groups. Randomisation into two groups would reduce the possibility of forming groups and prevent EME from being performed. (3) Furthermore, randomisation into a waiting list is not feasible because, in the case of EME, this would require at least 15 weeks, which would be an unnecessary additional patient burden. Regarding OT, this research project will support the further development of evidence-based practice and use of standardised treatment protocols, which are until to date lowly applied in the daily practice of OTs in Switzerland. Data from the survey on the implemented energy management strategies will inform OTs about the most effective as well as the most difficult strategies to implement and provide insights into predicting factors and successful behavioural changes. This kind of knowledge in OT was, until now, missing or is, at present, only subjectively and empirically. Furthermore, OT researchers are asked to contribute their perspective to the development and discussion of the science of habit because a better understanding of the factors that support changes in habits and routines is also relevant in other health areas (eg, pain management, ergonomic behavior, stress management and lifestyle behaviour). The findings of this study will support the training of OTs in general and especially those delivering self-management education and support their skills in coaching and empowering patients and offering a more informed perspective for persons with long COVID. This study will answer whether a higher effect justifies the effort of organising groups or if individual OTs are sufficiently empowering. At the same time, data from the cost-effective analysis will support rehabilitation managers, assurances and policymakers with data for informed decisions about group-based rehabilitation interventions. The results of this study will be published as articles in scientific journals from various fields and presented at national and international congresses. In addition, we will disseminate the results through the social media channels of the partner organisations (Altea Network and Long-COVID-Switzerland), media dedicated to practitioners (OT and medical staff) and general media. In the training courses for EME-leading OTs and workshops for other healthcare professionals (eg, nurses, physicians), which AW and RH are organising in all parts of Switzerland and the neighboring countries, the results of this study will be integrated, and participants will function as multiplicators of the new knowledge. |
Knowledge and beliefs of endocrine disruptors in pediatrics: all hands on deck! | 59e8908c-3775-40a0-9652-548926accd9e | 11225406 | Pediatrics[mh] | Introduction The impact of the environment on population health is a growing concern for public policy and health professionals. According to the World Health Organization , approximately 24% of global mortality is due to the environment. Endocrine disruptors (ED) contribute to this mortality as chemical compounds ubiquitously present in the environment. There are suspected to contribute to the development of chronic diseases such as diabetes, obesity, precocious puberty, and fertility . Their mechanisms of action are not fully understood, but may include mimetic hormone effects, antagonistic effects, and epigenetic effects . However, these mechanisms can be different between EDs or have a cumulative effect. Taken together, these uncertainties and complexity limit studies of high-level of evidence. Recommendations are to limit and decrease exposures as much as possible. Environmental safety is one parents’ primary concern, inducing many questions to Pediatric Health Professionals (PHP) in general, notably pediatricians, nurses, childcare assistants, and secretaries. Pregnancy and childhood are vulnerability windows, particularly in neonates, infants, and during puberty . Exposures during these periods are more at risk of engendering (long-term) health effects. Still, environmental exposure is a relatively recent concern: in France, dedicated teaching courses in medical schools are just emerging. As PHPs, we should help parents to adopt good health behavior to preserve their children health, but previous studies have reported a lack of knowledge on these topics among health professionals, including midwives, obstetricians, general practitioners and health care professionals . However, very little data are available specifically for PHPs , and parents . Dedicated ED questionnaires are scarce; specific knowledge or representation are less evaluated . Knowledge is not the only determinant of health behavior. Indeed, even though we all know that smoking kills people, some people keep smoking. Thus, an evaluation of knowledge and beliefs on ED is necessary to know from where we come and to improve both information and message delivery to the family. Our objectives were first to describe the knowledge and beliefs of both PHPs and parents, as well as their sources of information, using the same questionnaire, and second to search for factors of “better” answers. Methods We performed the STENDAL study, a cross-sectional quantitative study of parents and professionals at the Lyon Mother and Child Hospital, a tertiary pediatric university hospital (58,000 out-patient clinics, 68,000 hospitalizations and 85,000 visits at the pediatric emergency room yearly). We proposed the study to all parents who referred their children at the out-patient clinics (medical and chirurgical). A student was present in the waiting rooms during the study period, to directly give parents information and hand-delivered questionnaire after agreement to participate. Parents filled the questionnaire directly. All professionals from the PHP, including physicians, nurses, assistant nurses, secretaries, administrative and research staff received the questionnaire through an individual email sent to their professional mailbox. They had 2 months to respond, and one reminder was sent. A total of 408 questionnaires were given to parents between June 30, 2022 and October 24, 2022. All 1,580 PHP of the Lyon Mother and Child Hospital received a personal email the 3rd of May 2023, and one personal remember the 15th of May 2023. We constructed a 15-item standardized self-administered questionnaire with one multiple-choice question and 14 true/false questions. The questionnaire was the same for professionals and parents. The questions concerned theoretical knowledge (questions 0, 1, 6, 10, 11, and 14) and beliefs (questions 2, 3, 4, 5, 7, 8, 9, 12, and 13) regarding EDs. The score was calculated from 15 questions. A point was awarded for correct answers, and 0 for incorrect or missing responses. Six questions evaluated the belief dimension, seven questions evaluated knowledge, one evaluated knowledge and belief and one question two different aspects of belief. Questions 2, 9, and 14 evaluated the confidence in the “natural” products. Questionnaires were anonymous and simple demographic data (i.e., profession, reasons for consultation, age of professional, rural or urban residency) were collected at the beginning of the questionnaire survey. The study was approved by the Ethics Committee of Hospices Civils de Lyon (n° 2022022). Written information with correct answers were given to parents after receiving the questionnaire. An hospital meeting was organized to present the results of the survey to PHPs in October 2023. Descriptive statistics were used to describe the responses. Qualitative data are reported as percentage and quantitative data as median. Correct responses rates were compared using nonparametric Kruskal-Wallis and Mann Whitney U tests. p -values presented are not adjusted for multiple testing and are not inferential. Analysis was conducted with R statistical software version 4.2. Results and discussion 3.1 Results In total, 746 questionnaires were completed. The response rate was 28% ( n = 444) for PHP and 74% ( n = 302) for parents. The overall percentage of correct answers was 68%. It was significantly better in the PHPs’ group compared to the parents’ group (73% vs. 60%, respectively, p < 0.001). Proportions of correct answers to “knowledge” and “beliefs” questions were, respectively, 50 and 57% for parents, and 67 and 85% for PHPs. Rate of “good responses” to “belief” questions were 100% for medical doctor and 71.4% for the other professionals. The global and specific responses of the two groups are displayed in the radar chart. Questions 2, 9, and 14 related to confident in natural products have, respectively, 61, 27, and 45% of good answers in parents’ group and 84, 51, and 65% of good answers in PHPs’ group. The highest proportion of correct answers was for the question on cosmetic products (question 4): 94% in the parents’ group and 97% in the PHPs’ group ( p = NS). The lowest proportion of good answers was for the question on EDs mechanisms of action (question 11): 10% for parents and 11% for professionals ( p = NS). Overall, 66% of parents and 95% of PHPs had already heard of ED. This 66% of parents had a significantly better score than the others (67% vs. 53%, p < 0.001); similarly, this 95% of PHPs had a significantly better score (71% vs. 57%, p = 0.002). However, only 10% of parents and 5% of PHPs reported that they felt sufficiently informed, but they did not have a significantly better score than participants who felt insufficiently informed . We have observed that 20% of both parents and PHPs systematically screened the product composition, while 15% never screened it. However, this screening was not linked to the proportion of correct answers. By univariate analysis, factors associated with a higher proportion of correct answers for parents were rural location and higher socio-professional categories. For PHPs, these factors were age under 35 years and being a physician. Information sources were similar between parents and PHPs, the most frequent one being media (television or radio) and the web for 70% of parents and 80% of PHPs; PHPs used mostly “general public” resources. Only 20% of PHPs read scientific papers, and 4% have followed a training on EDs . 3.2 Discussion Knowledge on EDs is not optimal both for parents and PHPs. The main strengths of this study were a significant number of answers and an evaluation of both parents and PHPs with the same questionnaire. All professionals interacting with parents and children were represented, thus reinforcing the strength of the study. However, PHPs’ knowledge and beliefs were not optimal, and most of them were not trained on EDs. Our rates of responses are good, close, or better than other reports in the literature, i.e., 11–30% . Reasons for obtaining a satisfactory response rate are probably multiple and intricated: the increasing concern about environmental exposures, the availability of researchers for parents with a hand-delivered questionnaire, and the individual mail for the PHPs in a pediatric hospital in which the principal investigator of the study is the senior physician responsible for the pediatric research centre. Some factors were linked to the profile of “good responders.” For parents these were rural locations and higher socio-professional categories. In rural areas, parents and their children are exposed to specific pollutants such as pesticides and information on this type of environmental exposures is probably more spread. Higher socio-professional categories often have better level of education and better access to information. Furthermore, for the lower socio-professional categories, environmental exposures are not a priority in daily life, even though they are the population most exposed. For professionals, the profile of “good responders” was under 35 years of age and being a doctor. Young professionals are more sensitive to environmental exposures and climate change. Good responses from doctor are linked to better answers to beliefs questions (100% for medical doctor vs. 71.4% for other professionals). Even though they have not received a specific training on EDs, the training provided during their medical training has enabled doctors to acquire a critical mind, which may explain their better responses to beliefs questions. This single-center study has nevertheless several limitations, by design. As a result, only professionals and parents from patients followed in a tertiary university hospital participated. One may assume that liberal practitioners are more concerned and informed because they receive more questions from parents, with less severely ill children. Also, responders probably felt more concerned with the questions of environmental exposure than parents and PHPs who did not participate. Therefore, we could expect that responses in general populations will be even worse. In addition, we used a non-validated personal questionnaire. In our study, only 10% of parents and 5% of PHPs considered they are sufficiently informed about EDs. This result is congruent with a previous study reporting that only 11% of perinatal health professionals were sufficiently trained and informed . Another French study also found 82% of insufficiently informed professionals . The lack of specific training for health professionals on the topic has also been reported in many studies worldwide . The worst answers were about “natural” products. In fact, each question with the term “natural” automatically generated an analysis of “healthy” in parents’ mind but also, however in a lesser extent, in PHPs. As described in human health science publications, instructional messages are not an optimal way to inform both parents and PHPs . Dichotomist ideas such as “natural is good” and “industrial is bad” have to be avoided; beliefs must be explored in order to give better messages to the population. Moreover, it may be relevant to analyze in future studies in which state of mind PHPs who are also parents position themselves. More worrying is the way used by professionals to get information, as already descripted in 2001 . There is an urgent need for professionals to be better informed. To achieve this, specific training courses and recommendations from medical scientific societies are required. For example, the International Federation of Gynecology and Obstetric guidelines refer to the presence of heavy metal and EDs in prenatal vitamins and recommends reducing the exposure. These guidelines are easily accessible for the professionals. Conversely, information in the media is often perceived as stressful and incomprehensible by parents . In conclusion, we show that, despite the fact that the risk of environmental exposure and EDs is an increasing concern for parents, the specific knowledge remains scarce both for parents and PHPs. PHPs need to be trained on the topic, so as to provide optimal advice to families. Results In total, 746 questionnaires were completed. The response rate was 28% ( n = 444) for PHP and 74% ( n = 302) for parents. The overall percentage of correct answers was 68%. It was significantly better in the PHPs’ group compared to the parents’ group (73% vs. 60%, respectively, p < 0.001). Proportions of correct answers to “knowledge” and “beliefs” questions were, respectively, 50 and 57% for parents, and 67 and 85% for PHPs. Rate of “good responses” to “belief” questions were 100% for medical doctor and 71.4% for the other professionals. The global and specific responses of the two groups are displayed in the radar chart. Questions 2, 9, and 14 related to confident in natural products have, respectively, 61, 27, and 45% of good answers in parents’ group and 84, 51, and 65% of good answers in PHPs’ group. The highest proportion of correct answers was for the question on cosmetic products (question 4): 94% in the parents’ group and 97% in the PHPs’ group ( p = NS). The lowest proportion of good answers was for the question on EDs mechanisms of action (question 11): 10% for parents and 11% for professionals ( p = NS). Overall, 66% of parents and 95% of PHPs had already heard of ED. This 66% of parents had a significantly better score than the others (67% vs. 53%, p < 0.001); similarly, this 95% of PHPs had a significantly better score (71% vs. 57%, p = 0.002). However, only 10% of parents and 5% of PHPs reported that they felt sufficiently informed, but they did not have a significantly better score than participants who felt insufficiently informed . We have observed that 20% of both parents and PHPs systematically screened the product composition, while 15% never screened it. However, this screening was not linked to the proportion of correct answers. By univariate analysis, factors associated with a higher proportion of correct answers for parents were rural location and higher socio-professional categories. For PHPs, these factors were age under 35 years and being a physician. Information sources were similar between parents and PHPs, the most frequent one being media (television or radio) and the web for 70% of parents and 80% of PHPs; PHPs used mostly “general public” resources. Only 20% of PHPs read scientific papers, and 4% have followed a training on EDs . Discussion Knowledge on EDs is not optimal both for parents and PHPs. The main strengths of this study were a significant number of answers and an evaluation of both parents and PHPs with the same questionnaire. All professionals interacting with parents and children were represented, thus reinforcing the strength of the study. However, PHPs’ knowledge and beliefs were not optimal, and most of them were not trained on EDs. Our rates of responses are good, close, or better than other reports in the literature, i.e., 11–30% . Reasons for obtaining a satisfactory response rate are probably multiple and intricated: the increasing concern about environmental exposures, the availability of researchers for parents with a hand-delivered questionnaire, and the individual mail for the PHPs in a pediatric hospital in which the principal investigator of the study is the senior physician responsible for the pediatric research centre. Some factors were linked to the profile of “good responders.” For parents these were rural locations and higher socio-professional categories. In rural areas, parents and their children are exposed to specific pollutants such as pesticides and information on this type of environmental exposures is probably more spread. Higher socio-professional categories often have better level of education and better access to information. Furthermore, for the lower socio-professional categories, environmental exposures are not a priority in daily life, even though they are the population most exposed. For professionals, the profile of “good responders” was under 35 years of age and being a doctor. Young professionals are more sensitive to environmental exposures and climate change. Good responses from doctor are linked to better answers to beliefs questions (100% for medical doctor vs. 71.4% for other professionals). Even though they have not received a specific training on EDs, the training provided during their medical training has enabled doctors to acquire a critical mind, which may explain their better responses to beliefs questions. This single-center study has nevertheless several limitations, by design. As a result, only professionals and parents from patients followed in a tertiary university hospital participated. One may assume that liberal practitioners are more concerned and informed because they receive more questions from parents, with less severely ill children. Also, responders probably felt more concerned with the questions of environmental exposure than parents and PHPs who did not participate. Therefore, we could expect that responses in general populations will be even worse. In addition, we used a non-validated personal questionnaire. In our study, only 10% of parents and 5% of PHPs considered they are sufficiently informed about EDs. This result is congruent with a previous study reporting that only 11% of perinatal health professionals were sufficiently trained and informed . Another French study also found 82% of insufficiently informed professionals . The lack of specific training for health professionals on the topic has also been reported in many studies worldwide . The worst answers were about “natural” products. In fact, each question with the term “natural” automatically generated an analysis of “healthy” in parents’ mind but also, however in a lesser extent, in PHPs. As described in human health science publications, instructional messages are not an optimal way to inform both parents and PHPs . Dichotomist ideas such as “natural is good” and “industrial is bad” have to be avoided; beliefs must be explored in order to give better messages to the population. Moreover, it may be relevant to analyze in future studies in which state of mind PHPs who are also parents position themselves. More worrying is the way used by professionals to get information, as already descripted in 2001 . There is an urgent need for professionals to be better informed. To achieve this, specific training courses and recommendations from medical scientific societies are required. For example, the International Federation of Gynecology and Obstetric guidelines refer to the presence of heavy metal and EDs in prenatal vitamins and recommends reducing the exposure. These guidelines are easily accessible for the professionals. Conversely, information in the media is often perceived as stressful and incomprehensible by parents . In conclusion, we show that, despite the fact that the risk of environmental exposure and EDs is an increasing concern for parents, the specific knowledge remains scarce both for parents and PHPs. PHPs need to be trained on the topic, so as to provide optimal advice to families. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The studies involving humans were approved by the Ethics Committee of Hospices Civils de Lyon n° 2022022. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. AP: Conceptualization, Methodology, Project administration, Writing – original draft. TL: Data curation, Formal analysis, Writing – review & editing. LT: Investigation, Project administration, Writing – review & editing. GB: Investigation, Software, Writing – review & editing. NV: Investigation, Software, Writing – review & editing. BK: Supervision, Writing – review & editing. JB: Supervision, Writing – review & editing. |
Experimental Study on Noise-Reduced Propagation Characteristics of the Parametric Acoustic Array Field in a Neck Phantom | 10a3924b-69a5-4e7b-a06f-f31e4255323f | 11820233 | Surgical Procedures, Operative[mh] | The electrolarynx (EL) is a critical device for voice reconstruction in patients undergoing total laryngectomy, a surgical procedure that removes the larynx . This device compensates for the loss of vocal cords by using an external sound source, offering benefits like ease of use and the ability to produce continuous speech . Research shows that more than half of laryngectomy patients rely on EL as their primary means of communication within five years post-surgery . The EL functions by placing a vibrating membrane against the neck, allowing the sound produced to travel through surrounding tissues to the pharynx, thereby creating a substitute voice. However, a significant drawback is the mechanical noise (often referred to as “radiation noise”) generated by the device, which can reach 20–25 dB and severely interfere with speech intelligibility . Researchers have employed various strategies to eliminate radiation noise, including physical modifications to the EL and signal processing techniques. For instance, Madden et al. replaced the traditional motor with an eccentric motor, achieving a 20% improvement in speech intelligibility by reducing noise. However, maintaining clear and natural speech quality remains a challenge. Norton and Bernstein wrapped the EL in thick foam, which reduced radiation noise by approximately 5 dB. However, this modification made the device cumbersome and less user-friendly. Other studies have focused on signal processing methods, such as spectral subtraction, voice conversion, and adaptive filtering, which have shown promise in enhancing speech quality by minimizing background noise . Although these techniques enhance the clarity of the output, their lack of real-time performance often makes them unsuitable for spontaneous conversation. The primary source of radiation noise in electrolarynx (EL) devices originates from external sound sources. One potential solution is to relocate the sound source to the oral cavity or pharynx, a method that has been explored by several researchers. For instance, Takahashi et al. mounted a vibrating source on a denture; however, the resulting sound failed to produce natural voice quality. Huang et al. developed a sound-generating device that is placed on the upper jaw, using a 3D-printed speaker holder to secure the speaker to a tooth sleeve. This design allows users to modulate sound frequency and amplitude by adjusting lung pressure and mouth shape. Despite its advantages, the tooth sleeve can interfere with speech production and pose challenges related to stability and hygiene. Moreover, foreign objects in the mouth can further complicate articulation. Painter et al. explored an electromagnetic EL device for implantation in neck tissue, but this approach carries the risks associated with surgical procedures. Parametric acoustic array (PAA) technology offers a promising alternative to address these challenges. This technique, which has been successfully applied in fields such as underwater measurement , underwater communication , and parametric speakers , generates difference-frequency waves from high-frequency sound waves. This capability enables precise and focused sound generation, even in complex environments . Mills et al. demonstrated the feasibility of generating difference frequency signals in soft tissue, highlighting the potential of using PAA for internal voice source reconstruction. While simulations have provided valuable insights, they cannot fully replicate the complexity of real tissue environments. Experimental validation is therefore essential to confirm the feasibility of using PAA in practical applications. To bridge this gap, our study conducts experimental investigations using a tissue-mimicking phantom to explore the feasibility of generating voice sources within the human body through PAA technology. This study aims to experimentally investigate the use of modulated PAA technology to generate voice sources within a tissue-mimicking phantom that replicates the acoustic characteristics of human neck tissue. By comparing generated voice sources with natural voice and traditional EL outputs, this study seeks to establish the effectiveness of PAA technology in voice reconstruction. The findings aim to provide critical insights into the practical application of PAA technology for voice reconstruction in laryngectomy patients, with the potential to significantly improve their quality of life. 2.1. Experimental Platform The experimental platform was designed to investigate the propagation characteristics of the PAA difference-frequency sound field in a neck phantom. The setup comprises two main subsystems: the signal excitation system and the signal acquisition system, with a neck phantom serving as the medium for acoustic wave propagation. The complete experimental setup is illustrated in , which includes a schematic diagram ( a) and a photograph of the physical setup ( b). 2.1.1. Human Neck Tissue-Mimicking Phantom In this study, a polyvinyl alcohol (PVA) material (product No. 563900, Sigma-Aldrich) was used to fabricate an acoustic phantom that simulates human neck tissue. The PVA (molecular weight: 130,000, Sigma-Aldrich, Zwijndrecht, The Netherlands) was prepared by dissolving 20% wt PVA in a mixture of 80% wt dimethyl sulfoxide (DMSO, Sigma-Aldrich, Zwijndrecht, The Netherlands) and 20% wt Milli-Q water. The solution underwent a series of freezing and thawing cycles to achieve the desired acoustic properties, which were designed to approximate those of human tissue (speed of sound: 1616 m/s, attenuation coefficient: 1.69 dB/cm, B/A value: 11.7) . The phantom was shaped as a hollow cylinder, with a flat surface on the front for precise placement of the ultrasonic transducer. The height of the phantom was approximately 10 cm, and the outer diameter of the cylinder was designed to match the diameter of a human neck. The diameter of the central hole was chosen to resemble the size of the lower human vocal tract. Additionally, the thickness from the inner hole to the flat surface was made to approximate the thickness of human neck tissue . A circular hole, approximately 1 cm in diameter, was added to the rear of the phantom to allow the insertion of a microphone for measurement. 2.1.2. Signal Excitation System The signal excitation system was designed to generate stable ultrasonic signals. The excitation signal was produced by an arbitrary waveform generator (Analog Discovery 3, Digilent, WA, USA) and then amplified by a power amplifier (ATA3040, Aigtek, Xi’an, China) to drive the ultrasonic transducer (H2KA050 KA1CD00, Unictron, Taiwan). The transducer, with a center frequency of 50 kHz and a diameter of 5 cm, was positioned at the middle of the flat surface of the phantom, ensuring optimal transmission of acoustic waves into the medium. b illustrates the physical setup of the transducer and phantom. 2.2. Experimental Procedure The experiments were conducted in a quiet room to minimize external noise interference. The signal excitation system transmitted an excitation signal with a peak-to-peak voltage of 90 V, driving the ultrasonic transducer to emit ultrasound waves, which propagated through the neck phantom. The signal acquisition system captured 10 s of continuous audio data at a sampling rate of 204.8 kHz with 24-bit resolution. To assess the impact of the phantom’s acoustic properties, measurements were also taken in air after the phantom was removed. All measurements were repeated three times for reliability. Subsequently, the ultrasonic transducer was replaced with a commercial EL, and the same acquisition conditions were used to capture the sound emitted by the EL as it propagated through the phantom. 2.3. Excitation Signal The parametric array excitation signal used in this study was generated using the Amplitude Modulation (AM) method , as described by the following equation: (1) S t = ( A + m ( t ) ) × c o s ( 2 π f c t ) , where m ( t ) represents the envelope signal, s ( t ) is the resulting modulated signal, A is the amplitude of the carrier signal, and c o s ( 2 π f c t ) is the carrier signal with frequency f c . The envelope signal was generated using the Liljencrants–Fant (LF) glottal waveform model, which accurately simulates human glottal airflow during phonation . The glottal waveform frequency was set to 200 Hz, with a carrier frequency of 50 kHz corresponding to the central frequency of the ultrasonic transducer. The modulation depth was set to 100%, resulting in full modulation of the carrier signal by the glottal waveform. illustrates the predefined glottal waveform signal and the corresponding AM-modulated excitation signal. 2.4. Signal Processing and Parameter Evaluation The collected sound signals were subjected to a finite impulse response (FIR) band-pass filter with a passband of 20 Hz to 1000 Hz to extract the difference frequency signals. The extracted signal waveform was compared to the preset glottal waveform in the time domain. To quantify the similarity between the two signals, the Pearson correlation coefficient (r) was calculated. This was performed using MATLAB, with r computed based on the covariance of the variables normalized by their standard deviations: (2) r = ∑ x i − x ¯ y i − y ¯ ∑ x i − x ¯ 2 ∑ y i − y ¯ 2 where x i and y i represent the measured values of AS/TMS and LFS, respectively, and x ¯ and y ¯ are the mean values of AS/TMS and LFS. Additionally, the autoregressive (AR) power spectral density of the acquired signals was calculated using the AR Burg method, with an order of 190 chosen for its stability and accuracy in spectral estimation. The sound pressure levels (SPLs) recorded by the external and internal microphones were used to evaluate the intensity of the difference frequency signal and quantify radiation noise. The SPL difference (Δ L ) between the external microphone, which measures radiation noise, and the internal microphone, which measures the sound inside the phantom, serves as an indicator of noise leakage. A larger Δ L indicates less radiation noise. The SPL difference was calculated using the following formula: (3) ∆ L = S P L e x t − S P L i n t , To assess the statistical significance of the SPL differences between the two excitation sources (EL and PAA), a paired t -test was performed using a significance level of p < 0.001 . This stricter threshold was chosen to ensure robust conclusions in the context of this study. The experimental platform was designed to investigate the propagation characteristics of the PAA difference-frequency sound field in a neck phantom. The setup comprises two main subsystems: the signal excitation system and the signal acquisition system, with a neck phantom serving as the medium for acoustic wave propagation. The complete experimental setup is illustrated in , which includes a schematic diagram ( a) and a photograph of the physical setup ( b). 2.1.1. Human Neck Tissue-Mimicking Phantom In this study, a polyvinyl alcohol (PVA) material (product No. 563900, Sigma-Aldrich) was used to fabricate an acoustic phantom that simulates human neck tissue. The PVA (molecular weight: 130,000, Sigma-Aldrich, Zwijndrecht, The Netherlands) was prepared by dissolving 20% wt PVA in a mixture of 80% wt dimethyl sulfoxide (DMSO, Sigma-Aldrich, Zwijndrecht, The Netherlands) and 20% wt Milli-Q water. The solution underwent a series of freezing and thawing cycles to achieve the desired acoustic properties, which were designed to approximate those of human tissue (speed of sound: 1616 m/s, attenuation coefficient: 1.69 dB/cm, B/A value: 11.7) . The phantom was shaped as a hollow cylinder, with a flat surface on the front for precise placement of the ultrasonic transducer. The height of the phantom was approximately 10 cm, and the outer diameter of the cylinder was designed to match the diameter of a human neck. The diameter of the central hole was chosen to resemble the size of the lower human vocal tract. Additionally, the thickness from the inner hole to the flat surface was made to approximate the thickness of human neck tissue . A circular hole, approximately 1 cm in diameter, was added to the rear of the phantom to allow the insertion of a microphone for measurement. 2.1.2. Signal Excitation System The signal excitation system was designed to generate stable ultrasonic signals. The excitation signal was produced by an arbitrary waveform generator (Analog Discovery 3, Digilent, WA, USA) and then amplified by a power amplifier (ATA3040, Aigtek, Xi’an, China) to drive the ultrasonic transducer (H2KA050 KA1CD00, Unictron, Taiwan). The transducer, with a center frequency of 50 kHz and a diameter of 5 cm, was positioned at the middle of the flat surface of the phantom, ensuring optimal transmission of acoustic waves into the medium. b illustrates the physical setup of the transducer and phantom. In this study, a polyvinyl alcohol (PVA) material (product No. 563900, Sigma-Aldrich) was used to fabricate an acoustic phantom that simulates human neck tissue. The PVA (molecular weight: 130,000, Sigma-Aldrich, Zwijndrecht, The Netherlands) was prepared by dissolving 20% wt PVA in a mixture of 80% wt dimethyl sulfoxide (DMSO, Sigma-Aldrich, Zwijndrecht, The Netherlands) and 20% wt Milli-Q water. The solution underwent a series of freezing and thawing cycles to achieve the desired acoustic properties, which were designed to approximate those of human tissue (speed of sound: 1616 m/s, attenuation coefficient: 1.69 dB/cm, B/A value: 11.7) . The phantom was shaped as a hollow cylinder, with a flat surface on the front for precise placement of the ultrasonic transducer. The height of the phantom was approximately 10 cm, and the outer diameter of the cylinder was designed to match the diameter of a human neck. The diameter of the central hole was chosen to resemble the size of the lower human vocal tract. Additionally, the thickness from the inner hole to the flat surface was made to approximate the thickness of human neck tissue . A circular hole, approximately 1 cm in diameter, was added to the rear of the phantom to allow the insertion of a microphone for measurement. The signal excitation system was designed to generate stable ultrasonic signals. The excitation signal was produced by an arbitrary waveform generator (Analog Discovery 3, Digilent, WA, USA) and then amplified by a power amplifier (ATA3040, Aigtek, Xi’an, China) to drive the ultrasonic transducer (H2KA050 KA1CD00, Unictron, Taiwan). The transducer, with a center frequency of 50 kHz and a diameter of 5 cm, was positioned at the middle of the flat surface of the phantom, ensuring optimal transmission of acoustic waves into the medium. b illustrates the physical setup of the transducer and phantom. The experiments were conducted in a quiet room to minimize external noise interference. The signal excitation system transmitted an excitation signal with a peak-to-peak voltage of 90 V, driving the ultrasonic transducer to emit ultrasound waves, which propagated through the neck phantom. The signal acquisition system captured 10 s of continuous audio data at a sampling rate of 204.8 kHz with 24-bit resolution. To assess the impact of the phantom’s acoustic properties, measurements were also taken in air after the phantom was removed. All measurements were repeated three times for reliability. Subsequently, the ultrasonic transducer was replaced with a commercial EL, and the same acquisition conditions were used to capture the sound emitted by the EL as it propagated through the phantom. The parametric array excitation signal used in this study was generated using the Amplitude Modulation (AM) method , as described by the following equation: (1) S t = ( A + m ( t ) ) × c o s ( 2 π f c t ) , where m ( t ) represents the envelope signal, s ( t ) is the resulting modulated signal, A is the amplitude of the carrier signal, and c o s ( 2 π f c t ) is the carrier signal with frequency f c . The envelope signal was generated using the Liljencrants–Fant (LF) glottal waveform model, which accurately simulates human glottal airflow during phonation . The glottal waveform frequency was set to 200 Hz, with a carrier frequency of 50 kHz corresponding to the central frequency of the ultrasonic transducer. The modulation depth was set to 100%, resulting in full modulation of the carrier signal by the glottal waveform. illustrates the predefined glottal waveform signal and the corresponding AM-modulated excitation signal. The collected sound signals were subjected to a finite impulse response (FIR) band-pass filter with a passband of 20 Hz to 1000 Hz to extract the difference frequency signals. The extracted signal waveform was compared to the preset glottal waveform in the time domain. To quantify the similarity between the two signals, the Pearson correlation coefficient (r) was calculated. This was performed using MATLAB, with r computed based on the covariance of the variables normalized by their standard deviations: (2) r = ∑ x i − x ¯ y i − y ¯ ∑ x i − x ¯ 2 ∑ y i − y ¯ 2 where x i and y i represent the measured values of AS/TMS and LFS, respectively, and x ¯ and y ¯ are the mean values of AS/TMS and LFS. Additionally, the autoregressive (AR) power spectral density of the acquired signals was calculated using the AR Burg method, with an order of 190 chosen for its stability and accuracy in spectral estimation. The sound pressure levels (SPLs) recorded by the external and internal microphones were used to evaluate the intensity of the difference frequency signal and quantify radiation noise. The SPL difference (Δ L ) between the external microphone, which measures radiation noise, and the internal microphone, which measures the sound inside the phantom, serves as an indicator of noise leakage. A larger Δ L indicates less radiation noise. The SPL difference was calculated using the following formula: (3) ∆ L = S P L e x t − S P L i n t , To assess the statistical significance of the SPL differences between the two excitation sources (EL and PAA), a paired t -test was performed using a significance level of p < 0.001 . This stricter threshold was chosen to ensure robust conclusions in the context of this study. 3.1. Time-Domain Analysis The waveforms of the difference-frequency glottal wave obtained after excitation by the parametric array are shown in . a illustrates the waveform for five periods, with the first row representing the glottal wave obtained through the LF model calculation (envelope signal, LFS). The second and third rows show the difference-frequency signal waveforms captured at the transducer’s axial position in air (air signal, AS) and after propagation through the tissue-mimicking phantom (tissue-mimicking signal, TMS), respectively. From the time-domain signals, it can be observed that the modulated glottal wave signal, after propagating through both the air and tissue-mimicking media, retains a periodicity corresponding to the fundamental frequency (F0) of the original glottal wave signal. The restored waveforms closely resemble the original glottal wave signal. Pearson correlation analysis revealed a correlation coefficient of 0.9767 between AS and LFS, and 0.9438 between TMS and LFS. Compared to the LFS waveform, the difference-frequency waveforms (AS and TMS) show some distortion in their waveform shapes. To further analyze the differences, one period from the LFS, AS, and TMS waveforms, as well as the corresponding EL signal, were normalized and aligned by their signal peaks, as shown in b. The analysis indicates that the peak of all three waveforms occurs at approximately the 50% point of the signal period. However, compared to LFS, the rise time of AS and TMS is steeper. The trough of the LFS signal is stable, without high-frequency noise or unwanted frequency components. On the right side of the peak, at approximately 70% of the signal period, a second sharp peak appears in both AS and TMS signals, with the TMS signal showing a more pronounced peak. 3.2. Frequency-Domain Analysis The Burg AR power spectral density (PSD) curves of LFS, AS, and TMS signals are shown in . All three signals exhibit a dominant peak at the F0, with harmonic components appearing at integer multiples of the F0 (200 Hz, 400 Hz, 600 Hz, etc.). The energy of the F0 is the highest, while the harmonic components decrease in amplitude as the frequency increases. The primary energy is concentrated around 200 Hz, with harmonic components becoming progressively weaker at higher frequencies. The fourth harmonic in the TMS signal is notably smaller compared to the AS signal. The frequency spectra of AS and TMS signals show slight attenuation of higher harmonics compared to the LFS signal, with the TMS signal exhibiting more pronounced attenuation in the higher frequency range. 3.3. Radiation Noise Analysis As shown in a, the sound pressure level (SPL) of radiation noise measured with the EL as the excitation source was 81.20 dB (SD: 0.36 dB). In contrast, when using the PAA as the excitation source, the SPL of radiation noise was significantly lower at 24 dB (SD: 0.16 dB), with a difference that was statistically significant ( p < 0.0001). b illustrates the Δ L between the external and internal microphones. For the EL, the SPL difference was −16 dB (SD: 0.34 dB), whereas for the PAA, the SPL difference was −23 dB (SD: 0.15 dB). The SPL difference for the PAA was significantly higher than that for the EL ( p < 0.0001), indicating that less radiation noise was leaking outside the phantom when using the PAA. The waveforms of the difference-frequency glottal wave obtained after excitation by the parametric array are shown in . a illustrates the waveform for five periods, with the first row representing the glottal wave obtained through the LF model calculation (envelope signal, LFS). The second and third rows show the difference-frequency signal waveforms captured at the transducer’s axial position in air (air signal, AS) and after propagation through the tissue-mimicking phantom (tissue-mimicking signal, TMS), respectively. From the time-domain signals, it can be observed that the modulated glottal wave signal, after propagating through both the air and tissue-mimicking media, retains a periodicity corresponding to the fundamental frequency (F0) of the original glottal wave signal. The restored waveforms closely resemble the original glottal wave signal. Pearson correlation analysis revealed a correlation coefficient of 0.9767 between AS and LFS, and 0.9438 between TMS and LFS. Compared to the LFS waveform, the difference-frequency waveforms (AS and TMS) show some distortion in their waveform shapes. To further analyze the differences, one period from the LFS, AS, and TMS waveforms, as well as the corresponding EL signal, were normalized and aligned by their signal peaks, as shown in b. The analysis indicates that the peak of all three waveforms occurs at approximately the 50% point of the signal period. However, compared to LFS, the rise time of AS and TMS is steeper. The trough of the LFS signal is stable, without high-frequency noise or unwanted frequency components. On the right side of the peak, at approximately 70% of the signal period, a second sharp peak appears in both AS and TMS signals, with the TMS signal showing a more pronounced peak. The Burg AR power spectral density (PSD) curves of LFS, AS, and TMS signals are shown in . All three signals exhibit a dominant peak at the F0, with harmonic components appearing at integer multiples of the F0 (200 Hz, 400 Hz, 600 Hz, etc.). The energy of the F0 is the highest, while the harmonic components decrease in amplitude as the frequency increases. The primary energy is concentrated around 200 Hz, with harmonic components becoming progressively weaker at higher frequencies. The fourth harmonic in the TMS signal is notably smaller compared to the AS signal. The frequency spectra of AS and TMS signals show slight attenuation of higher harmonics compared to the LFS signal, with the TMS signal exhibiting more pronounced attenuation in the higher frequency range. As shown in a, the sound pressure level (SPL) of radiation noise measured with the EL as the excitation source was 81.20 dB (SD: 0.36 dB). In contrast, when using the PAA as the excitation source, the SPL of radiation noise was significantly lower at 24 dB (SD: 0.16 dB), with a difference that was statistically significant ( p < 0.0001). b illustrates the Δ L between the external and internal microphones. For the EL, the SPL difference was −16 dB (SD: 0.34 dB), whereas for the PAA, the SPL difference was −23 dB (SD: 0.15 dB). The SPL difference for the PAA was significantly higher than that for the EL ( p < 0.0001), indicating that less radiation noise was leaking outside the phantom when using the PAA. This study experimentally investigates the propagation of the PAA sound field through tissue-mimicking media to evaluate its potential for reconstructing glottal waveforms. The results confirm that the PAA effectively generates low-frequency difference waves within the tissue-mimicking media, retaining the envelope characteristics of the modulated signal after propagation. Notably, these difference-frequency waves, when transmitted through the medium, exhibit a higher degree of similarity to the human glottal waveform than the excitation signal generated by the EL. This finding demonstrates the feasibility of using the PAA to reconstruct glottal waveforms, offering a potential advantage over traditional EL devices, which produce signals that are significantly different from natural human phonation. The time–domain analysis revealed that the PAA emitted modulated glottal waveforms (LF model) maintaining periodicity with the same fundamental frequency (200 Hz) as the pre-defined signal, regardless of whether they propagated through air or tissue-mimicking media. The waveforms produced by PAA showed a high degree of similarity to the original glottal waveform, as evidenced by the Pearson correlation coefficients exceeding 0.9. In contrast, the waveform produced by the EL resembled a sharp, impulsive signal, rather than a smoothly modulated glottal waveform. This finding aligns with the simulations reported by Mills et al. but differs in that Mills’ approach used a frequency difference method with two transducers, which could only generate sine waves corresponding to the frequency difference between two excitation signals. This study, however, utilized a single transducer to modulate the excitation signal, allowing for more versatile waveform generation and yielding a signal closer to natural human phonation. Furthermore, the use of one transducer simplifies the design of future devices, making the system more portable and cost-effective. When comparing the LFS, AS, and TMS waveforms, the peak-to-peak value of the TMS waveform was significantly smaller than that of the AS waveform, possibly due to stronger attenuation of the sound by the tissue-mimicking medium. Additionally, the rise and fall of the TMS waveform were less smooth compared to the LFS waveform, with added peaks and valleys, which could be attributed to the harmonic components present in the original glottal signal. These harmonic components experienced some loss during propagation through the tissue-mimicking medium, a phenomenon that became more pronounced as the medium’s nonlinearity increased. This was further corroborated by the power spectral analysis. In the frequency domain, the AR power spectral density analysis indicated that, in addition to the F0, the LF model waveform contained several harmonic components. After propagation through both air and tissue-mimicking media, the parametric array sound field retained these harmonic components, with their energy gradually decreasing as the frequency increased, similar to the energy distribution observed in the original LF model. When compared to the EL-generated sound field, the PAA signal exhibited a frequency spectrum that more closely matched that of the LF model, emphasizing its potential to more accurately simulate human phonation. A key aspect of this study was the evaluation of radiation noise produced by the two excitation sources, using an external microphone placed behind the tissue-mimicking medium. The results presented in indicate a significant difference in radiation noise between the EL and the PAA. The SPL of the radiation noise generated by the PAA was substantially lower than that of the EL, owing to the directional nature of the PAA. Additionally, the simplified neck phantom used in this study may have led to some sound leakage at the openings of the trachea. In a more realistic human model, where the trachea is a closed tube, we would expect even lower radiation noise. Moreover, due to the low energy of the difference-frequency sound generated by the PAA, any leaked radiation noise would likely be imperceptible to the listener. While this study demonstrates the potential of the PAA for reconstructing glottal waveforms and reducing radiation noise, the glottal waveform frequency obtained (200 Hz) remains below the typical fundamental frequency range of normal human speech (60–500 Hz). This limitation is likely due to the output power of the transducer and the modulation method used. Future research should focus on enhancing the output power and improving the conversion efficiency of the difference-frequency signal to achieve a broader frequency range that better matches the natural human voice. Furthermore, the tissue-mimicking phantom in this study does not fully replicate the complex structure of human tissues, particularly regarding their nonlinear acoustic properties. Future studies should explore more anatomically accurate phantoms or in vivo experiments to further validate the feasibility of the parametric acoustic array for practical clinical applications, such as in improving the quality and naturalness of speech in patients with laryngectomy. The selection of a 5 cm diameter and a 50 kHz center frequency for the transducer in this study was driven by the specific requirements of PAA technology in generating glottal waves within the human body. The diameter was chosen to accommodate the limited area of the human neck while maximizing the energy output of the difference-frequency wave, as the emitting area directly influences the efficiency of PAA energy conversion. Similarly, the 50 kHz center frequency was selected to balance energy conversion efficiency and directivity, with lower frequencies providing higher efficiency but suffering from poor directivity. While the experimental results confirm the feasibility of generating glottal waves within the phantom, further optimization of transducer parameters remains an essential avenue for improving conversion efficiency. Future work will focus on refining transducer size and center frequency, exploring advanced modulation methods, and employing focused transducers to enhance power density at specific target locations, ensuring a stronger and more localized difference-frequency sound field for applications in the human body. This study provides compelling evidence that PAA technology is capable of reconstructing glottal waveforms with higher fidelity than traditional EL devices, offering significant advantages in both waveform accuracy and radiation noise reduction. Comparative analysis demonstrated a high degree of similarity between the PAA-generated signals and the model glottal waveforms. Additionally, the autoregressive spectral analysis confirmed that the PAA accurately reproduces essential spectral features of the glottal waveform, further supporting its potential for voice rehabilitation. The use of a single transducer to generate modulated signals makes this method more efficient and practical for future speech rehabilitation technologies. However, improvements in signal strength, frequency range, and system design are necessary to fully meet the demands of natural human speech. Future work will focus on optimizing transducer parameters, exploring advanced modulation techniques, and conducting clinical evaluations to ensure effective translation of this technology into practical applications. This study lays a strong foundation for advancing voice restoration solutions, with the potential to significantly improve the quality of life for individuals with total laryngectomy. |
New Antibiotics for Multidrug-Resistant Bacterial Strains: Latest Research Developments and Future Perspectives | e423df1d-b45d-4add-b895-390e4e59f470 | 8125338 | Pharmacology[mh] | Discoverer of penicillin Alexander Fleming, in December 1945, during his acceptance speech of the Nobel Prize in Medicine, announced the risk of the inevitable phenomenon of antibiotic resistance, already observed in laboratories, with the following words: “ it’s not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them…there is the danger that the ignorant man may easily under-dose himself and, by exposing his microbes to non-lethal quantities of the drug, make them resistant .” (A. Fleming, Penicillin, Nobel Lecture, 11 December 1945) Fleming’s predictions turned out to be accurate: The incorrect use, sometimes real abuse, of antibiotics, speeds up the development and spread of bacteria resistant to them. Considering the penicillin as an example, if bacteria are subjected to “non-lethal levels” of the antibiotic, they can use it as a signaling with regulatory functions. Bacteria can release β-lactamase enzymes, that hydrolyze the amide bond of the four-membered β-lactam ring resulting in the inactivation of the β-lactam antibiotic. The reported case is just one of the many defense mechanisms that bacteria have against antibiotics. Antibiotics have undoubtedly been a milestone in the history of humanity and modern medicine; they are an indispensable and life-saving weapon against numerous infectious diseases, including the ones associated with organ transplants, cancer chemotherapies, and intensive therapies. In the last century, research has produced many new antibiotics; however, since the 1990s, the number of antimicrobial agents discovered has been in sharp decline, with a simultaneous and worrying increase in the phenomenon of antibiotic resistance. Bacteria showing resistance to at least three different classes of antimicrobials, defined as multidrug resistant (MDR), have become common, especially in hospitals; there is a risk of entering a so-called “post-antibiotic era” in a few years, in which infections apparently under control easily turn into lethal threats. It is evident to everyone that antibiotic resistance is one of the main health problems nowadays, with a strong impact both clinically and economically. Pathogens such as methicillin-resistant Staphilococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) have become extremely difficult to eradicate. It is estimated that each year, more than 2.8 million people in the United States alone contract an infection resistant to traditional antibiotics, causing more than 35,000 deaths . In Europe, antibiotic resistance is responsible for about 33,000 deaths per year . Globally, pneumonia and blood infections that cause sepsis contribute heavily to infant mortality in the first five years of life. Approximately 30% of newborns with sepsis die from bacterial infections resistant to traditional antibiotics . In 2016, the World Health Organization (WHO) published a list of the world’s leading antibiotic-resistant bacteria, for which there is an urgent demand for new treatments . The aim is certainly to help countries accelerate national surveillance, control, and research activities for new active ingredients. The list is divided into three categories, each describing the risk associated with the antibiotic-resistant bacterial species: Critical, high, and medium. Mycobacterium (including M. tuberculosis , responsible for 1.8 million deaths per year worldwide) has not been included in this list because it is a long-established threat. Note that Gram-negative bacteria pose a looming danger. Pathogens of the genera Acinetobacter , Pseudomonas , and Enterobacteriaceae (including Klebsiella pneumoniae , Escherichia coli , Enterobacter spp., Serratia spp., Proteus spp., Providencia spp., and Morganella spp.) are the most feared in hospitals, nursing homes, and aged care facilities, where related infections can be lethal. These pathogens have already developed resistance to carbapenems, which are extremely powerful antibiotics, often used as life-saving drugs in hospitalized patients. In fact, the rapid increase in the number of infections caused by carbapenem-resistant Enterobacteriaceae (CRE), which produce carbapenemases (especially K. pneumoniae carbapenemase) capable of hydrolyzing and inactivating carbapenems and β-lactams, is alarming. Of high priority are VRE, MRSA, Helicobacter pylori (the first risk factor for stomach cancer), Campylobacter spp. (responsible for the highest number of food contaminations in Europe), Salmonella spp. (food poisoning), and Neisseria gonorrhoeae (causes gonorrhea, a sexually transmitted disease). Finally, Streptococcus pneumoniae (responsible for the majority of community-acquired pneumonia), Haemophilus influenzae (related to respiratory infections), and Shigella spp. (transmitted through water or foods contaminated with feces, causes dysentery) are placed in the category of medium priority. In addition, it should be pointed out that the potential spread of resistant organisms also has a negative impact on the health of subjects not directly exposed to certain antibiotics. Faced with this scenario, which is no longer science fiction but a reality, there is an urgent need for new antibacterial active ingredients in order to ensure effective treatments against infections resistant to traditional antibiotics. In recent years, awareness of the issue of antibiotic resistance has increased, including in the political field: In 2017, the G20 countries decided to intensify global collaboration on this issue to stimulate the R&D of antimicrobial molecules, also starting from existing antibiotics. Since 2017, eight new antibiotics have been approved by the FDA, including one for the treatment of multidrug-resistant tuberculosis: Most of these drugs were developed from traditional molecules and target Enterobacteriaceae resistant to carbapenems and other pathogens considered dangerous by WHO . The following paper examines from a chemical and clinical point of view the most promising new compounds still under preclinical and clinical investigation, including the active ingredients that entered Phase III last year and the agents of pharmacological interest that gained market authorization between 2017 and 2020. The latest results of the research are shown together with the strategies necessary to stem the problem of antibiotic resistance, concluding with perspectives for the future.
Antibiotic resistance threatens modern medicine, and above all the effectiveness of a decisive and prompt global health response to infectious diseases, due to systematic abuse and excessive use of antibiotics in human medicine and food production. Indeed, the massive or inappropriate use of such drugs in humans, animals or agriculture results in the emergence of drug-resistant microorganisms evolved under this strong selective pressure. In 2015, aware of the huge problem of antibiotic resistance, the WHO decided to adopt the Global Action Plan on Antimicrobial Resistance, based on five strict objectives: To improve awareness and understanding of antimicrobial resistance; to strengthen knowledge and the amount of data; to reduce the incidence of infections through effective hygiene measures; to optimize the use of antimicrobial drugs in human and animal health; and to increase investment in new drugs, diagnostic tools, vaccines, and other interventions . In addition to the WHO, there are other associations such as the Food and Agriculture Organization of the United Nations and the World Organization for Animal Health that give ample space to the fight against antibiotic resistance. The use of antibiotics in veterinary medicine is extremely important: It is necessary to strengthen the regulatory system for medicated food and feed, mainly used in intensive farming, in order to prevent the onset of infections due to the large number of animals raised in situations of confinement. To this end, the surveillance and monitoring systems for resistant bacteria and the indiscriminate use of antibiotics have multiplied, not only in human medicine, but also in veterinary. In general, it is good practice to avoid the repeated use of the same molecule and to increase patient compliance with correct drug dosages and timing. Regarding this, in 2013, the European Centre for Disease Prevention and Control (ECDC) published a paper reviewing procedures and guidelines to improve the compliance of health professionals with regard to the timing, dosage, and duration of peri-operative antibiotic prophylaxis for the prevention of infections in surgical rooms . New molecules are therefore vital to overcoming the resistances that have developed as well as the need to empower the use of existing antibiotics and to promote the study of increasingly valid diagnostic tests for the identification of resistant bacteria and for determining antibiotic sensitivity.
Bacteria are able to inactivate antibiotics through numerous molecular mechanisms : (a) Production of inactivating enzymes: The antibiotic loses its biological activity as it is precisely inactivated by specific enzymes produced by the bacterium. This happens, for example, in the case of β-lactam antibiotics that are hydrolyzed by β-lactamases. Enterobacter spp. produce extended-spectrum β-lactamases (ESBL) with the same inactivating function, becoming difficult to eradicate. Other enzymes capable of inactivating certain antibiotics are acetyltransferase, phosphotransferase, and adenyltransferase. (b) Changes and alterations in the antibiotic target: This happens, for example, in resistance to erythromycin, wherein the methylation of an adenine residue in the peptidyl-transferase of r-RNA 23S decreases its affinity for the antibiotic without damaging protein synthesis. Another important case is the modification of penicillin binding proteins (PBPs) by MRSA. (c) Reduced cellular permeability: The penetration of an antibiotic can be reduced by structural changes in the cell’s surface casings. In Gram-negatives, the resistance may be due to an alteration or quantitative decrease in porines, or proteins through which many antibiotics penetrate. They delay the incoming flow of numerous antibiotics thanks to different mechanisms that include limitation in relation to molecular size, hydrophobicity, and charge repulsion, thus contributing to the intrinsic resistance of many microorganisms. This is the case for Pseudomonas aeruginosa , which shows resistance to imipenem. (d) Increased outflow: Antibiotics taken-up into bacteria cell are removed by energy-driven drug efflux systems. Activation of alternative metabolic pathways: The case of sulfamidics is explanatory. Bacteria treated with sulfamidics, in fact, still manage to synthesize folic acid through alternative metabolic pathways.
Currently, the search for new antimicrobial active ingredients is largely led by small- and medium-sized enterprises, as large pharmaceutical companies continue to abandon such projects. In fact, the return on investment for antibiotics that have been marketed in recent decades has been rather negative. In 2011, an analysis entitled “Challenges of Antibacterial Discovery”, published in the journal American Society for Microbiology , referred to a “discovery void” that had persisted since 1987 without significant patents or advances . Unfortunately, it is difficult to obtain incentives to develop and study new antibiotics, for various reasons. Many of the molecules selected in the laboratory that are directed against enzyme targets quickly end up developing resistance in the pathogens examined. An active substance is considered innovative if it does not show the phenomenon of cross-resistance to existing antibiotics. In this context, cross-resistance is defined as resistance within the same class of antibiotics, which can be quantified by systematic, in vitro susceptibility tests to genetically determined pathogens. If sufficient information on cross-resistance is absent or not available, an active substance is considered innovative if it belongs to a new class of antibiotics (new scaffold or pharmacophore), if it has a new target or binding site that has never been present before, or if it shows a new mechanism of action. Antibiotics discovered in the so-called “golden age” of the last century were generally very complex natural products with numerous molecular targets, so the phenomenon of resistance was less common during clinical trials. The dosages of the most widely used antibiotics are usually in the range of hundreds of milligrams per day, so it is necessary that these active ingredients be extremely selective for bacterial targets in order to avoid toxic effects for the patient. However, some newly discovered molecules, active against multidrug-resistant bacteria, are not selective and can consequently cause important side effects. For example, three antibiotics, telithromycin, temafloxacin, and trovofloxacin, which were approved in the 1990s and early 2000s, were removed from the market due to serious adverse reactions, as they were not selective . In addition, new antibiotics are usually administered in the hospital as a “last resort” in patients with complicated and multidrug-resistant infections not treatable with traditional antibiotics. It is therefore not difficult to understand why pharmaceutical companies prefer to avoid investing in the development of new antibiotics. However, there are small start-ups that propose new approaches and a greater commitment to R&D. In addition, research involving public investments aimed at the development of new antibiotics has increased in recent years: The Global Antibiotic Research and Development Partnership was created in 2016. Aware of the need to ensure the availability of antibiotics even for patients undergoing chemotherapy or organ transplants, many countries around the world are implementing different initiatives to stimulate the research of innovative antibiotics. The new findings are not powerful enough weapons to combat the current challenges of antibiotic resistance, but it can be interesting to discuss the latest developments and highlight the compounds that appear most significant according to clinical studies. The current framework for pharmaceutical research and the development of new antimicrobial drugs is outlined by two 2020 reports: “Antibacterial agents in clinical development: An analysis of the antibacterial clinical development pipeline” and “Antibacterial agents in preclinical development” , both compiled by the WHO’s Antibacterial Resistance Division. Eight new antibacterial active ingredients, including one for the treatment of tuberculosis, have been approved since 2017. Pretomanid, an agent against multidrug-resistant tuberculosis, was developed by the non-profit organization TB Alliance. About half of the newly approved antibiotics target the carbapenem-resistant Enterobacteriaceae (CRE), oxacillinase-48-producing Enterobacteriaceae (OXA-48), and β-lactamase-producing Enterobacteriaceae (ESBL). Sixty products are in clinical development (as of 2020), including ten biological drugs. Among these different products under evaluation, 32 antibiotics are active against the most dangerous pathogens included in the WHO’s 2016 list (WHO priority pathogens), and many of them consist of combinations of new β-lactams and β-lactam inhibitors. Twelve antibiotics in clinical development target at least one of the critical Gram-negative pathogens. Antibiotics are still unable to treat carbapenem-resistant Acinetobacter baumannii and P. aeruginosa , even though the research on agents against tuberculosis and Clostridium difficile has made considerable progress . Since 2019, the inhalation formulation of murepavadin (a polypeptide antibiotic), whose clinical trial regarding the intravenous formulation had been discontinued due to suspected nephrotoxicity, has been under development . Murepavadin is the only potential treatment against Gram-negative bacteria that can meet all of the criteria of innovation, including the absence of cross-resistance within the same class of antibiotics. However, if a compound does not meet all the criteria of innovation, it does not necessarily mean that it lacks therapeutic utility for particular categories of patients. Since the 2018 update, many new compounds have entered Phase I of clinical development. The two new oral inhibitors of topoisomerase (zoliflodacin and gepotidacin) have successfully passed Phase II clinical trials, entering Phase III. Lefamulin (new pleuromotilin) and the combination relebactam/imipenem/cilastatin have been approved by the FDA. In addition, worth mentioning is the approval of cefiderocol, a β-lactam antibiotic active against the three critical priority pathogens, by the FDA for complicated urinary tract infections. The largest proportion of Phase III antibiotics come from existing classes, especially β-lactams, fluoroquinolones, macrolides, oxazolidinones, and topoisomerase inhibitors. The most promising compounds are examined below. 4.1. Zoliflodacin in the Treatment of Multidrug-Resistant N. gonorrhoeae The bacterium N. gonorrhoeae , resistant to third-generation cephalosporins and fluoroquinolones, is included in the category of high priority pathogens: There is an urgent need for new antibiotics that can overcome such resistance. N. gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted disease that in women can remain asymptomatic for long periods but which is responsible, in severe cases, for severe complications such as infertility, ectopic pregnancies, and neonatal blindness. In recent years, infections with N. gonorrhoeae resistant to penicillin and cephalosporins such as cefixime and ceftriaxone (usually used as the last therapy available in combination with azithromycin) have increased disproportionately. A recent report from the CDC—Centers for Disease Control and Prevention—documented over 500,000 new cases of gonorrhea in the United States during 2018 . Since 2019, the compound zoliflodacin is in Phase III for the treatment of multidrug-resistant N. gonorrhoeae , developed by Entasis Therapeutics in collaboration with the Global Antibiotic Research Development Program . It is the first synthesized antibiotic belonging to the class of spiropyrimidinetrions. It has a unique mechanism of action: It inhibits type II bacterial topoisomerase by binding to a different site than that of fluoroquinolones. The minimal inhibitory concentration (MIC) value together with pharmacokinetic parameters are regarded to have the greatest importance in the optimization of targeted antibiotic therapy . The MIC50 provides the so-called “intrinsic activity” of an antimicrobial, while the MIC90, which is calculated on the basis of larger, inter-center studies, is a reflection of different resistance mechanisms of the species under investigation. Zoliflodacin shows a very low resistance frequency and is active not only against multidrug-resistant N. gonorrhoeae with a MIC between 0.002 and 0.25 μg/mL but also against some troublesome Gram-positive and Gram-negative bacteria. From a chemical point of view, the formula of zoliflodacin is based on a new benzisoxazole scaffold and contains the pyrimidinetrione spirocyclic pharmacophore, which gives its name to this innovative class of antibiotics. The structure activity relationship (SAR) of this molecule was developed using in vitro tests showing the mechanism of inhibition of DNA gyrase and antibacterial activity. Several compounds in the benzisoxazole series have good activity against quinolone-resistant pathogens, including S. aureus , S. pneumoniae , and H. influenzae . The insertion of a substituent (4-methyl-1,3-oxazolidin-2-one) in position 3 of the benzisoxazole ring, provides derivatives with excellent antibacterial activity and better pharmacokinetic profile, an example is zoliflodacin, the most promising in the series of spiropyrimidinetriones. Topoisomerase DNA are enzymes that control the three-dimensional conformation of DNA. Topoisomerases I and II are distinguished on the basis of their ability to cause single- or double-chain ruptures in DNA. DNA gyrase and topoisomerase IV are the two type II topoisomerases present in bacteria. Their different roles are fundamental in DNA replication. These enzymes are the target of the fluoroquinolone class. DNA gyrase is composed of two subunits, GyrA (97 kDa) and GyrB (90 kDa); the active form being an A2B2 heterotetramer able to introduce negative supercoils into the DNA molecules. This process of supercoiling is crucial to allow DNA to re-enter newly created cells. Zoliflodacin, as ciprofloxacin (fluoroquinolone antibiotic), has the ability to inhibit bacterial topoisomerases much more selectively than mammalian topoisomerases, blocking supercoiling catalyzed by DNA gyrase (in Gram-negative bacteria) and the development of the double helix mediated by topoisomerase IV (in Gram-positive bacteria). Blocking such mechanisms leads to the death of the bacterium. In addition, zoliflodacin stabilizes the enzyme–DNA complex for both gyrase and topoisomerase IV. In particular, the primary target of the antibiotic is the GyrB subunit of gyrase, unlike ciprofloxacin, which instead fits mainly into the pocket present in the GyrA subunit (bond with Ser83 and Asp87) . N. gonorrhoeae mutants resistant to zoliflodacin are not observed, even when bacteria are exposed to combinations between zoliflodacin and antibiotics already in use, such as ceftriaxone, doxycycline, and gentamycin. Zoliflodacin does not show the phenomenon of cross-resistance with fluoroquinolones currently on the market . It also has activities against S. aureus (MRSA), Mycoplasma genitalium , Moraxella catarrhalis (MIC90 of 0.25 μg/mL), vancomycin-resistant H. influenzae (MIC90 of 0.5 μg/mL), and Enterococcus faecalis (MIC90 of 1 μg/mL). Combining zoliflodacin with other antibiotics such as tetracyclines, ceftriaxone, or gentamycin increases its effectiveness against N. gonorroheae . Zoliflodacin has significantly lower MIC values with respect to other antibiotics on the market, with higher percentages of susceptible bacterial strains . The in vitro tests to measure the inhibition of growth of myeloid and erythroid cell lines in mammals showed no genotoxicity or bone marrow toxicity for zoliflodacin at the highest concentrations tested. This suggests that this antibiotic presents reduced risks of hematologic toxicity compared with some fourth-generation fluoroquinolones (such as gemifloxacin), which have been associated with bone marrow suppression, anemia, and leukopenia. In addition, it has been shown that zoliflodacin is a powerful and very selective inhibitor of bacterial topoisomerases . Two Phase I studies were conducted by administering individual doses for zoliflodacin to healthy volunteers . The first study concerned the safety and tolerability of the molecule; the second focused, instead, on pharmacokinetic parameters (ADME). Phase II studies involved administering the antibiotic (single dose of 2 or 3 g) to men and women aged 18 to 55 with symptoms of urogenital gonorrhea. The data revealed the effectiveness of zoliflodacin against rectal infections, urogenital infections, and gonorrhea. The company Entasis Therapeutics announced the entry of the molecule into Phase III in 2019; the final results are expected in 2021 . Another antibiotic inhibitor of topoisomerase II, gepotidacin, active against Gram-positive and Gram-negative cocci, is currently in Phase III for the treatment of urinary infections and gonorrhea . 4.2. C. difficile Infection: Focus on the New Compounds Ridinilazole and Bezlotoxumab C . difficile infection (CDI) is the most common health-related nosocomial infection in the world, associated with high hospital costs. This is a looming threat to public health: It was estimated that in 2014, the costs of hospitalizing patients with recurrent CDI in the United States reached $6500 a day , which makes us aware of the enormous scale of the problem. Such infections are frequent, especially in nursing homes and stays for the elderly. After an initial episode of CDI, the risk of recurring infections developing increases exponentially; it can occur in 35%–65% of patients who have already contracted the pathogen . Even after a course of therapy with metronidazole or vancomycin, 25% of patients suffer from a second infection. The patient is therefore in a sort of vicious cycle with continuous relapses: Those who have had an infection previously are much more predisposed to develop others later. Primary C. difficile infection is associated with a mortality range of 3 to 36%, values that rise by 33% in the case of recurrent infections . The guidelines provide numerous treatments for primary infection, but options for the treatment of recurrent infections appear very limited. The objective in the clinical field is the management of the prevention of such relapses. More specifically, C. difficile is an anaerobic, Gram-positive, spore-forming, toxin-producing bacillus, causing infections ranging from medium-sized diarrhea to much more complicated and severe diseases such as enterocolitis and pseudomembranous colitis. It is present in healthy gut and vaginal microbiota. The spread of spores by symptomatic and asymptomatic patients determines the rapid spread of the pathogen. The spores, resistant to heat, radiation and alcohol-based disinfectants, are in fact persistent: The introduction of such spores by oro–fecal means is a kind of a foreman towards gut colonization by C. difficile . Spores travel from the stomach to the intestine where, under optimal conditions (high levels of colic acid in bile salts and lower levels of chenodesoxycholic acids), they turn into vegetative cells, which then colonize and proliferate in the colon, starting the infection itself. A natural and innate defense against C. difficile is gut microflora. However, the imbalance in microbiota that usually occurs after a course of antibiotics predisposes to the occurrence of infection. The main condition, which allows the development of CDI, is in fact linked to the destruction of the normal microbial flora of the colon due not only to antibiotic therapy but also chemotherapy. All classes of antibiotics are predisposing factors for the development of CDI, however antibiotics such as ampicillin, cephalosporins and clindamycin are the most risky for the onset of CDI. The best strategy for eradicating the bacterium would be to discontinue ongoing antibiotic therapy, although this is not always possible. Metronidazole (belonging to the nitro-imidazole class) has for years been the first-line treatment in the care of primary and non-severe CDI, but frequent resistance and increased failure rates, with an average of around 25% and peaks of 50%, have made it less effective . Currently, vancomycin (a glycopeptide) is the preferred antibiotic in the treatment of both primary and recurrent infections. Despite this, the two drugs still showed negative effects on the gut microbiota. Another compound, fidaxomicin, represents a powerful option with minimal risk of damage to bacterial microflora . However, the very high costs of treatment with fidaxomicin drastically limit its use in hospitals. Other antibiotics used as the last possible option are tigecycline, teicoplanin, rifaximine, and bacitracin. There are now six new antibiotics under development versus C. difficile , the most promising being ridinilazole , in Phase III of clinical development. The common problem is always the treatment of recurrent infections, for which few compounds are really active. Clinical evidence also demonstrates the effectiveness of other therapies such as immunotherapy and fecal transplantation (fecal microbiota transplant), for which further studies are needed to confirm effectiveness . Ridinilazole is a synthetic antibiotic of the class of bis-benzimidazoles, discovered by Summit Therapeutics for the treatment of C. difficile : It showed rapid bactericidal activity . From the promising results of Phase II, it was evident that patients who were given ridinilazole had a greater clinical response in the eradication of C. difficile compared to patients who received vancomycin. From a chemical point of view , it is composed of a double benzimidazole (hence the name of the bis-benzimidazoles class), each one bonded to a pyridinic ring. Ridinilazole has a unique mechanism of action: It probably inhibits the cell division of the bacterium, binding to the minor groove of DNA. Transcriptome analyses have, in fact, highlighted an altered expression of C. difficile genes involved precisely in cell division following exposure to the antibiotic . After oral administration, it is poorly absorbed by the gastrointestinal lumen. Selective activity against C. difficile, demonstrated in vitro, together with limited systemic absorption and reduced action against gut microflora, make ridinilazole almost an ideal drug for the treatment of CDI. MIC values were lower than metronidazole and vancomycin, comparable to those of fidaxomycin. No ridinilazole-resistant strains were highlighted. The results related to the activity on the microbiota were very positive, as the microflora remained almost unchanged after treatment with this drug, unlike vancomycin therapy (which led to a drastic decrease in Bifidobacteria ) or the newest fidaxomycin therapy. In in vitro models of C. difficile -affected bowels, the antibiotic also showed only one activity against toxins A and B produced by the bacterium as well as decreased levels of interleukin (IL)-8. This is an important advantage; it means that it is potentially able to reduce the gut inflammation of the patient (present in the most severe forms of CDI). This is not the case in treatments with metronidazole and vancomycin, which have no action against toxins produced by gut cells. Phase II studies investigated the efficacy and safety of the antibiotic, always compared with vancomycin and metronidazole: Ridinilazole had side effects (especially in the gastrointestinal tract) comparable to those of vancomycin, but to a lesser extent. Phase III results (coming in 2021) will help outline the use profile and value of this drug. In the 2019 analysis “Antibacterial agents in clinical development: An analysis of the antibacterial clinical development pipeline”, by the WHO , ten biological drugs are reported, including monoclonal and polyclonal antibodies used as a support for existing therapies; however, their potential uses in mono-therapy have yet to be investigated. The only monoclonal antibody, whose target is C. difficile , included in the report, is bezlotoxumab, approved by the FDA in 2016 and now marketed in the United States under the name Zinplava ® . Recent studies have focused on new compounds preventing recurrent CDI, for which valid alternatives are still lacking, especially those targeting the virulence factors involved in the pathogenicity of the infection. The most severe forms of CDI are regulated by the expression of genes that control the main functions of toxin production (toxins A and B genes), toxin expression (toxin R), the release (toxin E), and toxin synthesis (toxin C) . The production of toxins is the virulence factor that contributes most to the infection. Non-pathogenic strains of C. difficile produce spores but do not cause symptomatic infections. In symptomatic infections, vegetative cells release toxins, resulting in CDI. Following the failure of actoxumab, a monoclonal antibody against toxin A, research has focused on compounds capable of blocking toxin B, which is responsible for the most severe pathological effects. Hence, bezlotoxumab (Zinplava ® ), a human monoclonal antibody directed against toxin B, showed promising results during Phase III. Bezlotoxumab is approved for the prevention of recurrent CDI in adults, administered in an intravenous formulation (10 mg/kg infusion as a single dose) in combination with an antibiotic treatment against C. difficile ; it is, in fact, not effective in monotherapy. The results of Phase I and Phase II clinical trials showed significant benefits and reduced incidence of recurrent CDI (decreased by 40% in 12 weeks compared to placebo) . No adverse events have emerged in healthy volunteers, not even drug-resistant bacterial strains. Based on Phase III data, in 2016, the FDA approved the use of bezlotoxumab in combination with antibiotics in the prevention of recurrent CDI.
The bacterium N. gonorrhoeae , resistant to third-generation cephalosporins and fluoroquinolones, is included in the category of high priority pathogens: There is an urgent need for new antibiotics that can overcome such resistance. N. gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted disease that in women can remain asymptomatic for long periods but which is responsible, in severe cases, for severe complications such as infertility, ectopic pregnancies, and neonatal blindness. In recent years, infections with N. gonorrhoeae resistant to penicillin and cephalosporins such as cefixime and ceftriaxone (usually used as the last therapy available in combination with azithromycin) have increased disproportionately. A recent report from the CDC—Centers for Disease Control and Prevention—documented over 500,000 new cases of gonorrhea in the United States during 2018 . Since 2019, the compound zoliflodacin is in Phase III for the treatment of multidrug-resistant N. gonorrhoeae , developed by Entasis Therapeutics in collaboration with the Global Antibiotic Research Development Program . It is the first synthesized antibiotic belonging to the class of spiropyrimidinetrions. It has a unique mechanism of action: It inhibits type II bacterial topoisomerase by binding to a different site than that of fluoroquinolones. The minimal inhibitory concentration (MIC) value together with pharmacokinetic parameters are regarded to have the greatest importance in the optimization of targeted antibiotic therapy . The MIC50 provides the so-called “intrinsic activity” of an antimicrobial, while the MIC90, which is calculated on the basis of larger, inter-center studies, is a reflection of different resistance mechanisms of the species under investigation. Zoliflodacin shows a very low resistance frequency and is active not only against multidrug-resistant N. gonorrhoeae with a MIC between 0.002 and 0.25 μg/mL but also against some troublesome Gram-positive and Gram-negative bacteria. From a chemical point of view, the formula of zoliflodacin is based on a new benzisoxazole scaffold and contains the pyrimidinetrione spirocyclic pharmacophore, which gives its name to this innovative class of antibiotics. The structure activity relationship (SAR) of this molecule was developed using in vitro tests showing the mechanism of inhibition of DNA gyrase and antibacterial activity. Several compounds in the benzisoxazole series have good activity against quinolone-resistant pathogens, including S. aureus , S. pneumoniae , and H. influenzae . The insertion of a substituent (4-methyl-1,3-oxazolidin-2-one) in position 3 of the benzisoxazole ring, provides derivatives with excellent antibacterial activity and better pharmacokinetic profile, an example is zoliflodacin, the most promising in the series of spiropyrimidinetriones. Topoisomerase DNA are enzymes that control the three-dimensional conformation of DNA. Topoisomerases I and II are distinguished on the basis of their ability to cause single- or double-chain ruptures in DNA. DNA gyrase and topoisomerase IV are the two type II topoisomerases present in bacteria. Their different roles are fundamental in DNA replication. These enzymes are the target of the fluoroquinolone class. DNA gyrase is composed of two subunits, GyrA (97 kDa) and GyrB (90 kDa); the active form being an A2B2 heterotetramer able to introduce negative supercoils into the DNA molecules. This process of supercoiling is crucial to allow DNA to re-enter newly created cells. Zoliflodacin, as ciprofloxacin (fluoroquinolone antibiotic), has the ability to inhibit bacterial topoisomerases much more selectively than mammalian topoisomerases, blocking supercoiling catalyzed by DNA gyrase (in Gram-negative bacteria) and the development of the double helix mediated by topoisomerase IV (in Gram-positive bacteria). Blocking such mechanisms leads to the death of the bacterium. In addition, zoliflodacin stabilizes the enzyme–DNA complex for both gyrase and topoisomerase IV. In particular, the primary target of the antibiotic is the GyrB subunit of gyrase, unlike ciprofloxacin, which instead fits mainly into the pocket present in the GyrA subunit (bond with Ser83 and Asp87) . N. gonorrhoeae mutants resistant to zoliflodacin are not observed, even when bacteria are exposed to combinations between zoliflodacin and antibiotics already in use, such as ceftriaxone, doxycycline, and gentamycin. Zoliflodacin does not show the phenomenon of cross-resistance with fluoroquinolones currently on the market . It also has activities against S. aureus (MRSA), Mycoplasma genitalium , Moraxella catarrhalis (MIC90 of 0.25 μg/mL), vancomycin-resistant H. influenzae (MIC90 of 0.5 μg/mL), and Enterococcus faecalis (MIC90 of 1 μg/mL). Combining zoliflodacin with other antibiotics such as tetracyclines, ceftriaxone, or gentamycin increases its effectiveness against N. gonorroheae . Zoliflodacin has significantly lower MIC values with respect to other antibiotics on the market, with higher percentages of susceptible bacterial strains . The in vitro tests to measure the inhibition of growth of myeloid and erythroid cell lines in mammals showed no genotoxicity or bone marrow toxicity for zoliflodacin at the highest concentrations tested. This suggests that this antibiotic presents reduced risks of hematologic toxicity compared with some fourth-generation fluoroquinolones (such as gemifloxacin), which have been associated with bone marrow suppression, anemia, and leukopenia. In addition, it has been shown that zoliflodacin is a powerful and very selective inhibitor of bacterial topoisomerases . Two Phase I studies were conducted by administering individual doses for zoliflodacin to healthy volunteers . The first study concerned the safety and tolerability of the molecule; the second focused, instead, on pharmacokinetic parameters (ADME). Phase II studies involved administering the antibiotic (single dose of 2 or 3 g) to men and women aged 18 to 55 with symptoms of urogenital gonorrhea. The data revealed the effectiveness of zoliflodacin against rectal infections, urogenital infections, and gonorrhea. The company Entasis Therapeutics announced the entry of the molecule into Phase III in 2019; the final results are expected in 2021 . Another antibiotic inhibitor of topoisomerase II, gepotidacin, active against Gram-positive and Gram-negative cocci, is currently in Phase III for the treatment of urinary infections and gonorrhea .
C . difficile infection (CDI) is the most common health-related nosocomial infection in the world, associated with high hospital costs. This is a looming threat to public health: It was estimated that in 2014, the costs of hospitalizing patients with recurrent CDI in the United States reached $6500 a day , which makes us aware of the enormous scale of the problem. Such infections are frequent, especially in nursing homes and stays for the elderly. After an initial episode of CDI, the risk of recurring infections developing increases exponentially; it can occur in 35%–65% of patients who have already contracted the pathogen . Even after a course of therapy with metronidazole or vancomycin, 25% of patients suffer from a second infection. The patient is therefore in a sort of vicious cycle with continuous relapses: Those who have had an infection previously are much more predisposed to develop others later. Primary C. difficile infection is associated with a mortality range of 3 to 36%, values that rise by 33% in the case of recurrent infections . The guidelines provide numerous treatments for primary infection, but options for the treatment of recurrent infections appear very limited. The objective in the clinical field is the management of the prevention of such relapses. More specifically, C. difficile is an anaerobic, Gram-positive, spore-forming, toxin-producing bacillus, causing infections ranging from medium-sized diarrhea to much more complicated and severe diseases such as enterocolitis and pseudomembranous colitis. It is present in healthy gut and vaginal microbiota. The spread of spores by symptomatic and asymptomatic patients determines the rapid spread of the pathogen. The spores, resistant to heat, radiation and alcohol-based disinfectants, are in fact persistent: The introduction of such spores by oro–fecal means is a kind of a foreman towards gut colonization by C. difficile . Spores travel from the stomach to the intestine where, under optimal conditions (high levels of colic acid in bile salts and lower levels of chenodesoxycholic acids), they turn into vegetative cells, which then colonize and proliferate in the colon, starting the infection itself. A natural and innate defense against C. difficile is gut microflora. However, the imbalance in microbiota that usually occurs after a course of antibiotics predisposes to the occurrence of infection. The main condition, which allows the development of CDI, is in fact linked to the destruction of the normal microbial flora of the colon due not only to antibiotic therapy but also chemotherapy. All classes of antibiotics are predisposing factors for the development of CDI, however antibiotics such as ampicillin, cephalosporins and clindamycin are the most risky for the onset of CDI. The best strategy for eradicating the bacterium would be to discontinue ongoing antibiotic therapy, although this is not always possible. Metronidazole (belonging to the nitro-imidazole class) has for years been the first-line treatment in the care of primary and non-severe CDI, but frequent resistance and increased failure rates, with an average of around 25% and peaks of 50%, have made it less effective . Currently, vancomycin (a glycopeptide) is the preferred antibiotic in the treatment of both primary and recurrent infections. Despite this, the two drugs still showed negative effects on the gut microbiota. Another compound, fidaxomicin, represents a powerful option with minimal risk of damage to bacterial microflora . However, the very high costs of treatment with fidaxomicin drastically limit its use in hospitals. Other antibiotics used as the last possible option are tigecycline, teicoplanin, rifaximine, and bacitracin. There are now six new antibiotics under development versus C. difficile , the most promising being ridinilazole , in Phase III of clinical development. The common problem is always the treatment of recurrent infections, for which few compounds are really active. Clinical evidence also demonstrates the effectiveness of other therapies such as immunotherapy and fecal transplantation (fecal microbiota transplant), for which further studies are needed to confirm effectiveness . Ridinilazole is a synthetic antibiotic of the class of bis-benzimidazoles, discovered by Summit Therapeutics for the treatment of C. difficile : It showed rapid bactericidal activity . From the promising results of Phase II, it was evident that patients who were given ridinilazole had a greater clinical response in the eradication of C. difficile compared to patients who received vancomycin. From a chemical point of view , it is composed of a double benzimidazole (hence the name of the bis-benzimidazoles class), each one bonded to a pyridinic ring. Ridinilazole has a unique mechanism of action: It probably inhibits the cell division of the bacterium, binding to the minor groove of DNA. Transcriptome analyses have, in fact, highlighted an altered expression of C. difficile genes involved precisely in cell division following exposure to the antibiotic . After oral administration, it is poorly absorbed by the gastrointestinal lumen. Selective activity against C. difficile, demonstrated in vitro, together with limited systemic absorption and reduced action against gut microflora, make ridinilazole almost an ideal drug for the treatment of CDI. MIC values were lower than metronidazole and vancomycin, comparable to those of fidaxomycin. No ridinilazole-resistant strains were highlighted. The results related to the activity on the microbiota were very positive, as the microflora remained almost unchanged after treatment with this drug, unlike vancomycin therapy (which led to a drastic decrease in Bifidobacteria ) or the newest fidaxomycin therapy. In in vitro models of C. difficile -affected bowels, the antibiotic also showed only one activity against toxins A and B produced by the bacterium as well as decreased levels of interleukin (IL)-8. This is an important advantage; it means that it is potentially able to reduce the gut inflammation of the patient (present in the most severe forms of CDI). This is not the case in treatments with metronidazole and vancomycin, which have no action against toxins produced by gut cells. Phase II studies investigated the efficacy and safety of the antibiotic, always compared with vancomycin and metronidazole: Ridinilazole had side effects (especially in the gastrointestinal tract) comparable to those of vancomycin, but to a lesser extent. Phase III results (coming in 2021) will help outline the use profile and value of this drug. In the 2019 analysis “Antibacterial agents in clinical development: An analysis of the antibacterial clinical development pipeline”, by the WHO , ten biological drugs are reported, including monoclonal and polyclonal antibodies used as a support for existing therapies; however, their potential uses in mono-therapy have yet to be investigated. The only monoclonal antibody, whose target is C. difficile , included in the report, is bezlotoxumab, approved by the FDA in 2016 and now marketed in the United States under the name Zinplava ® . Recent studies have focused on new compounds preventing recurrent CDI, for which valid alternatives are still lacking, especially those targeting the virulence factors involved in the pathogenicity of the infection. The most severe forms of CDI are regulated by the expression of genes that control the main functions of toxin production (toxins A and B genes), toxin expression (toxin R), the release (toxin E), and toxin synthesis (toxin C) . The production of toxins is the virulence factor that contributes most to the infection. Non-pathogenic strains of C. difficile produce spores but do not cause symptomatic infections. In symptomatic infections, vegetative cells release toxins, resulting in CDI. Following the failure of actoxumab, a monoclonal antibody against toxin A, research has focused on compounds capable of blocking toxin B, which is responsible for the most severe pathological effects. Hence, bezlotoxumab (Zinplava ® ), a human monoclonal antibody directed against toxin B, showed promising results during Phase III. Bezlotoxumab is approved for the prevention of recurrent CDI in adults, administered in an intravenous formulation (10 mg/kg infusion as a single dose) in combination with an antibiotic treatment against C. difficile ; it is, in fact, not effective in monotherapy. The results of Phase I and Phase II clinical trials showed significant benefits and reduced incidence of recurrent CDI (decreased by 40% in 12 weeks compared to placebo) . No adverse events have emerged in healthy volunteers, not even drug-resistant bacterial strains. Based on Phase III data, in 2016, the FDA approved the use of bezlotoxumab in combination with antibiotics in the prevention of recurrent CDI.
As of 2017, eight new antibiotics have been approved by the FDA, including one for the treatment of multidrug-resistant tuberculosis. The full list can be found in the “Agents that obtained market authorization” section of the “Antibacterial agents in clinical development: An analysis of the antibacterial clinical development pipeline” . Derivatives of existing antibiotic classes, such as the tetracycline derivatives eravacycline and omadacycline as well as new β-lactams, prevail by far. Most of the approved compounds target carbapenem-resistant Enterobacteriaceae and other pathogens (of high and medium priority) included in the WHO’s list. Both omadacycline and eravacycline are derivatives of tetracyclines. Omadacycline is a semisynthetic drug and has activities against Gram-positives, including difficult to eradicate MRSA and some Gram-negatives. It is approved in the treatment of community-acquired pneumonia (CAP). Eravacycline, on the other hand, is totally synthetic and approved in the treatment of complicated intra-abdominal infections. The results of further studies must be awaited to better delineate the clinical profile of these antibiotics. Furthermore, a promising new combination of β-lactam antibiotic and β-lactamase inhibitor, presenting activities against K. pneumonia carbapenemase (KPC), has been approved. This is the combination meropenem and vaborbactam (a β-lactamase inhibitor). However, new treatment options for carbapenem-resistant A. baumannii (CRAB) and carbapenem-resistant P. aeruginosa (CRPA) are still lacking. The sole antibiotic approved in recent years against tuberculosis is pretomanid, a nitroimidazo-oxazine developed by the TB Alliance organization. It is, together with bedaquiline and linezolid, a completely innovative treatment for adult patients with extremely resistant tuberculosis (XDR) and pulmonary multidrug resistant (MDR) tuberculosis. Most antibiotics that have obtained trade authorization are effective in treating complicated urinary infections and intra-abdominal infections. Companies involved in the research for new antibiotics have highlighted some difficulties; an example is the case of Achaogen, a biotechnology company that developed the aminoglycosydic plazomicin, approved in 2018, and went bankrupt a few months later despite receiving $2.4 million from Boston University’s CARB-X project for the development of the new drug . For all eight antibiotics newly authorized by the FDA, obviously, post-marketing data are not yet available, and further studies are needed to define their therapeutic profile and adequacy of use in particular categories of patients. 5.1. Tetracycline Derivatives: Eravacycline Eravacycline is a fully synthetic fluorocycline belonging to the tetracycline class, developed by Tetraphase Pharmaceuticals and approved by the EMA and FDA in 2018, for the treatment of complicated intra-abdominal infections (cIAI). It is marketed under the name Xerava ® . cIAI normally extend into the area of the abdomen (peritoneal cavity, mesentery) and result in localized or diffuse peritonytes . Especially if not treated, such infections are associated with significant mortality, and it is in fact necessary to start antibiotic therapy as soon as possible, which becomes fundamental, and in some cases, lifesaving. Usually, a large number of enteric microorganisms responsible for symptomatology are involved, such as Enterobacteriaceae ( K. pneumoniae , E. coli ), Enterococcus spp., and Bacteroides spp. The increase in MDR pathogens belonging to the aforementioned species is a very serious global health problem that threatens the treatment of such intra-abdominal infections. Eravacycline and relebactam with imipenem/cilastatin combination (Recarbrio ® , approved in 2019) are included in the list of eight antibiotics authorized from 2017 to 2020 and constitute the most recent and innovative treatment options for patients with cIAI. Eravacycline was specifically developed to overcome the acquired resistance phenomenon experienced with traditional tetracyclines . The two primary mechanisms that confer the resistance of pathogens to tetracyclines are, in fact, the acquisition of genes encoding some efflux pumps and the presence of ribosomal protection proteins (RPPs) . Various types of efflux pumps are present in the Gram-positive and Gram-negative bacteria; the most represented efflux pumps are encoded by tet(A) and tet(B) genes in Gram-negative and by tet(K) and tet(L) in Gram-positive bacteria. First generation tetracyclines are more easily inactivated by efflux pumps in contrast to second generation tetracyclines (doxycycline and minocycline) or third generation tetracyclines (tigecycline), which are not sensitive to the actions of the pumps themselves. Efflux consists of actively reducing the concentration of the antibiotic within the bacterial cell thanks to the inducible synthesis of membrane proteins encoded by genes (tetA and tetB) placed on plasmids or transposons. These proteins weaken the interactions between the tetracyclines and the binding site on the 30S ribosomal subunit. In fact, tetracyclines act by inhibiting protein synthesis, blocking the transfer of acyl-tRNA to that subunit. RPP also makes pathogens resistant to first and second generation tetracyclines, with less effect on the antibacterial activity of the latest generation tetracyclines. There are also other mechanisms of acquired resistance to tetracyclines such as mutations in the 16S RNA subunit; however, they are much less common than efflux pumps and ribosomal proteins. Third generation tetracyclines (also called glycylcyclines), which include tigecycline and the new eravacycline, allow for overcoming the main resistances to tetracyclines: Efflux pumps do not recognize these molecules, as they have a substituent in position 9 of the tetracycle . This is the key difference from previous generations of tetracyclines. Moreover, they are also insensitive to the action of ribosomal protection proteins. Eravacycline retains the pharmacophore characteristic of tetracyclines; however, it exhibits two unique changes in ring D at position C7 (addition of a fluorine atom) and at C9 (addition of a pyrrolidine acetamide group) . The fluorine is not present in the tigecycline structure, which has a tertiary amino group in its place. As a result of such substitutions in positions 7 and 9, eravacycline has activities against Gram-positive and Gram-negative bacterial strains that, in vitro, resulted in various mechanisms resistant to first- and second-generation tetracyclines. Like other tetracyclines, eravacycline performed its antibacterial activity by reversibly binding to the ribosomal subunit 30S, blocking the entry of molecules of the aminoacyl-tRNA complex. Compared to traditional tetracyclines, however, the link of eravacycline with ribosome is much stronger because it is able to recognize multiple attack sites, stabilizing the complex that forms. The compounds belonging to the first and second generation are bacteriostatic; however, eravacycline also has in vitro bactericidal activity against certain strains of A. baumannii , E. coli , and K. pneumoniae . Eravacycline showed potent in vitro activity as determined by MIC90 against a broad spectrum of Gram-positive pathogens, including E. faecalis and E. faecium , both resistant to vancomycin, and S. aureus (MRSA), as well as against Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae . Such pathogens are among those responsible for cIAI, so the drug has proven effective in clinical trials. Promising is the detected activity of eravacycline against isolated strains of A. baumannii MDR and resistant to carbapenems. The MIC90 values of eravacycline measured for numerous Gram-positive and Gram-negative pathogens are clearly lower than those of antibiotics such as imipenem and vancomycin, remaining lower even in comparison with tigecycline. In addition, there is no cross-resistance mechanism between eravacycline and other classes of antibacterials such as fluoroquinolones, penicillins, cephalosporins, and carbapenems. The antibiotic is metabolized mainly by CYP3A4; therefore, the concomitant intake of eravacycline with strong inducers of this cytochrome (rifampicin, phenobarbital, carbamazepine, phenytoin, to name a few), accelerates the metabolism of the antibacterial drug, decreasing its plasma concentration. Eravacycline in intravenous form has been approved since 2018 as Xerava ® in numerous countries, including the United States and some European states, for the treatment of cIAI, in adult patients. The recommended dosage is 1 mg/kg administered every 12 h for 4 to 14 days depending on the prescribed therapy . In two double-blind clinical trials, the efficacy of eravacycline was not inferior to that of ertapenem and meropenem in terms of clinical response and acceptability. High doses of intravenous tetracyclines can cause microvescicular liver steatosis with lactic acidosis and severe liver dysfunction (LASH syndrome), but this complication has not been reported with the use of third-generation intravenous tetracyclines (eravacycline, tigecycline, omadacycline). Given the wide spectrum of activity against clinically relevant common pathogens (including those expressing mechanisms of acquired resistance to tetracyclines) and the increased in vitro potency along with a better tolerability profile compared to tigecycline, eravacycline is an innovative option for the treatment of cIAI, especially against bacterial species resistant to traditional antibiotics. 5.2. Fourth-Generation Fluoroquinolones: Delafloxacin Fluoroquinolones are effective antibiotics, used in therapy for over 50 years. However, the increase in resistance cases and some recorded adverse effects have severely limited their use. The last approved fluoroquinolonic, delafloxacin, is the only anionic (non-zwitterionic) antibiotic in this class. The particular molecular structure of the drug has given greater in vitro activity against many Gram-positive pathogens, including quinolone-resistant strains. Delafloxacin was developed by Melinta Therapeutics and then approved by the FDA in 2017 for the treatment of acute bacterial skin and skin structure infections (ABSSSI), marketed under the name Baxdela ® . Such infections are associated with significant morbidity and mortality. Numerous Gram-positive and Gram-negative bacteria have been identified as etiological agents. However, the most dangerous pathogen for ABSSSI worldwide, is S. aureus , followed by other Gram-positive ( Enterococcus spp., Streptococcus pyogenes ) and Gram-negative ( P. aeruginosa and E. coli ) bacteria, more commonly found in surgical site infections . A serious problem is the presence of pathogens resistant to traditional antibiotics, especially MRSA Staphylococchi : This contributes to the increase in mortality as well as the hospital costs related to such infections. Current guidelines in treatment offer a variety of antibiotics and therapeutic strategies, depending on the type—purulent (abscesses) or non-purulent (erysipelas, necrotizing infections)—and the severity of infection. Usually, in the treatment of infections caused by methycillin-sensitive S. aureus (MSSA), it is recommended to use oxacillin or other β-lactamases-resistant penicillins or cephalozoline in the case of specific allergy to penicillins. If the etiological agent is MRSA, other more powerful antibiotics are administered such as vancomycin, linezolid, daptomycin, or ceftarolines. Sometimes, in cases of both MRSA and MSSA, more “dated” antibiotics are also used: clindamycin, minocycline, or the combination trimetoprim-sulfametoxazole. However, all these antibiotics are associated with limitations such as high levels of resistance (clindamycin and minocycline), high hospital costs and possible toxicity (linezolid), decreased sensitivity that involves using higher doses (vancomycin), and increased risk of developing C. difficile (clindamycin) infections. Therefore, new active antibiotics against resistant pathogens that cause ABSSSI have been studied, especially for infections caused by MRSA. The most recently approved antibiotics include dalbavancin, tedizolid, oritavancin, and delafloxacin. Another concern is S . aureus’ ability to survive in the acidic environment of the skin. The survival of bacteria depends on the expression of an enzyme that gives resistance to polyamines (anti-inflammatory compounds promoting tissue regeneration and wound healing). Polyamines are present in the skin acid environment and are toxic to Staphylococcus . Moreover, the bacteria are able to adopt specific behaviors that play an important role in the pathogenesis of infections such as their organization in biofilm . The consequence is that the requirements of the new compounds are not only their activity against resistant strains but also their stability in the acid pH of the skin. Delafloxacin has an increased activity in acidic mediums . In addition, it shows promising efficacy on a wide spectrum of Gram-positive and Gram-negative bacteria involved in major acute skin infections. Delafloxacin differs from other fluoroquinolones in the absence of a basic group in position C7; as a consequence, this molecule is a weak acid, and at a neutral pH, it is an anion and not zwitterion, as are most of the antibiotics belonging to the same class. Moreover, in position C8, a chlorine atom is added, which acts as an electron-attractor group on the aromatic ring, improving polarity to the compound as well as increased activity and stability. Finally, thanks to the voluminous heteroaromatic substitution in N1 (instead of the cyclopropyl present in ciprofloxacin and moxifloxacin), delafloxacin has a larger molecular structure compared to that of other fluoroquinolones. Due to the lack of a basic group in C7, the only ionizing group is the carboxyl in C3. At a neutral pH of 7.4, the predominant form (98.5%) is anionic delafloxacin (COO − ), while at a slightly acidic pH of 5.2, the neutral form prevails (62.7%) ( a) . These modifications have a direct impact on the activity of the antibiotic and may explain the increased potency at an acidic pH compared to other fluoroquinolones (second and third generation: ciprofloxacin and levofloxacin, but also fourth generation: moxifloxacin), for which activity decreases drastically in an acidic medium. Delafloxacin also has lower MIC values than those of traditional fluoroquinolones against a wide spectrum of Gram-positive pathogens. Prior to delafloxacin, the most-recent fluoroquinolonic antibiotic was finafloxacin ( b), which was approved in 2014 for the treatment of acute otitis and has substantial differences compared to delafloxacin: It changes the group in C7 (more basic), there is no chlorine atom in C8, and it retains the cyclopropyl in N1 as in other fluoroquinolones. Quinolones inhibit bacterial DNA and topoisomerase IV. The structural peculiarities of delafloxacin allow it to bind with equal affinity both to DNA gyrase and topoisomerase IV of Gram-positive ( S. aureus ) and Gram-negative ( E. coli ) bacteria. This reduces the likelihood of resistance, which requires the accumulation of multiple mutations at the level of both enzymes. In the study promoted by CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) in 2016, it was found that delafloxacin had the lowest MIC values toward MSSA, MRSA, and S. aureus compared to other antibiotics such as levofloxacin, ceftaroline, ciprofloxacin, clindamycin, linezolid, and oxacillin . Delafloxacin showed MIC values equal to the fifth part of those of ciprofloxacin against Enterobacteriaceae ( E. coli ) isolated from the urine of patients suffering from urinary infections. Another in vitro study showed the efficacy of delafloxacin in combination with caspofungin against many Gram-positive infections. Caspofungin is an antifungal drug inhibiting the synthesis of the polysaccharide components of the bacterial biofilm of S. aureus . As mentioned before, delafloxacin is active at an acidic pH. This has been demonstrated by comparing this antibiotic and other fluoroquinolones at several pH values. Moreover, in vitro studies have confirmed the effectiveness of delafloxacin in fluoroquinolonic-resistant bacterial strains: It is bactericidal against E. coli in 6 h and S. aureus in 10 h. It has also been found to be more active than other antibiotics against Gram-negative pathogens such as H. influenzae , N. gonorrhoeae , Legionell a spp., P. aeruginosa , and H. pylori . Fluoroquinolones have collected a long history of adverse events, including tendinitis, swelling and tendon injuries, memory problems, muscle pain or weakness, peripheral neuropathy, and exacerbations of myasthenia gravis. As a result, in the United States, many fluoroquinolones on the market, including delafloxacin, carry a boxed warning on the outer packaging and in the package leaflet about these effects . The EMA has also added restrictions on the use of such antibiotics, which must be administered only to particular infections (to “severe infections for which no other antibiotics can be used”) . The FDA reported that peripheral neuropathies and effects on the nervous system were observed during the clinical development of delafloxacin as well as diarrhea associated with C. difficile ; however, these effects were not recorded more frequently in the cohort receiving the antibiotic than in the comparison cohort . Numerous studies have been carried out to examine the specific side effects related to fluoroquinolone class membership. In clinical models, delafloxacin, compared to moxifloxacin, has not been related to heart disorders (such as QT stretching) even at doses higher than therapeutic ones . Moreover, another study was conducted on photosensitivity, commonly associated with the presence of the halogen substituent in position C8 (as it is for lomefloxacin, which has a fluorine in C8), in which delafloxacin was found not to be phototoxic, despite the presence of a chlorine atom in C8 . However, the safety profile of delafloxacin will be further demonstrated through large-scale use. Based on pharmacological data, delafloxacin (Baxdela ® ) shows favorable properties: it maintains 60% bioavailability after oral administration, it is a mild inhibitor of cytochrome P450 3A, it interacts with few other drugs, and there is no cross-resistance with fluoroquinolones currently on the market. It is available in both tablets and an intravenous formulation for the treatment of acute skin infections, and it could also be effective in respiratory infections. Of course, this must be confirmed by further studies in the coming years. 5.3. New Β-Lactams in the Treatment of Multidrug-Resistant Enterobacteriaceae Infections: The Meropenem–Vaborbactam Combination Recently, there has been a continuous increase in the number of infections due to Gram-negative pathogens, especially carbapenem-resistant Enterobacteriaceae , included by the WHO in the list of critical priority pathogens that pose a looming danger to world health. The family Enterobacteriaceae consists precisely of Gram-negative bacteria whose natural habitat is the animal intestine and they are responsible for a long list of gut and urinary infections. The main mechanism of resistance of these pathogens to many traditional antibiotics is attributable to the production of carbapenemases, or enzymes that include a variety of beta-lactamases capable of hydrolyzing both carbapenems and penicillins, some cephalosporins, and aztreonam. The activity of these enzymes, with rare exceptions, is not blocked by classic β-lactamase inhibitors such as clavulanic acid, tazobactam, and sulbactam. KPC is the most-frequently produced enzyme by pathogens belonging to the Enterobacteriaceae species. Such CRE-producing pathogens are often isolated from patients’ urine, which is not surprising, as Enterobacteria are responsible for most of complicated urinary tract infections (cUTI), especially those associated with high mortality. In fact, invasive infections caused by CRE result in mortality of between 26% and 44% . The production of carbapenemases is not the only mechanism that Enterobacteria possess to develop resistance: There are also efflux pumps, enzymatic degradations, mutations at the porine level, and alterations of the target site. As a result, treatment options for CRE infections are unfortunately limited. In some cases, no first-line antibiotics are active against such pathogens, and the only treatments available are polymyxins and aminoglycosides or rather old antibiotics in addition to the rediscovery of colistine, the toxic effects of which are by no means negligible. It is clear that new compounds are needed to treat Gram-negative bacteria infections, mainly CRE. Β-lactams are a class of antibiotics with absolutely established use. They attack peptidoglycan biosynthesis, interrupting the formation of the bacterial cell wall through covalent binding to PBPs. The group includes penicillin, cephalosporins, carbapenems, and monobactams. The emergence of β-lactamase-producing bacteria has made many of these antibiotics ineffective; moreover, the spread of extended spectrum β-lactamases (ESBLs) also gives resistance to third-generation, broad-spectrum cephalosporins such as ceftriaxone and ceftazidime. Class B β-lactamases contain a zinc ion at the active site of the enzyme. The other classes of β-lactamase (type A, C, and D) are serine β-lactamases. The main strategy to stem the hydrolysis of antibiotics belonging to this class is to combine a β-lactam and a β-lactamase inhibitor (BLI) such as clavulanic acid, tazobactam, or sulbactam. The latter are able to inhibit the aforementioned ESBLs; however, they have no activity towards carbapenemases. Recently, some combinations of β-lactamase inhibitors with carbapenems or cephalosporins have been approved, including ceftolozane with tazobactam and ceftazidime with avibactam. Taniborbactam/cefepime (in clinical development) and cefiderocol (already approved) cover all classes of β-lactamases, including class D, produced by A. baumannii . In 2015, the combination of ceftazidime (a broad-spectrum, third-generation cephalosporin) with avibactam (Zavicefta ® and Avycaz ® ) , was approved for the treatment of cUTI and cIAI. This combination is active in vitro and inhibits class A (e.g., KPC) and Class D (e.g., OXA-48) carbapenamases. Retrospective studies have shown a decrease in mortality from CRE infections and an increased survival rate of 92% with ceftazidime/avibactam, compared to 55% mortality observed using the combination of colistine, aminoglycosides, and carbapenems . Although these data are promising and encouraging, cases of resistance in Enterobacteria treated with ceftazidime and avibactam are already reported. This shows the huge need for new active compounds against CRE along with conscious and appropriate use of existing antibiotics. The combination of the carbapenemic antibiotic meropenem and vaborbactam , a new β-lactamase inhibitor based on the boron acid formula, has powerful in vitro activity against Enterobacteria producing KPC . In 2018, this association received marketing authorization for the treatment of cUTI, including acute pyelonephritis, cIAI, and hospital-acquired pneumonia (HAP), including assisted ventilation pneumonia (VAP). It was developed by Rempex Pharmaceuticals and marketed as Vabomere ® . From a chemical point of view, meropenem is a 1-β-methyl carbapenem. It is produced by total chemical synthesis. Unlike imipenem, it has a carbon methyl group (β) at position 1 as well as a different carbon substitution at position 2. The side chain linked to C2 is, in fact, much more cluttered than that of the imipenem. This justifies the greater stability of meropenem compared to hydrolysis by the enzyme human renal dehydropeptidase-1 (DHP-1), which is why it does not require co-administration with cilastatin (which was indispensable in the case of imipenem). Moreover, it is stable even in the presence of β-lactamases, including penicillinase and cephalosporinase, thanks to the presence of 6-trans-hydroxyethyl. Meropenem is marketed under the name Merrem ® for parenteral use. It has been authorized in the European Union since the 1990s . Vaborbactam is a new inhibitor of β-lactamases whose cyclic pharmacophore is based on the structure of boronic acid. It strengthens the activity of meropenem alone. The boronic ester allows the compound to assume a particular conformation that can selectively inhibit β-lactamases as compared to mammalian serine-proteases. In particular, the portion derived from boron mimics the tetrahedral intermediate that is formed as a result of the interaction between the hydrolytic enzymes such as metallo-β-lactamases (class B) or serine β-lactamases (class A, C, and D) and the β-lactam antibiotic. In this way, the enzyme binds to vaborbactam instead of inactivating the antibiotic. In vitro experiments were conducted to explore the SAR of vaborbactam with the aim of finding the best substitutes to enhance the activity of meropenem: In particular, the addition of the thienyl-acetyl group in position 2 of the ring proved to be very promising. Vaborbactam inhibits many class A and C β-lactamases and carbapenemases, and it is especially essential that it is effective against KPC. Vaborbactam manages to enter the outer membrane of the bacterium K. pneumonia by exploiting the porines OmpK35 and OmpK36 . Meropenem is a broad-spectrum, bactericidal carbapenem with activity to many MDR pathogens, and it remains stable even in the presence of extended spectrum β-lactamases (ESBL). Vaborbactam alone, on the other hand, has no antibacterial activity. For strains of Escherichia coli that produce carbapenemases, the values of MIC for the combination meropenem with vaborbactam and for meropenem alone were both ≤0.03 mg/L . The addition of vaborbactam did not improve the effectiveness of meropenem against Acinetobacter spp. or P. aeruginosa because the resistance of such bacterial species to carbapenems was multifactorial: It was not only caused by the production of β-lactamases but also depended on other mechanisms (one of them was the expression of efflux pumps). The combination showed, however, powerful in vitro activity against numerous strains of Enterobacteria , including carbapenem-resistant K. pneumonia . In fact, in the presence of CRE, vaborbactam greatly enhanced the effectiveness of meropenem alone. On 9 July 2020, the R&D division of Menarini Ricerche Group announced the publication of an abstract that reported the latest evidence deriving from the clinical studies on meropenem/vaborbactam (marketed as Vaborem ® in the European Union and as Vabomere ® in the USA) . Based on the TANGO I (Targeting Antibiotic Non-susceptible Gram-negative Organisms) clinical study, which compared meropenem/vaborbactam with the piperacillin-tazobactam association, Vabomere ® was initially approved by the FDA for cUTI, including pyelonephritis, in adult patients. In this randomized Phase 3 study, Vabomere ® was administered in monotherapy to patients with confirmed or suspected CRE infections and was compared with the best available treatment, which consisted mainly of monotherapy or combinations of multiple antibiotics (polymyxin B, colistine, carbapenems, aminoglycosides, thygecycline, or ceftazidime/avibactam). During the study, the association of meropenem and vaborbactam showed a considerable reduction in mortality and an improvement in clinical safety (decreased adverse events, such as nephrotoxicity) and tolerability and was shown to be an effective therapeutic option for the treatment of HABP/VABP (bacterial pneumonia associated with the ventilator) and bacteriemia from CRE. Clinical studies have shown the good tolerability of the combination of meropenem and vaborbactam; the most common side effects recorded in TANGO I were headaches, diarrhea, and nausea. Meropenem/vaborbactam could represent a turning point in the fight against Gram-negative infections that are difficult to treat, as it addresses the important medical issue of carbapenem-resistant Enterobacteria . It should be considered a first-line treatment for the treatment of infections from KPC-producing pathogens, with use restricted to these particular infections. Further results and future work will make it possible to define the role of this combination of antibiotics, which is certainly an additional weapon to combat the growth of resistance to carbapenems in Enterobacteria . Relebactam is an active β-lactamase inhibitor against class A (including KPC) and class C β-lactamases. The structure is similar to that of avibactam. In vitro studies have shown that the addition of relebactam to the combination of imipenem/cilastatin restores the activity of the same association against strains of Enterobacteriaceae that produce KPC, normally not sensitive to imipenem . Phase II studies have shown the effectiveness and tolerability of the association of imipenem and relebactam in the treatment of cIAI, cUTI, and acute pyelonephritis. Phase III was completed in 2018 . Developed by Merck & Co., the drug containing imipenem monohydrate, sodium cilastatin, and relebactam monohydrate is marketed in the European Union under the brand name Recarbrio ® ; this medicinal product requires additional clinical monitoring because of the absolutely promising in vitro results’ lack of extended clinical data. This combination could represent a valid alternative in the treatment of complicated, carbapenem-resistant Enterobacteriaceae infections, especially KPC producers, together with the aforementioned meropenem/vaborbactam association. 5.4. New Aminoglycosides in the Treatment of Infection Caused by Multidrug-Resistant Enterobacteriaceae: Plazomicin Aminoglycosides are historical antibiotics, used in therapy for many years. They are irreversibly bound to a ribosomal site consisting of three proteins of subunit 50S (mechanism of action of streptomycin) and possibly other proteins of subunit 30S (all other aminoglycosides). As a result, they block the ribosome on the starting codon (AUG), which results in the detachment of the ribosomal complex and an incomplete synthesis of the protein. They are bactericidal antibiotics on Gram-negative aerobes and some Gram-positive and Mycobacteria spp. Parenteral use is limited to serious infections with Gram-negative bacteria and as antitubercular agents; in fact, many aminoglycosides have nephrotoxicity and ototoxicity when administered through this route. The onset of antibiotic resistant phenomena of this class is occurring more and more often. The most common resistance mechanism consists of the production of enzymes (acetyltransferase, phosphorylase, adenosyltransferase) that inactivate the antibiotic through conjugation reactions at the expense of amine and oxidyl functions, making it less akin to binding sites in the bacterial ribosome. Susceptibility to these enzymes is different in various aminoglycosides: It is minimal in amikacin and netilmycin (both of semisyntetic origin), thanks to the presence of substitutes that sterically interfere with the binding to the inactivation enzyme. There are also ribosomal modifications that produce high resistance: These are methylations of specific bases (guanine) of rRNA in subunit 16S. Enzymatic resistance to aminoglycosides is very common in the Enterobacteriaceae species. Plazomicin is a new aminoglycoside that derives from the modification of sisomicin (a specific antibiotic against Gram-negative infections for which gentamicin, the first-choice molecule, did not give the desired effects) . Plazomicin, specifically in Enterobacteria spp., blocks most of the Aminoglycoside Modifying Enzymes (AME) inactivating aminoglycosidic antibiotics. This is due to the innovative chemical structure of plazomicin compared to other aminoglycosides: It differs considerably from the structures of gentamycin and tobramycin but gets closer to that of amikacin. Plazomicin belongs to the group of 2-deoxystreptamines in addition to amikacin, gentamycin, and tobramycin. As in amikacin and plazomicin, the aminocyclitol is substituted in positions 1’, 4’, and 6’. In particular, to block inactivating enzymes, the substituent (hydroxyethyl group) in position 6’, is bulkier compared with the other antibiotics of this class. Moreover, between positions 4′ and 5′, there is a double bond that is not present in the structures of other aminoglycosides except for netilmycin, an unsaturated derivative of gentamycin that is endowed with less ototoxicity. In position 1, there is hydroxy aminobutyric acid already inserted in the structure of amikacin; it specifically prevents adenylation and phosphorylation, resulting in an increase in antibiotic potency and spectrum enlargement. In vitro studies report that resistance to plazomicin may occur by methylation of ribosomal subunit 16S. Plazomicin is a broad-spectrum antibiotic, with activities against many Gram-positive and Gram-negative bacteria, including CRE and KPC Enterobacteria , which are pathogens that produce ESBL, and strains of E. coli not sensitive to aminoglycoside gentamycin . It is more potent than antibiotics belonging to the same class against KPC Enterobacteria . In fact, among strains of KPC-producing K. pneumoniae , the measured values of MIC50 of plazomycin were 0.5 mg/L, while gentamycin had a MIC50 of 8 mg/L and amikacin and tobramycin more than 32 mg/L . Plazomicin demonstrated promising efficacy and safety in Phase II results in the treatment of urinary infections, which allowed two Phase III studies to begin. The first is the EPIC clinical trial (Evaluating Plazomicin in cUTI) where plazomicin was administered in an intravenous formulation to adult patients hospitalized with cUTI or acute pyelonephritis, both caused by Enterobacteriaceae . The second is the CARE (Combating Antibiotic-Resistant Enterobacteriaceae ) a randomized study evaluating the efficacy and safety of plazomicin-based combination therapy compared with colistin-based combination therapy for the treatment of patients with invasive, CRE-involved infections such as ventilator-associated pneumonia (VAP), hospital-acquired pneumonia (HAP), and cUTI. In both studies, plazomicin proved to be well-tolerated. There was, however, reversible ototoxicity in a patient involved in the EPIC study. Developed by Achaogen, plazomicin was approved by the FDA in the United States in 2018 with the name of Zemdri ® as alternative for the treatment of cUTI and pyelonephritis caused by Enterobacteriaceae spp. (including E. coli , K. pneumoniae , Proteus mirabilis , and Enterobacter cloacae ). In Europe, plazomicin has not yet received marketing authorization. 5.5. Siderophore Cephalosporins: Cefiderocol Cephalosporins belong to the class of β-lactam antibiotics, and they were discovered in 1945 by the Italian, Giuseppe Brotzu, who was the rector of the University of Cagliari in Sardinia, Italy. The mechanism of action is identical to that of penicillins: They act by blocking the synthesis of the bacterial wall. There are five generations of cephalosporins, each characterized by a precise antimicrobial spectrum that becomes wider and wider reaching the fifth generation, also active on MRSA. In fact, the compounds belonging to the latter generation (ceftobiprole, ceftarolin, ceftolozane) have been developed to specifically combat MDR bacterial strains. Ceftobiprole, used to treat community-acquired pneumonia, is effective against methycillin-resistant Staphylococchi . Ceftolozane, combined with the β-lactamase inhibitor tazobactam (Zerbaxa ® ), is highly dedicated to carbapenem-resistant Enterobacteriaceae and P. aeruginosa . Cefiderocol is part of the siderophore cephalosporins, a new class of drugs, of which this antibiotic was the first to be approved, by the FDA in 2019 and by EMA in April 2020, for cUTI caused by Gram-negative, community-acquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia (VABP) . Siderophores are molecules with the marked properties of chelate ions, especially iron; they are produced and released by numerous bacterial species to facilitate the transport of ions into the cell, as required for supporting biological functions and bacterial growth. Siderophores all share the same structure: a functional unit that binds iron (transferrin or lactoferrin) and a peptide that interacts with a receptor present on the surface of the bacterial membrane. Research on siderophores has suggested that their involvement in the active transport of the bacterial cell makes pathogens more sensitive to antibiotics associated with siderophore groups; in fact, the measured MIC values are lower than traditional antibiotics. In the 1980s, researchers from some companies began to develop synthetic β-lactam antibiotics functionalized with siderophores, which showed powerful in vitro antibacterial activity against numerous Gram-negative bacteria, including P. aeruginosa . The siderophore group of these molecules seizes iron from the external environment. The iron–siderophore–antibiotic complex binds to the iron transporter outside the bacterial membrane, and it is actively transported inside the bacterial cell, bypassing the pathogen’s defense systems. This mechanism is called the “Trojan horse strategy” and allows for exploiting the iron transporter, improving the penetration of the antibiotic. In addition, the development of intrinsic and acquired resistance mechanisms is avoided. The first compounds involving the conjugation of cephalosporin–siderophore portions such as cefetecol (GR69153) and M-14659 (specific anti-Pseudomonas cephalosporin) could not pass the early clinical stages, despite their powerful in vitro activity. In the 1990s, the Japanese company Shionogi & Co developed the cephalosporin S-9096, which showed powerful activity against P. aeruginosa . This compound presents a new catechol moiety also found in natural siderophores produced by E. coli and P. aeruginosa (i.e., enterobactine and pyoverdine), however, S-9096 didn’t pass the clinical stages due to low stability and potential cardiotoxicity. Shionogi’s researchers initiated new research on siderophoric cephalosporins in the early 2000s, when antibiotic resistance had increased exponentially from 20 years earlier and few therapeutic alternatives were available. . The challenge was to translate the great in vitro activity shown by the first siderophore cephalosporin into the development of products with in vivo activity and good pharmacokinetic and pharmacodynamic properties. The SAR of natural and cephalosporin-conjugated siderophores leaded to the development of cefiderocol (S-649266) bearing a catechol moiety. In vitro studies have shown that this compound is up to 100 more stable to the action of different types of carbapenemases than ceftazidime. The structure of cefiderocol is similar to that of cefepime, a fourth-generation cephalosporin: Both have a pyrrolydinic group bound to the chain in C3, which results in quaternary ammonium. They are zwitterions; this allows them to penetrate better into both the Gram-positive and Gram-negative. An additional (carboxypropyl)oximine chain and an aminothiazole ring (common to many broad-spectrum cephalosporins) increase their antibacterial activity to Gram-negatives. Carboxylic acid in the C7 side-chain improves the permeability of cefiderocol in the outer membrane. Oxime and dimetyl groups, on the other hand, increase stability toward hydrolysis by β-lactamases. The main distinction between cefiderocol and the other cephalosporins examined (ceftazidime and cefepime) of previous generations lies in the substitute in position 3: this gives siderophore properties. Cefiderocol contains a portion consisting of a chlorocatechol (chloro-chloro-3,4-dihydroxibenzoic acid) covalently bound, via a particular linker, to the nitrogen of the pyrrolidine ring, to form a quaternary ammonium cation. The additional catechol portion allows achieving high plasma concentrations of cefiderocol compared to those of ceftazidyme and cefepime, thanks to the ability of the two hydroxyl groups to chelate the iron ion (Fe3 + ) and, consequently, exploit the transporter of the same ion . All these structural changes compared with the oldest cephalosporines give cefiderocol a strong stability against β-lactamases, including carbapenemases, while maintaining a high affinity toward the molecular target, the PBPs. It is transported inside the bacterial cell through iron transport systems located on the outer membrane of gram-negatives. Once the complex has passed the outer membrane, cefiderocol dissociates and, like other β-lactam antibiotics, inhibits PBPs, resulting in the death of the bacterium that can no longer synthesize the cell wall. The active transport of the cefiderocol–iron complex not only contributes to making this antibiotic available within the periplasmic space where PBPs are located but also overcomes the problems related to the low permeability of the drug due to the bacterial outflow pumps that tend to expel it. In fact, cefiderocol also maintains effectiveness in cases of up-regulation of efflux pumps, which is one of the mechanisms of resistance developed by pathogens against carbapenemases. A major point in favor of cefiderocol is structural stability against a wide range of serine and metallo-β-lactamases such as KPC, oxacillin carbapenemase (OXA), and New Delhi metallo-β-lactamase (NDM) . The results of a study on this topic showed that bacteria could potentially more easily develop resistance to ceftazidime and other third and fourth generation cephalosporins than cefiderocol, and there was no cross-resistance between it and the other cephalosporins. Although rather limited information is still available, recent studies suggest that resistance to cefiderocol may occur due to genetic mutations at the iron carrier level; the molecular mechanisms underlying these considerations remain to be clarified . Cefiderocol has an absolutely unique antibacterial spectrum against a wide variety of clinically relevant Gram-negative strains, including not only pathogens belonging to Enterobacteriaceae ( Klebsiella spp., Shigella flexneri , Salmonella spp., Vibrio spp., and Yersinia spp.), but also against bacterial species such as Acinetobacter spp., Pseudomonas spp., and Burkholderia spp. . It also has activities against pathogens such as Haemophilus spp. that cause respiratory tract infections. Moreover, this antibiotic shows powerful in vitro activity, with low MIC values on several multidrug-resistant Gram-negative strains and β-lactamase producers strains (including ESBL, serine and metallo-carbapenemase). Cefiderocol is very promising, also, for the treatment of carbapenem-resistant strains, which are considered critical priorities by the WHO. This is demonstrated by the results reported in the Global Surveillance Study , which collected data from three consecutive SIDERO-WT annual studies from patients in Europe and North America. Almost all (96.2%) isolated strains of Enterobacteriaceae , A. baumannii , P. aeruginosa , and Stenotrophomonas maltophilia are sensitive to less than 4 mg/L of cefiderocol. Gaussian distribution links the percentage of strains to the MIC value of cefiderocol. Unlike previous siderophore cephalosporins, the very good in vitro activity of cefiderocol is supported by in vivo clinical efficacy. Consequently, cefiderocol has been approved as an injectable drug and in addition, is the first cephalosporin capable of treating A. baumannii infections. In 2019, the FDA announced its approval under the trade name Fetroja ® for the treatment of adult patients with cUTI, including kidney infections caused by sensitive, Gram-negative microorganisms, which have limited treatment options or no alternative. In 2020 the indication was added for the treatment of HABP and VABP caused by the following bacteria: A. baumannii , E. coli , Enterobacter cloacae , K. pneumoniae , P. aeruginosa , and Serratia marcescens . Following that, cefiderocol was also approved by the EMA in April 2020. The safety and efficacy results of the pilot study on patients with cUTI showed that 72.6% had a resolution of symptoms and eradication of bacteria seven days after treatment as compared to 54.6% in patients who received an alternative antibiotic. Fetroja ® received from the FDA the designation of a Qualified Infectious Disease Product (QIDP), which is given to antibacterial and antifungal products intended to treat serious or life-threatening infections. The real novelty compared to the other newly approved antibiotics (including the combination already seen of meropenem/vaborbactam) is its ability to overcome all three mechanisms of development of resistance to β-lactams, namely the production of bacterial β-lactamases, the up-regulation of efflux pumps, and the modification of porines. 5.6. Treatment of Multidrug-Resistant Tuberculosis: Pretomanid Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Koch bacillus); it is categorized as pulmonary tuberculosis, generalized tuberculosis (tubercular sepsis), and extra-pulmonary tuberculosis. Mycobacteria are acid-resistant bacilli with an elaborate wall that are characterized by the presence of several unusual lipids. It is believed that, to date, approximately a third of the world’s population suffers from latent tuberculosis, and in addition, about 2 million people die each year as a result of infection . Tuberculosis is also the leading cause of death in individuals with HIV. Of the 10 million TB cases recorded in 2019, at least 500,000 were resistant to rifampicin or the rifampicin–isoniazide combination, using two of the most widely used frontline drugs. The biggest problem at present lies in the presence of multidrug-resistant forms of tuberculosis (MDR-TBC), with a mortality rate approaching 50%. Numerous awareness campaigns have been conducted, including the “Stop TB” strategy of the World Health Organization, which aims, by 2030, to eradicate the tuberculosis epidemic. Achieving this will be extremely challenging, but also stimulating. To eradicate mycobacterium, it is necessary to use therapies combined with at least two drugs to which the bacillus is sensitive in order to reduce the selection of mutant strains and, at the same time, generate a synergistic effect. Combined therapy should continue for a long time, for a minimum of 6 months, with an inevitable incidence of side effects and frequent interactions with other drugs. If the first-line drugs (isoniazid, rifampicin, ethambutol, pyrazinamide) have not been effective, especially due to the onset of resistance phenomena, second-line drugs (para-aminosalicylic acid, ethionamide, thyoacetazone, amikacin, and many others) are used. Unfortunately, there are many forms of TBC resistant to conventional treatment that constitute a real threat to world public health. Resistant tuberculosis is classified as MDR-TBC when there is no response to rifampicin and isoniazid (frontline drugs); extensively resistant (XDR-TBC) in the event that the administration of three or more second-line drugs is not effective (generally not resistant to fluoroquinolones and at least to another second-line injectable drug); and totally drug-resistant (TDR-TBC), i.e., not treatable with any of the drugs that currently exist. The treatment of MDR-TBC consists of taking multiple drug therapy for a period of at least 21 months. Aminoglycoside antibiotics such as capreomycin and kanamycin can be used as well as fluoroquinolones such as ofloxacin and moxifloxacin and in some cases, cycloserine as well. The antibiotic linezolid (oxazolidinone) is often prescribed in severe cases of multidrug-resistant tuberculosis, but there are numerous side effects related to the drug. It is clear that, especially for the most difficult forms of tuberculosis to treat, new drugs are needed that also manage to reduce the overall duration of treatment and are also compatible with antiretroviral drugs administered to HIV-positive patients who contract a M. tuberculosis infection. Currently, 12 new active ingredients against M. tuberculosis are in clinical development, 7 of which meet the criterion of innovation that provides for the absence of cross-resistance, while 6 antibiotics are able to meet all four criteria. Eight promising compounds are in Phase II and III . The molecular targets that these drugs inhibit are multiple and diverse: the enzyme DprE1 (decaprenylphosphoryl-β- d -ribose 2-epimerase), is important for the synthesis of the cell wall of the mycobacterium, and the enzyme leucyl-tRNA synthetase (LeuRS) is necessary for protein synthesis. The most recently approved drugs for the treatment of multidrug-resistant pulmonary tuberculosis (MDR-TBC) are bedaquiline (approved in 2012) and delamanid (2014). Bedaquiline (marketed as Sirturo ® ) is, chemically, a diarylquinoline . This compound is an absolutely innovative drug, as it presents an unprecedented mechanism of action: It inhibits the ATPases proton pump that supplies ATP to the mycobacterium. Further data are needed to determine whether the benefits of bedaquiline outweigh its risks and, consequently, to define its role in the management of MDR-TB. Nitroimidazoles are heterocyclic nitro-derivatives. In the 1990s, it was observed that metronidazole (5-nitroimidazole), belonging to this class, had moderate bactericidal activity against M. tuberculosis in anaerobic conditions. Subsequent studies led to the discovery of other nitroimidazoles, starting with the formula of metronidazole, which were more effective against mycobacteria. The 2-nitroimidazole replaced in positions 1 and 5 were the first nitroimidazolic compounds with antitubercular activity . They are currently one of the most promising classes of antituberculosis agents in clinical research. Delamanid (Deltyba ® , OPC-67683 in clinical development, ), approved by the FDA in 2014, is a 6-nitro-2,3-dihydro-imidazo-oxazole belonging to the class of nitroimidazoles and works by blocking the synthesis of the mycolic acids that make up the cell wall of M. tuberculosis. Delamanid has also been considered effective for the form XDR-TBC (extensively resistant), which is very difficult to treat and for which there are limited treatment options; it is common especially in India and southeast Asian countries. This is an important achievement. In August 2019, the FDA approved pretomanid (Dovprela ® , PA-824 in clinical development, ), the first antitubercular bicyclic nitroimidazo-oxazine successfully developed and registered by TB Alliance, a non-profit organization founded in South Africa in 2000 . The suffix “preto” comes from the city of Pretoria, South Africa, where the drug was developed. In 2020, the drug also received marketing approval from EMA, in a combination regimen with bedaquiline and linezolid (BPaL regimen), to be taken for only 6 months (a real revolution compared to existing therapies) for the treatment of XDR tuberculosis in adults and MDR tuberculosis that did not respond to other conventional antibiotics. This regimen was effective in 89% of the cases recorded in the clinical trial, which assessed the use of the same antibiotics in the MDR and XDR forms of tuberculosis. Moreover, it is also included in the new BPaMZ regimen, consisting of bedaquine, pretomanid, moxifloxacin, and pyrazinamide. The mechanism of action is very complex. Mycobacterium can live in both aerobic conditions and hypoxia. Under aerobic conditions, the drug inhibits the biosynthesis of mycobacterium proteins and lipids; in particular, pretomanid blocks the transformation of hydroximicolic acid into ketomycolate (i.e., mycolic acids that, together with arabinogalattans and lipoarabinomannans, make up the wall of mycobacterium), with subsequent accumulation of hydroximicolic acid and depletion of ketomycolates . Moreover, pretomanid also blocks the cellular respiratory processes of mycobacterium in an anaerobic environment through the release of nitric oxide, which kills M. tuberculosis . Thus, pretomanid is effective on both replication and latent M. tuberculosis cells, aerobically and anaerobically. The mechanism of action is therefore completely innovative. This was observed in laboratory experiments: Pretomanid-treated bacteria showed, in vitro, a different pattern of metabolites (especially with regard to the metabolic pathways of fatty acids, proteins, and the pentose-phosphate) than bacteria that received other antitubercular antibiotics . The SAR of pretomanid shows that the enantiomer S is the most active; moreover, the presence of a nitro group in position 2 of the imidazole ring, the lipophilic tail in position 6 of the oxazinic ring, and the rigidity of the bicyclic system are crucial for antitubercular activity. These important portions are also found in other nitroimidazole antibiotics (CGI-1734 and TBA-354, in phase I clinical development). Delamanid has notable affinities with pretomanid. Both delamanid and pretomanid are lipophilic, as required to penetrate the wall of the mycobacterium. Pretomanid is available in tablets for the treatment of pulmonary MDR and XDR tuberculosis; however, it is not active against extra-pulmonary tuberculosis, a particular form that fortunately represents only 5% of all existing TBC forms. Thanks to the latest drugs, the most aggressive and severe forms of tuberculosis resistant to traditional drugs are more treatable. Tuberculosis is the infectious disease that has caused the greatest number of deaths ever, that’s why research has witnessed remarkable growth, also thanks to the growing investments and collaborations promoted and stimulated by the United Nations General Assembly and the TB Alliance.
Eravacycline is a fully synthetic fluorocycline belonging to the tetracycline class, developed by Tetraphase Pharmaceuticals and approved by the EMA and FDA in 2018, for the treatment of complicated intra-abdominal infections (cIAI). It is marketed under the name Xerava ® . cIAI normally extend into the area of the abdomen (peritoneal cavity, mesentery) and result in localized or diffuse peritonytes . Especially if not treated, such infections are associated with significant mortality, and it is in fact necessary to start antibiotic therapy as soon as possible, which becomes fundamental, and in some cases, lifesaving. Usually, a large number of enteric microorganisms responsible for symptomatology are involved, such as Enterobacteriaceae ( K. pneumoniae , E. coli ), Enterococcus spp., and Bacteroides spp. The increase in MDR pathogens belonging to the aforementioned species is a very serious global health problem that threatens the treatment of such intra-abdominal infections. Eravacycline and relebactam with imipenem/cilastatin combination (Recarbrio ® , approved in 2019) are included in the list of eight antibiotics authorized from 2017 to 2020 and constitute the most recent and innovative treatment options for patients with cIAI. Eravacycline was specifically developed to overcome the acquired resistance phenomenon experienced with traditional tetracyclines . The two primary mechanisms that confer the resistance of pathogens to tetracyclines are, in fact, the acquisition of genes encoding some efflux pumps and the presence of ribosomal protection proteins (RPPs) . Various types of efflux pumps are present in the Gram-positive and Gram-negative bacteria; the most represented efflux pumps are encoded by tet(A) and tet(B) genes in Gram-negative and by tet(K) and tet(L) in Gram-positive bacteria. First generation tetracyclines are more easily inactivated by efflux pumps in contrast to second generation tetracyclines (doxycycline and minocycline) or third generation tetracyclines (tigecycline), which are not sensitive to the actions of the pumps themselves. Efflux consists of actively reducing the concentration of the antibiotic within the bacterial cell thanks to the inducible synthesis of membrane proteins encoded by genes (tetA and tetB) placed on plasmids or transposons. These proteins weaken the interactions between the tetracyclines and the binding site on the 30S ribosomal subunit. In fact, tetracyclines act by inhibiting protein synthesis, blocking the transfer of acyl-tRNA to that subunit. RPP also makes pathogens resistant to first and second generation tetracyclines, with less effect on the antibacterial activity of the latest generation tetracyclines. There are also other mechanisms of acquired resistance to tetracyclines such as mutations in the 16S RNA subunit; however, they are much less common than efflux pumps and ribosomal proteins. Third generation tetracyclines (also called glycylcyclines), which include tigecycline and the new eravacycline, allow for overcoming the main resistances to tetracyclines: Efflux pumps do not recognize these molecules, as they have a substituent in position 9 of the tetracycle . This is the key difference from previous generations of tetracyclines. Moreover, they are also insensitive to the action of ribosomal protection proteins. Eravacycline retains the pharmacophore characteristic of tetracyclines; however, it exhibits two unique changes in ring D at position C7 (addition of a fluorine atom) and at C9 (addition of a pyrrolidine acetamide group) . The fluorine is not present in the tigecycline structure, which has a tertiary amino group in its place. As a result of such substitutions in positions 7 and 9, eravacycline has activities against Gram-positive and Gram-negative bacterial strains that, in vitro, resulted in various mechanisms resistant to first- and second-generation tetracyclines. Like other tetracyclines, eravacycline performed its antibacterial activity by reversibly binding to the ribosomal subunit 30S, blocking the entry of molecules of the aminoacyl-tRNA complex. Compared to traditional tetracyclines, however, the link of eravacycline with ribosome is much stronger because it is able to recognize multiple attack sites, stabilizing the complex that forms. The compounds belonging to the first and second generation are bacteriostatic; however, eravacycline also has in vitro bactericidal activity against certain strains of A. baumannii , E. coli , and K. pneumoniae . Eravacycline showed potent in vitro activity as determined by MIC90 against a broad spectrum of Gram-positive pathogens, including E. faecalis and E. faecium , both resistant to vancomycin, and S. aureus (MRSA), as well as against Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae . Such pathogens are among those responsible for cIAI, so the drug has proven effective in clinical trials. Promising is the detected activity of eravacycline against isolated strains of A. baumannii MDR and resistant to carbapenems. The MIC90 values of eravacycline measured for numerous Gram-positive and Gram-negative pathogens are clearly lower than those of antibiotics such as imipenem and vancomycin, remaining lower even in comparison with tigecycline. In addition, there is no cross-resistance mechanism between eravacycline and other classes of antibacterials such as fluoroquinolones, penicillins, cephalosporins, and carbapenems. The antibiotic is metabolized mainly by CYP3A4; therefore, the concomitant intake of eravacycline with strong inducers of this cytochrome (rifampicin, phenobarbital, carbamazepine, phenytoin, to name a few), accelerates the metabolism of the antibacterial drug, decreasing its plasma concentration. Eravacycline in intravenous form has been approved since 2018 as Xerava ® in numerous countries, including the United States and some European states, for the treatment of cIAI, in adult patients. The recommended dosage is 1 mg/kg administered every 12 h for 4 to 14 days depending on the prescribed therapy . In two double-blind clinical trials, the efficacy of eravacycline was not inferior to that of ertapenem and meropenem in terms of clinical response and acceptability. High doses of intravenous tetracyclines can cause microvescicular liver steatosis with lactic acidosis and severe liver dysfunction (LASH syndrome), but this complication has not been reported with the use of third-generation intravenous tetracyclines (eravacycline, tigecycline, omadacycline). Given the wide spectrum of activity against clinically relevant common pathogens (including those expressing mechanisms of acquired resistance to tetracyclines) and the increased in vitro potency along with a better tolerability profile compared to tigecycline, eravacycline is an innovative option for the treatment of cIAI, especially against bacterial species resistant to traditional antibiotics.
Fluoroquinolones are effective antibiotics, used in therapy for over 50 years. However, the increase in resistance cases and some recorded adverse effects have severely limited their use. The last approved fluoroquinolonic, delafloxacin, is the only anionic (non-zwitterionic) antibiotic in this class. The particular molecular structure of the drug has given greater in vitro activity against many Gram-positive pathogens, including quinolone-resistant strains. Delafloxacin was developed by Melinta Therapeutics and then approved by the FDA in 2017 for the treatment of acute bacterial skin and skin structure infections (ABSSSI), marketed under the name Baxdela ® . Such infections are associated with significant morbidity and mortality. Numerous Gram-positive and Gram-negative bacteria have been identified as etiological agents. However, the most dangerous pathogen for ABSSSI worldwide, is S. aureus , followed by other Gram-positive ( Enterococcus spp., Streptococcus pyogenes ) and Gram-negative ( P. aeruginosa and E. coli ) bacteria, more commonly found in surgical site infections . A serious problem is the presence of pathogens resistant to traditional antibiotics, especially MRSA Staphylococchi : This contributes to the increase in mortality as well as the hospital costs related to such infections. Current guidelines in treatment offer a variety of antibiotics and therapeutic strategies, depending on the type—purulent (abscesses) or non-purulent (erysipelas, necrotizing infections)—and the severity of infection. Usually, in the treatment of infections caused by methycillin-sensitive S. aureus (MSSA), it is recommended to use oxacillin or other β-lactamases-resistant penicillins or cephalozoline in the case of specific allergy to penicillins. If the etiological agent is MRSA, other more powerful antibiotics are administered such as vancomycin, linezolid, daptomycin, or ceftarolines. Sometimes, in cases of both MRSA and MSSA, more “dated” antibiotics are also used: clindamycin, minocycline, or the combination trimetoprim-sulfametoxazole. However, all these antibiotics are associated with limitations such as high levels of resistance (clindamycin and minocycline), high hospital costs and possible toxicity (linezolid), decreased sensitivity that involves using higher doses (vancomycin), and increased risk of developing C. difficile (clindamycin) infections. Therefore, new active antibiotics against resistant pathogens that cause ABSSSI have been studied, especially for infections caused by MRSA. The most recently approved antibiotics include dalbavancin, tedizolid, oritavancin, and delafloxacin. Another concern is S . aureus’ ability to survive in the acidic environment of the skin. The survival of bacteria depends on the expression of an enzyme that gives resistance to polyamines (anti-inflammatory compounds promoting tissue regeneration and wound healing). Polyamines are present in the skin acid environment and are toxic to Staphylococcus . Moreover, the bacteria are able to adopt specific behaviors that play an important role in the pathogenesis of infections such as their organization in biofilm . The consequence is that the requirements of the new compounds are not only their activity against resistant strains but also their stability in the acid pH of the skin. Delafloxacin has an increased activity in acidic mediums . In addition, it shows promising efficacy on a wide spectrum of Gram-positive and Gram-negative bacteria involved in major acute skin infections. Delafloxacin differs from other fluoroquinolones in the absence of a basic group in position C7; as a consequence, this molecule is a weak acid, and at a neutral pH, it is an anion and not zwitterion, as are most of the antibiotics belonging to the same class. Moreover, in position C8, a chlorine atom is added, which acts as an electron-attractor group on the aromatic ring, improving polarity to the compound as well as increased activity and stability. Finally, thanks to the voluminous heteroaromatic substitution in N1 (instead of the cyclopropyl present in ciprofloxacin and moxifloxacin), delafloxacin has a larger molecular structure compared to that of other fluoroquinolones. Due to the lack of a basic group in C7, the only ionizing group is the carboxyl in C3. At a neutral pH of 7.4, the predominant form (98.5%) is anionic delafloxacin (COO − ), while at a slightly acidic pH of 5.2, the neutral form prevails (62.7%) ( a) . These modifications have a direct impact on the activity of the antibiotic and may explain the increased potency at an acidic pH compared to other fluoroquinolones (second and third generation: ciprofloxacin and levofloxacin, but also fourth generation: moxifloxacin), for which activity decreases drastically in an acidic medium. Delafloxacin also has lower MIC values than those of traditional fluoroquinolones against a wide spectrum of Gram-positive pathogens. Prior to delafloxacin, the most-recent fluoroquinolonic antibiotic was finafloxacin ( b), which was approved in 2014 for the treatment of acute otitis and has substantial differences compared to delafloxacin: It changes the group in C7 (more basic), there is no chlorine atom in C8, and it retains the cyclopropyl in N1 as in other fluoroquinolones. Quinolones inhibit bacterial DNA and topoisomerase IV. The structural peculiarities of delafloxacin allow it to bind with equal affinity both to DNA gyrase and topoisomerase IV of Gram-positive ( S. aureus ) and Gram-negative ( E. coli ) bacteria. This reduces the likelihood of resistance, which requires the accumulation of multiple mutations at the level of both enzymes. In the study promoted by CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) in 2016, it was found that delafloxacin had the lowest MIC values toward MSSA, MRSA, and S. aureus compared to other antibiotics such as levofloxacin, ceftaroline, ciprofloxacin, clindamycin, linezolid, and oxacillin . Delafloxacin showed MIC values equal to the fifth part of those of ciprofloxacin against Enterobacteriaceae ( E. coli ) isolated from the urine of patients suffering from urinary infections. Another in vitro study showed the efficacy of delafloxacin in combination with caspofungin against many Gram-positive infections. Caspofungin is an antifungal drug inhibiting the synthesis of the polysaccharide components of the bacterial biofilm of S. aureus . As mentioned before, delafloxacin is active at an acidic pH. This has been demonstrated by comparing this antibiotic and other fluoroquinolones at several pH values. Moreover, in vitro studies have confirmed the effectiveness of delafloxacin in fluoroquinolonic-resistant bacterial strains: It is bactericidal against E. coli in 6 h and S. aureus in 10 h. It has also been found to be more active than other antibiotics against Gram-negative pathogens such as H. influenzae , N. gonorrhoeae , Legionell a spp., P. aeruginosa , and H. pylori . Fluoroquinolones have collected a long history of adverse events, including tendinitis, swelling and tendon injuries, memory problems, muscle pain or weakness, peripheral neuropathy, and exacerbations of myasthenia gravis. As a result, in the United States, many fluoroquinolones on the market, including delafloxacin, carry a boxed warning on the outer packaging and in the package leaflet about these effects . The EMA has also added restrictions on the use of such antibiotics, which must be administered only to particular infections (to “severe infections for which no other antibiotics can be used”) . The FDA reported that peripheral neuropathies and effects on the nervous system were observed during the clinical development of delafloxacin as well as diarrhea associated with C. difficile ; however, these effects were not recorded more frequently in the cohort receiving the antibiotic than in the comparison cohort . Numerous studies have been carried out to examine the specific side effects related to fluoroquinolone class membership. In clinical models, delafloxacin, compared to moxifloxacin, has not been related to heart disorders (such as QT stretching) even at doses higher than therapeutic ones . Moreover, another study was conducted on photosensitivity, commonly associated with the presence of the halogen substituent in position C8 (as it is for lomefloxacin, which has a fluorine in C8), in which delafloxacin was found not to be phototoxic, despite the presence of a chlorine atom in C8 . However, the safety profile of delafloxacin will be further demonstrated through large-scale use. Based on pharmacological data, delafloxacin (Baxdela ® ) shows favorable properties: it maintains 60% bioavailability after oral administration, it is a mild inhibitor of cytochrome P450 3A, it interacts with few other drugs, and there is no cross-resistance with fluoroquinolones currently on the market. It is available in both tablets and an intravenous formulation for the treatment of acute skin infections, and it could also be effective in respiratory infections. Of course, this must be confirmed by further studies in the coming years.
Recently, there has been a continuous increase in the number of infections due to Gram-negative pathogens, especially carbapenem-resistant Enterobacteriaceae , included by the WHO in the list of critical priority pathogens that pose a looming danger to world health. The family Enterobacteriaceae consists precisely of Gram-negative bacteria whose natural habitat is the animal intestine and they are responsible for a long list of gut and urinary infections. The main mechanism of resistance of these pathogens to many traditional antibiotics is attributable to the production of carbapenemases, or enzymes that include a variety of beta-lactamases capable of hydrolyzing both carbapenems and penicillins, some cephalosporins, and aztreonam. The activity of these enzymes, with rare exceptions, is not blocked by classic β-lactamase inhibitors such as clavulanic acid, tazobactam, and sulbactam. KPC is the most-frequently produced enzyme by pathogens belonging to the Enterobacteriaceae species. Such CRE-producing pathogens are often isolated from patients’ urine, which is not surprising, as Enterobacteria are responsible for most of complicated urinary tract infections (cUTI), especially those associated with high mortality. In fact, invasive infections caused by CRE result in mortality of between 26% and 44% . The production of carbapenemases is not the only mechanism that Enterobacteria possess to develop resistance: There are also efflux pumps, enzymatic degradations, mutations at the porine level, and alterations of the target site. As a result, treatment options for CRE infections are unfortunately limited. In some cases, no first-line antibiotics are active against such pathogens, and the only treatments available are polymyxins and aminoglycosides or rather old antibiotics in addition to the rediscovery of colistine, the toxic effects of which are by no means negligible. It is clear that new compounds are needed to treat Gram-negative bacteria infections, mainly CRE. Β-lactams are a class of antibiotics with absolutely established use. They attack peptidoglycan biosynthesis, interrupting the formation of the bacterial cell wall through covalent binding to PBPs. The group includes penicillin, cephalosporins, carbapenems, and monobactams. The emergence of β-lactamase-producing bacteria has made many of these antibiotics ineffective; moreover, the spread of extended spectrum β-lactamases (ESBLs) also gives resistance to third-generation, broad-spectrum cephalosporins such as ceftriaxone and ceftazidime. Class B β-lactamases contain a zinc ion at the active site of the enzyme. The other classes of β-lactamase (type A, C, and D) are serine β-lactamases. The main strategy to stem the hydrolysis of antibiotics belonging to this class is to combine a β-lactam and a β-lactamase inhibitor (BLI) such as clavulanic acid, tazobactam, or sulbactam. The latter are able to inhibit the aforementioned ESBLs; however, they have no activity towards carbapenemases. Recently, some combinations of β-lactamase inhibitors with carbapenems or cephalosporins have been approved, including ceftolozane with tazobactam and ceftazidime with avibactam. Taniborbactam/cefepime (in clinical development) and cefiderocol (already approved) cover all classes of β-lactamases, including class D, produced by A. baumannii . In 2015, the combination of ceftazidime (a broad-spectrum, third-generation cephalosporin) with avibactam (Zavicefta ® and Avycaz ® ) , was approved for the treatment of cUTI and cIAI. This combination is active in vitro and inhibits class A (e.g., KPC) and Class D (e.g., OXA-48) carbapenamases. Retrospective studies have shown a decrease in mortality from CRE infections and an increased survival rate of 92% with ceftazidime/avibactam, compared to 55% mortality observed using the combination of colistine, aminoglycosides, and carbapenems . Although these data are promising and encouraging, cases of resistance in Enterobacteria treated with ceftazidime and avibactam are already reported. This shows the huge need for new active compounds against CRE along with conscious and appropriate use of existing antibiotics. The combination of the carbapenemic antibiotic meropenem and vaborbactam , a new β-lactamase inhibitor based on the boron acid formula, has powerful in vitro activity against Enterobacteria producing KPC . In 2018, this association received marketing authorization for the treatment of cUTI, including acute pyelonephritis, cIAI, and hospital-acquired pneumonia (HAP), including assisted ventilation pneumonia (VAP). It was developed by Rempex Pharmaceuticals and marketed as Vabomere ® . From a chemical point of view, meropenem is a 1-β-methyl carbapenem. It is produced by total chemical synthesis. Unlike imipenem, it has a carbon methyl group (β) at position 1 as well as a different carbon substitution at position 2. The side chain linked to C2 is, in fact, much more cluttered than that of the imipenem. This justifies the greater stability of meropenem compared to hydrolysis by the enzyme human renal dehydropeptidase-1 (DHP-1), which is why it does not require co-administration with cilastatin (which was indispensable in the case of imipenem). Moreover, it is stable even in the presence of β-lactamases, including penicillinase and cephalosporinase, thanks to the presence of 6-trans-hydroxyethyl. Meropenem is marketed under the name Merrem ® for parenteral use. It has been authorized in the European Union since the 1990s . Vaborbactam is a new inhibitor of β-lactamases whose cyclic pharmacophore is based on the structure of boronic acid. It strengthens the activity of meropenem alone. The boronic ester allows the compound to assume a particular conformation that can selectively inhibit β-lactamases as compared to mammalian serine-proteases. In particular, the portion derived from boron mimics the tetrahedral intermediate that is formed as a result of the interaction between the hydrolytic enzymes such as metallo-β-lactamases (class B) or serine β-lactamases (class A, C, and D) and the β-lactam antibiotic. In this way, the enzyme binds to vaborbactam instead of inactivating the antibiotic. In vitro experiments were conducted to explore the SAR of vaborbactam with the aim of finding the best substitutes to enhance the activity of meropenem: In particular, the addition of the thienyl-acetyl group in position 2 of the ring proved to be very promising. Vaborbactam inhibits many class A and C β-lactamases and carbapenemases, and it is especially essential that it is effective against KPC. Vaborbactam manages to enter the outer membrane of the bacterium K. pneumonia by exploiting the porines OmpK35 and OmpK36 . Meropenem is a broad-spectrum, bactericidal carbapenem with activity to many MDR pathogens, and it remains stable even in the presence of extended spectrum β-lactamases (ESBL). Vaborbactam alone, on the other hand, has no antibacterial activity. For strains of Escherichia coli that produce carbapenemases, the values of MIC for the combination meropenem with vaborbactam and for meropenem alone were both ≤0.03 mg/L . The addition of vaborbactam did not improve the effectiveness of meropenem against Acinetobacter spp. or P. aeruginosa because the resistance of such bacterial species to carbapenems was multifactorial: It was not only caused by the production of β-lactamases but also depended on other mechanisms (one of them was the expression of efflux pumps). The combination showed, however, powerful in vitro activity against numerous strains of Enterobacteria , including carbapenem-resistant K. pneumonia . In fact, in the presence of CRE, vaborbactam greatly enhanced the effectiveness of meropenem alone. On 9 July 2020, the R&D division of Menarini Ricerche Group announced the publication of an abstract that reported the latest evidence deriving from the clinical studies on meropenem/vaborbactam (marketed as Vaborem ® in the European Union and as Vabomere ® in the USA) . Based on the TANGO I (Targeting Antibiotic Non-susceptible Gram-negative Organisms) clinical study, which compared meropenem/vaborbactam with the piperacillin-tazobactam association, Vabomere ® was initially approved by the FDA for cUTI, including pyelonephritis, in adult patients. In this randomized Phase 3 study, Vabomere ® was administered in monotherapy to patients with confirmed or suspected CRE infections and was compared with the best available treatment, which consisted mainly of monotherapy or combinations of multiple antibiotics (polymyxin B, colistine, carbapenems, aminoglycosides, thygecycline, or ceftazidime/avibactam). During the study, the association of meropenem and vaborbactam showed a considerable reduction in mortality and an improvement in clinical safety (decreased adverse events, such as nephrotoxicity) and tolerability and was shown to be an effective therapeutic option for the treatment of HABP/VABP (bacterial pneumonia associated with the ventilator) and bacteriemia from CRE. Clinical studies have shown the good tolerability of the combination of meropenem and vaborbactam; the most common side effects recorded in TANGO I were headaches, diarrhea, and nausea. Meropenem/vaborbactam could represent a turning point in the fight against Gram-negative infections that are difficult to treat, as it addresses the important medical issue of carbapenem-resistant Enterobacteria . It should be considered a first-line treatment for the treatment of infections from KPC-producing pathogens, with use restricted to these particular infections. Further results and future work will make it possible to define the role of this combination of antibiotics, which is certainly an additional weapon to combat the growth of resistance to carbapenems in Enterobacteria . Relebactam is an active β-lactamase inhibitor against class A (including KPC) and class C β-lactamases. The structure is similar to that of avibactam. In vitro studies have shown that the addition of relebactam to the combination of imipenem/cilastatin restores the activity of the same association against strains of Enterobacteriaceae that produce KPC, normally not sensitive to imipenem . Phase II studies have shown the effectiveness and tolerability of the association of imipenem and relebactam in the treatment of cIAI, cUTI, and acute pyelonephritis. Phase III was completed in 2018 . Developed by Merck & Co., the drug containing imipenem monohydrate, sodium cilastatin, and relebactam monohydrate is marketed in the European Union under the brand name Recarbrio ® ; this medicinal product requires additional clinical monitoring because of the absolutely promising in vitro results’ lack of extended clinical data. This combination could represent a valid alternative in the treatment of complicated, carbapenem-resistant Enterobacteriaceae infections, especially KPC producers, together with the aforementioned meropenem/vaborbactam association.
Plazomicin Aminoglycosides are historical antibiotics, used in therapy for many years. They are irreversibly bound to a ribosomal site consisting of three proteins of subunit 50S (mechanism of action of streptomycin) and possibly other proteins of subunit 30S (all other aminoglycosides). As a result, they block the ribosome on the starting codon (AUG), which results in the detachment of the ribosomal complex and an incomplete synthesis of the protein. They are bactericidal antibiotics on Gram-negative aerobes and some Gram-positive and Mycobacteria spp. Parenteral use is limited to serious infections with Gram-negative bacteria and as antitubercular agents; in fact, many aminoglycosides have nephrotoxicity and ototoxicity when administered through this route. The onset of antibiotic resistant phenomena of this class is occurring more and more often. The most common resistance mechanism consists of the production of enzymes (acetyltransferase, phosphorylase, adenosyltransferase) that inactivate the antibiotic through conjugation reactions at the expense of amine and oxidyl functions, making it less akin to binding sites in the bacterial ribosome. Susceptibility to these enzymes is different in various aminoglycosides: It is minimal in amikacin and netilmycin (both of semisyntetic origin), thanks to the presence of substitutes that sterically interfere with the binding to the inactivation enzyme. There are also ribosomal modifications that produce high resistance: These are methylations of specific bases (guanine) of rRNA in subunit 16S. Enzymatic resistance to aminoglycosides is very common in the Enterobacteriaceae species. Plazomicin is a new aminoglycoside that derives from the modification of sisomicin (a specific antibiotic against Gram-negative infections for which gentamicin, the first-choice molecule, did not give the desired effects) . Plazomicin, specifically in Enterobacteria spp., blocks most of the Aminoglycoside Modifying Enzymes (AME) inactivating aminoglycosidic antibiotics. This is due to the innovative chemical structure of plazomicin compared to other aminoglycosides: It differs considerably from the structures of gentamycin and tobramycin but gets closer to that of amikacin. Plazomicin belongs to the group of 2-deoxystreptamines in addition to amikacin, gentamycin, and tobramycin. As in amikacin and plazomicin, the aminocyclitol is substituted in positions 1’, 4’, and 6’. In particular, to block inactivating enzymes, the substituent (hydroxyethyl group) in position 6’, is bulkier compared with the other antibiotics of this class. Moreover, between positions 4′ and 5′, there is a double bond that is not present in the structures of other aminoglycosides except for netilmycin, an unsaturated derivative of gentamycin that is endowed with less ototoxicity. In position 1, there is hydroxy aminobutyric acid already inserted in the structure of amikacin; it specifically prevents adenylation and phosphorylation, resulting in an increase in antibiotic potency and spectrum enlargement. In vitro studies report that resistance to plazomicin may occur by methylation of ribosomal subunit 16S. Plazomicin is a broad-spectrum antibiotic, with activities against many Gram-positive and Gram-negative bacteria, including CRE and KPC Enterobacteria , which are pathogens that produce ESBL, and strains of E. coli not sensitive to aminoglycoside gentamycin . It is more potent than antibiotics belonging to the same class against KPC Enterobacteria . In fact, among strains of KPC-producing K. pneumoniae , the measured values of MIC50 of plazomycin were 0.5 mg/L, while gentamycin had a MIC50 of 8 mg/L and amikacin and tobramycin more than 32 mg/L . Plazomicin demonstrated promising efficacy and safety in Phase II results in the treatment of urinary infections, which allowed two Phase III studies to begin. The first is the EPIC clinical trial (Evaluating Plazomicin in cUTI) where plazomicin was administered in an intravenous formulation to adult patients hospitalized with cUTI or acute pyelonephritis, both caused by Enterobacteriaceae . The second is the CARE (Combating Antibiotic-Resistant Enterobacteriaceae ) a randomized study evaluating the efficacy and safety of plazomicin-based combination therapy compared with colistin-based combination therapy for the treatment of patients with invasive, CRE-involved infections such as ventilator-associated pneumonia (VAP), hospital-acquired pneumonia (HAP), and cUTI. In both studies, plazomicin proved to be well-tolerated. There was, however, reversible ototoxicity in a patient involved in the EPIC study. Developed by Achaogen, plazomicin was approved by the FDA in the United States in 2018 with the name of Zemdri ® as alternative for the treatment of cUTI and pyelonephritis caused by Enterobacteriaceae spp. (including E. coli , K. pneumoniae , Proteus mirabilis , and Enterobacter cloacae ). In Europe, plazomicin has not yet received marketing authorization.
Cephalosporins belong to the class of β-lactam antibiotics, and they were discovered in 1945 by the Italian, Giuseppe Brotzu, who was the rector of the University of Cagliari in Sardinia, Italy. The mechanism of action is identical to that of penicillins: They act by blocking the synthesis of the bacterial wall. There are five generations of cephalosporins, each characterized by a precise antimicrobial spectrum that becomes wider and wider reaching the fifth generation, also active on MRSA. In fact, the compounds belonging to the latter generation (ceftobiprole, ceftarolin, ceftolozane) have been developed to specifically combat MDR bacterial strains. Ceftobiprole, used to treat community-acquired pneumonia, is effective against methycillin-resistant Staphylococchi . Ceftolozane, combined with the β-lactamase inhibitor tazobactam (Zerbaxa ® ), is highly dedicated to carbapenem-resistant Enterobacteriaceae and P. aeruginosa . Cefiderocol is part of the siderophore cephalosporins, a new class of drugs, of which this antibiotic was the first to be approved, by the FDA in 2019 and by EMA in April 2020, for cUTI caused by Gram-negative, community-acquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia (VABP) . Siderophores are molecules with the marked properties of chelate ions, especially iron; they are produced and released by numerous bacterial species to facilitate the transport of ions into the cell, as required for supporting biological functions and bacterial growth. Siderophores all share the same structure: a functional unit that binds iron (transferrin or lactoferrin) and a peptide that interacts with a receptor present on the surface of the bacterial membrane. Research on siderophores has suggested that their involvement in the active transport of the bacterial cell makes pathogens more sensitive to antibiotics associated with siderophore groups; in fact, the measured MIC values are lower than traditional antibiotics. In the 1980s, researchers from some companies began to develop synthetic β-lactam antibiotics functionalized with siderophores, which showed powerful in vitro antibacterial activity against numerous Gram-negative bacteria, including P. aeruginosa . The siderophore group of these molecules seizes iron from the external environment. The iron–siderophore–antibiotic complex binds to the iron transporter outside the bacterial membrane, and it is actively transported inside the bacterial cell, bypassing the pathogen’s defense systems. This mechanism is called the “Trojan horse strategy” and allows for exploiting the iron transporter, improving the penetration of the antibiotic. In addition, the development of intrinsic and acquired resistance mechanisms is avoided. The first compounds involving the conjugation of cephalosporin–siderophore portions such as cefetecol (GR69153) and M-14659 (specific anti-Pseudomonas cephalosporin) could not pass the early clinical stages, despite their powerful in vitro activity. In the 1990s, the Japanese company Shionogi & Co developed the cephalosporin S-9096, which showed powerful activity against P. aeruginosa . This compound presents a new catechol moiety also found in natural siderophores produced by E. coli and P. aeruginosa (i.e., enterobactine and pyoverdine), however, S-9096 didn’t pass the clinical stages due to low stability and potential cardiotoxicity. Shionogi’s researchers initiated new research on siderophoric cephalosporins in the early 2000s, when antibiotic resistance had increased exponentially from 20 years earlier and few therapeutic alternatives were available. . The challenge was to translate the great in vitro activity shown by the first siderophore cephalosporin into the development of products with in vivo activity and good pharmacokinetic and pharmacodynamic properties. The SAR of natural and cephalosporin-conjugated siderophores leaded to the development of cefiderocol (S-649266) bearing a catechol moiety. In vitro studies have shown that this compound is up to 100 more stable to the action of different types of carbapenemases than ceftazidime. The structure of cefiderocol is similar to that of cefepime, a fourth-generation cephalosporin: Both have a pyrrolydinic group bound to the chain in C3, which results in quaternary ammonium. They are zwitterions; this allows them to penetrate better into both the Gram-positive and Gram-negative. An additional (carboxypropyl)oximine chain and an aminothiazole ring (common to many broad-spectrum cephalosporins) increase their antibacterial activity to Gram-negatives. Carboxylic acid in the C7 side-chain improves the permeability of cefiderocol in the outer membrane. Oxime and dimetyl groups, on the other hand, increase stability toward hydrolysis by β-lactamases. The main distinction between cefiderocol and the other cephalosporins examined (ceftazidime and cefepime) of previous generations lies in the substitute in position 3: this gives siderophore properties. Cefiderocol contains a portion consisting of a chlorocatechol (chloro-chloro-3,4-dihydroxibenzoic acid) covalently bound, via a particular linker, to the nitrogen of the pyrrolidine ring, to form a quaternary ammonium cation. The additional catechol portion allows achieving high plasma concentrations of cefiderocol compared to those of ceftazidyme and cefepime, thanks to the ability of the two hydroxyl groups to chelate the iron ion (Fe3 + ) and, consequently, exploit the transporter of the same ion . All these structural changes compared with the oldest cephalosporines give cefiderocol a strong stability against β-lactamases, including carbapenemases, while maintaining a high affinity toward the molecular target, the PBPs. It is transported inside the bacterial cell through iron transport systems located on the outer membrane of gram-negatives. Once the complex has passed the outer membrane, cefiderocol dissociates and, like other β-lactam antibiotics, inhibits PBPs, resulting in the death of the bacterium that can no longer synthesize the cell wall. The active transport of the cefiderocol–iron complex not only contributes to making this antibiotic available within the periplasmic space where PBPs are located but also overcomes the problems related to the low permeability of the drug due to the bacterial outflow pumps that tend to expel it. In fact, cefiderocol also maintains effectiveness in cases of up-regulation of efflux pumps, which is one of the mechanisms of resistance developed by pathogens against carbapenemases. A major point in favor of cefiderocol is structural stability against a wide range of serine and metallo-β-lactamases such as KPC, oxacillin carbapenemase (OXA), and New Delhi metallo-β-lactamase (NDM) . The results of a study on this topic showed that bacteria could potentially more easily develop resistance to ceftazidime and other third and fourth generation cephalosporins than cefiderocol, and there was no cross-resistance between it and the other cephalosporins. Although rather limited information is still available, recent studies suggest that resistance to cefiderocol may occur due to genetic mutations at the iron carrier level; the molecular mechanisms underlying these considerations remain to be clarified . Cefiderocol has an absolutely unique antibacterial spectrum against a wide variety of clinically relevant Gram-negative strains, including not only pathogens belonging to Enterobacteriaceae ( Klebsiella spp., Shigella flexneri , Salmonella spp., Vibrio spp., and Yersinia spp.), but also against bacterial species such as Acinetobacter spp., Pseudomonas spp., and Burkholderia spp. . It also has activities against pathogens such as Haemophilus spp. that cause respiratory tract infections. Moreover, this antibiotic shows powerful in vitro activity, with low MIC values on several multidrug-resistant Gram-negative strains and β-lactamase producers strains (including ESBL, serine and metallo-carbapenemase). Cefiderocol is very promising, also, for the treatment of carbapenem-resistant strains, which are considered critical priorities by the WHO. This is demonstrated by the results reported in the Global Surveillance Study , which collected data from three consecutive SIDERO-WT annual studies from patients in Europe and North America. Almost all (96.2%) isolated strains of Enterobacteriaceae , A. baumannii , P. aeruginosa , and Stenotrophomonas maltophilia are sensitive to less than 4 mg/L of cefiderocol. Gaussian distribution links the percentage of strains to the MIC value of cefiderocol. Unlike previous siderophore cephalosporins, the very good in vitro activity of cefiderocol is supported by in vivo clinical efficacy. Consequently, cefiderocol has been approved as an injectable drug and in addition, is the first cephalosporin capable of treating A. baumannii infections. In 2019, the FDA announced its approval under the trade name Fetroja ® for the treatment of adult patients with cUTI, including kidney infections caused by sensitive, Gram-negative microorganisms, which have limited treatment options or no alternative. In 2020 the indication was added for the treatment of HABP and VABP caused by the following bacteria: A. baumannii , E. coli , Enterobacter cloacae , K. pneumoniae , P. aeruginosa , and Serratia marcescens . Following that, cefiderocol was also approved by the EMA in April 2020. The safety and efficacy results of the pilot study on patients with cUTI showed that 72.6% had a resolution of symptoms and eradication of bacteria seven days after treatment as compared to 54.6% in patients who received an alternative antibiotic. Fetroja ® received from the FDA the designation of a Qualified Infectious Disease Product (QIDP), which is given to antibacterial and antifungal products intended to treat serious or life-threatening infections. The real novelty compared to the other newly approved antibiotics (including the combination already seen of meropenem/vaborbactam) is its ability to overcome all three mechanisms of development of resistance to β-lactams, namely the production of bacterial β-lactamases, the up-regulation of efflux pumps, and the modification of porines.
Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Koch bacillus); it is categorized as pulmonary tuberculosis, generalized tuberculosis (tubercular sepsis), and extra-pulmonary tuberculosis. Mycobacteria are acid-resistant bacilli with an elaborate wall that are characterized by the presence of several unusual lipids. It is believed that, to date, approximately a third of the world’s population suffers from latent tuberculosis, and in addition, about 2 million people die each year as a result of infection . Tuberculosis is also the leading cause of death in individuals with HIV. Of the 10 million TB cases recorded in 2019, at least 500,000 were resistant to rifampicin or the rifampicin–isoniazide combination, using two of the most widely used frontline drugs. The biggest problem at present lies in the presence of multidrug-resistant forms of tuberculosis (MDR-TBC), with a mortality rate approaching 50%. Numerous awareness campaigns have been conducted, including the “Stop TB” strategy of the World Health Organization, which aims, by 2030, to eradicate the tuberculosis epidemic. Achieving this will be extremely challenging, but also stimulating. To eradicate mycobacterium, it is necessary to use therapies combined with at least two drugs to which the bacillus is sensitive in order to reduce the selection of mutant strains and, at the same time, generate a synergistic effect. Combined therapy should continue for a long time, for a minimum of 6 months, with an inevitable incidence of side effects and frequent interactions with other drugs. If the first-line drugs (isoniazid, rifampicin, ethambutol, pyrazinamide) have not been effective, especially due to the onset of resistance phenomena, second-line drugs (para-aminosalicylic acid, ethionamide, thyoacetazone, amikacin, and many others) are used. Unfortunately, there are many forms of TBC resistant to conventional treatment that constitute a real threat to world public health. Resistant tuberculosis is classified as MDR-TBC when there is no response to rifampicin and isoniazid (frontline drugs); extensively resistant (XDR-TBC) in the event that the administration of three or more second-line drugs is not effective (generally not resistant to fluoroquinolones and at least to another second-line injectable drug); and totally drug-resistant (TDR-TBC), i.e., not treatable with any of the drugs that currently exist. The treatment of MDR-TBC consists of taking multiple drug therapy for a period of at least 21 months. Aminoglycoside antibiotics such as capreomycin and kanamycin can be used as well as fluoroquinolones such as ofloxacin and moxifloxacin and in some cases, cycloserine as well. The antibiotic linezolid (oxazolidinone) is often prescribed in severe cases of multidrug-resistant tuberculosis, but there are numerous side effects related to the drug. It is clear that, especially for the most difficult forms of tuberculosis to treat, new drugs are needed that also manage to reduce the overall duration of treatment and are also compatible with antiretroviral drugs administered to HIV-positive patients who contract a M. tuberculosis infection. Currently, 12 new active ingredients against M. tuberculosis are in clinical development, 7 of which meet the criterion of innovation that provides for the absence of cross-resistance, while 6 antibiotics are able to meet all four criteria. Eight promising compounds are in Phase II and III . The molecular targets that these drugs inhibit are multiple and diverse: the enzyme DprE1 (decaprenylphosphoryl-β- d -ribose 2-epimerase), is important for the synthesis of the cell wall of the mycobacterium, and the enzyme leucyl-tRNA synthetase (LeuRS) is necessary for protein synthesis. The most recently approved drugs for the treatment of multidrug-resistant pulmonary tuberculosis (MDR-TBC) are bedaquiline (approved in 2012) and delamanid (2014). Bedaquiline (marketed as Sirturo ® ) is, chemically, a diarylquinoline . This compound is an absolutely innovative drug, as it presents an unprecedented mechanism of action: It inhibits the ATPases proton pump that supplies ATP to the mycobacterium. Further data are needed to determine whether the benefits of bedaquiline outweigh its risks and, consequently, to define its role in the management of MDR-TB. Nitroimidazoles are heterocyclic nitro-derivatives. In the 1990s, it was observed that metronidazole (5-nitroimidazole), belonging to this class, had moderate bactericidal activity against M. tuberculosis in anaerobic conditions. Subsequent studies led to the discovery of other nitroimidazoles, starting with the formula of metronidazole, which were more effective against mycobacteria. The 2-nitroimidazole replaced in positions 1 and 5 were the first nitroimidazolic compounds with antitubercular activity . They are currently one of the most promising classes of antituberculosis agents in clinical research. Delamanid (Deltyba ® , OPC-67683 in clinical development, ), approved by the FDA in 2014, is a 6-nitro-2,3-dihydro-imidazo-oxazole belonging to the class of nitroimidazoles and works by blocking the synthesis of the mycolic acids that make up the cell wall of M. tuberculosis. Delamanid has also been considered effective for the form XDR-TBC (extensively resistant), which is very difficult to treat and for which there are limited treatment options; it is common especially in India and southeast Asian countries. This is an important achievement. In August 2019, the FDA approved pretomanid (Dovprela ® , PA-824 in clinical development, ), the first antitubercular bicyclic nitroimidazo-oxazine successfully developed and registered by TB Alliance, a non-profit organization founded in South Africa in 2000 . The suffix “preto” comes from the city of Pretoria, South Africa, where the drug was developed. In 2020, the drug also received marketing approval from EMA, in a combination regimen with bedaquiline and linezolid (BPaL regimen), to be taken for only 6 months (a real revolution compared to existing therapies) for the treatment of XDR tuberculosis in adults and MDR tuberculosis that did not respond to other conventional antibiotics. This regimen was effective in 89% of the cases recorded in the clinical trial, which assessed the use of the same antibiotics in the MDR and XDR forms of tuberculosis. Moreover, it is also included in the new BPaMZ regimen, consisting of bedaquine, pretomanid, moxifloxacin, and pyrazinamide. The mechanism of action is very complex. Mycobacterium can live in both aerobic conditions and hypoxia. Under aerobic conditions, the drug inhibits the biosynthesis of mycobacterium proteins and lipids; in particular, pretomanid blocks the transformation of hydroximicolic acid into ketomycolate (i.e., mycolic acids that, together with arabinogalattans and lipoarabinomannans, make up the wall of mycobacterium), with subsequent accumulation of hydroximicolic acid and depletion of ketomycolates . Moreover, pretomanid also blocks the cellular respiratory processes of mycobacterium in an anaerobic environment through the release of nitric oxide, which kills M. tuberculosis . Thus, pretomanid is effective on both replication and latent M. tuberculosis cells, aerobically and anaerobically. The mechanism of action is therefore completely innovative. This was observed in laboratory experiments: Pretomanid-treated bacteria showed, in vitro, a different pattern of metabolites (especially with regard to the metabolic pathways of fatty acids, proteins, and the pentose-phosphate) than bacteria that received other antitubercular antibiotics . The SAR of pretomanid shows that the enantiomer S is the most active; moreover, the presence of a nitro group in position 2 of the imidazole ring, the lipophilic tail in position 6 of the oxazinic ring, and the rigidity of the bicyclic system are crucial for antitubercular activity. These important portions are also found in other nitroimidazole antibiotics (CGI-1734 and TBA-354, in phase I clinical development). Delamanid has notable affinities with pretomanid. Both delamanid and pretomanid are lipophilic, as required to penetrate the wall of the mycobacterium. Pretomanid is available in tablets for the treatment of pulmonary MDR and XDR tuberculosis; however, it is not active against extra-pulmonary tuberculosis, a particular form that fortunately represents only 5% of all existing TBC forms. Thanks to the latest drugs, the most aggressive and severe forms of tuberculosis resistant to traditional drugs are more treatable. Tuberculosis is the infectious disease that has caused the greatest number of deaths ever, that’s why research has witnessed remarkable growth, also thanks to the growing investments and collaborations promoted and stimulated by the United Nations General Assembly and the TB Alliance.
Only two antibiotics of the eight approved since 2017 represent a new chemical scaffold . The remaining antibiotics are actually derivatives of existing classes of compounds that bring benefits and advantages over traditional antibiotics. The eight new antibiotics all have activities against ESBL (extended spectrum β-lactamase) enzymes; most of them are effective against carbapenem-resistant Enterobacteria (KPC producers), while very few compounds are active against carbapenem-resistant P. aeruginosa and multidrug-resistant A. baumannii . Unfortunately, there are still an extremely limited number of therapeutic alternatives for the latter. These antibiotics are mainly used in the treatment of cUTI and cIAI. Further scientific evidence is needed to assess their actual effectiveness in the treatment of other infections. Note that the combination of vaborbactam, meropenem, and plazomycin was included in the WHO Model List of Essential Medicines. There is significant progress in research: The number of new effective antibiotics against Gram-negative bacteria has increased. Most of the compounds approved and in clinical development from 2017 to today, whose targets are pathogens included in the list drawn up by the WHO in 2016 (critical priority, high, and medium), consist of combinations between a β-lactam and a β-lactamase inhibitor. Cefiderocol is the only antibiotic that is active against all three pathogens of critical priority, along with the compound called SPR-206 phase I (an analogue of polymyxins with an excellent antibacterial spectrum). At the end of 2020, there were 43 antibiotics in clinical development, of which, 15 were Phase I, 13 in Phase II, and 13 in Phase III. As many as 19 antibiotics are shown to be effective in vitro in the treatment of infections caused by pathogens of the so-called ESKAPE group, an acronym that includes the Enterococcus faecium , S. aureus , K. pneumoniae , A. baumannii , P. aeruginosa , and Enterobacter species, responsible for the six main nosocomial infections related to care . It is, of course, essential that the new antibiotics developed do not have cross-resistance with other existing compounds. In fact, the search for new antibacterial drugs that result from the modification of traditional antibiotics is also based on knowledge of cross-resistance mechanisms. However, finding innovative chemical structures with new targets and binding sites is very difficult and yields fewer results than other approaches . Furthermore, in addition to the small and large molecules that have been analyzed in this review, there are other potentially effective non-traditional approaches such as fecal bacteriotherapy (also called fecal microbiome transplantation) in the treatment of recurrent C. difficile infections. Other non-traditional approaches (such as immunomodulators and phage products) have not yet entered clinical development due to considerable obstacles. Unfortunately, unfavorable market trends remain: Although public investments in the development of new antibiotics have increased slightly in recent years (mainly from Germany, the United Kingdom, and the United States, thanks to organizations such as BARDA, CARB-X, and GARDP), private investment has fallen further. Many pharmaceutical companies are abandoning research in this area, not least because of the high costs involved in the clinical development of a new antibiotic. In view of the rather long time required for clinical development, 11 new antibiotics are expected to be approved in the next five years, while many compounds are likely to remain stagnant in Phases II and III due to the costs involved. 6.1. Has the SARS-CoV-2 Pandemic Affected Antibiotic Resistance? A study conducted by the American non-profit organization The Pew Charitable Trusts, published in March 2021, examines the data of about 6000 SARS-CoV-2 positive and hospitalized patients in the United States, analyzing the period from February to July 2020 . The data collected show that half of patients (52%) received antibiotic treatment in the first six months of the pandemic, increasing to 90% in March and April. On the other hand, 36% of hospital admissions led to the use of several antibiotics at the same time. Antibiotics were prescribed, in fact, to prevent bacterial infections secondary to viral infection, often even before the bacterial infection was confirmed. Another factor that contributed to worsening the situation was the difficulty, on the part of medical staff, in distinguishing bacterial pneumonia from SARS-CoV-2 viral pneumonia, evident especially in the first months of the emergency, when knowledge about SARS-CoV-2 was extremely limited. The results suggest that, most likely, there was an excessive prescription of such drugs: Many patients, in fact, did not need antibiotic treatment. Bacterial infections can actually occur in patients diagnosed with viral infections, resulting in further deterioration of the clinical condition of the patient and complicating the therapy. The 20% of SARS-CoV-2 positive patients examined were affected by bacterial pneumonia, and in particular, community-acquired pneumonia. In 96% of the COVID-19 cases, antibiotics were administered within the first 48 h of admission to hospital. Few patients received other antibiotics in the period after 48 h after admission. However, considering patients who were given at least one antibiotic, only 33% had a confirmed diagnosis of community-acquired bacterial pneumonia. This means that in the remaining cases (67%), the antibiotic was prescribed unnecessarily, helping to fuel the phenomenon of antibiotic resistance. Moreover, the study showed that the most frequently used antibiotics were macrolide azithromycin (used for more than 50% of hospital admissions), ceftriaxone (42%), vancomycin (25%), and the piperacillin-tazobactam association (23%). These antibiotics are commonly prescribed precisely for the treatment of bacterial pneumonia. Azithromycin has been administered in many subjects with interstitial pneumonia from SARS-CoV-2, as it is usually used to eradicate Legionella or Chlamydia , which can cause a similar pneumonia. It should be added that some patients (29%) have been treated with antibiotics that can increase the risk of contracting the pathogen C. difficile . The massive use of antibiotics during the pandemic, especially those with a broad antibacterial spectrum, risks hindering and slowing down the progress and results achieved by research in recent years. Some situations and particular factors can favor or prevent the transmission of MDR organisms: A study reported in the Journal of Hospital Infection from 2020 analyzes the potential impact of the SARS-CoV-2 pandemic on hospital transmission of these pathogens . It is even more evident, given the current delicate situation, that the efforts of recent years will soon have to lead to the development of more and more antibiotics effective against multidrug-resistant organisms. However, it is not only antibiotics that are being cited: Recently, numerous research groups are focusing on new therapeutic approaches, which are one more weapon in the fight against antibiotic resistance. 6.2. Nanomedicine for Treatment of Infective Diseases A possible strategy could be the destruction of the extracellular matrix that constitutes the bacterial biofilm (aggregations of microorganisms that form surface-adherent films). About 60% of microbial infections are associated with biofilm formation, as the bacteria organized in that structure are able to resist multiple antibiotics and the host’s immune system. The destruction of the biofilm leads to the release of bacteria that, therefore, regain sensitivity to the action of antibiotics. Research groups are currently studying polymeric lipid nanoparticles involving the conjugation of ramnolipids (biosurfactants secreted by the pathogen P. aeruginosa ) and polymer nanoparticles in order to combat the resistance of H. pylori bacterial biofilm to commonly used antibiotics . This system contains clarithromycin encapsulated in a polymeric core of chitosan; above all, it has antibacterial properties, also managing to prevent the formation of biofilm and bacterial adhesion. By the same principle, rhamnolipid-coated silver and iron oxide nanoparticles have been developed, which have been shown to be effective in eradicating S. aureus and P. aeruginosa biofilms . Other structures that have been evaluated for their potential as release systems for antimicrobial drugs are crystalline liquid non-lamellar nanoparticles; they are made up of several amphiphilic structures with a large surface and are able to encapsulating both hydrophilic and hydrophobic drugs . An example is the positively charged nanoparticles containing rifampicin, which showed lower MIC values respect to non-encapsulated rifampicin by inhibiting the growth of S. aureus . There are also combinations between nanoparticles and natural compounds: Rodenak-Kladniew examined the incorporation of chitosan and eugenol (a natural phenolic compound) within a lipid matrix containing the antibiotic ofloxacin . The results showed increased bactericidal action against P. aeruginosa and S. aureus . Among the new systems for the release of antibiotics is the use of polymeric materials that respond to pH and the presence of enzymes at the site of infection, which allow the release of the active ingredient precisely where the infection occurs. Other approaches under study are antimicrobial oligonucleotides and photodynamic therapy . A system has been developed that involves loading the antibiotic ciprofloxacin into photoactivable liposomes; the authors report that about 90% of the active substance was released in less than 30 s . Combined therapy is often preferred to monotherapy to treat multidrug-resistant strains because the simultaneous use of several antibiotics with synergistic action allows, in fact, avoiding antibiotic resistance, increasing the antimicrobial spectrum, and decreasing the side effects of therapy. Co-encapsulation of several antibiotics into nanosystems can offer significant benefits: Research groups have manufactured liposomes with ciprofloxacin and colistine to treat P. aeruginosa infections . In vitro results have shown that combined therapy is more effective than monotherapy. Nano-antibiotics are another promising line of research. The transformation of therapeutic agents into corresponding structures at the order of the nanoscale can modify their chemical–physical properties, increase the bioavailability of the drug, and improve its interaction and penetration into the bacterial wall and therefore its effectiveness against resistant strains. Clarithromycin formulations in nanocrystals have shown activity against multidrug-resistant H. pylori : Nanocrystals allow the drug to be directed to the desired site with a better therapeutic profile than clarithromycin suspension and powder . Some nanostructured systems containing antibiotics and antimicrobial peptides are currently in clinical trials . For example, numerous inhalation formulations of liposomal ciprofloxacin are in Phase I, II, and III, while a formulation of liposomal amikacin for the treatment of recurrent P. aeruginosa infections in patients with cystic fibrosis is already in Phase III of clinical development. In nanomedicine, the advantages of using liposomes as antibiotic carriers range from the reduction of toxicity to the improvement of pharmacokinetic parameters and in particular, of biodistribution. The fusion of liposomal vesicles with the external membrane of the bacterium allows a better release of the antibiotic and a better penetration into the bacterial cell. Although nanostructured systems are more traditionally used in oncology and cancer immunotherapy, they could also represent a revolution in antibiotic delivery, where much remains to be discovered. It is clear that translating such antibiotic-loaded nanostructured systems into clinical practice requires further investigation and efforts to combat antibiotic resistance, which today is something of a “silent” pandemic. Thanks to the use of nanoparticles, it will be possible to overcome the resistance mechanisms: These structures allow a better internalization of antibiotics, both hydrophilic and hydrophobic, that are not enzymatically inactivated and selectively reach the site of infection. As reported in numerous studies, the use of nanoparticles loaded with antibiotics or antimicrobial peptides shows significant reductions in MIC values compared to the corresponding values expected from the use of non-encapsulated active ingredients. In this way, it is possible to inhibit the development of antibiotic resistance mechanisms. Despite the promising results obtained in vitro, there are still few formulations included in clinical trials, also due to the high costs of these preparations. In 2014, Jim O’Neill published an article commissioned by the British government entitled “ Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations ”, in which there was a completely catastrophic projection: The author estimated, in fact, that by 2050, there will be approximately 10 million deaths per year caused by antibiotic resistance, even higher than the sum of deaths from cancer and diabetes . This prediction has also been mentioned in numerous other publications, including the media and health authorities. To achieve that tragic value, the model used in the report multiplied the number of bloodstream infections nationwide by national resistance rates, as reported by the European Antimicrobial Resistance Surveillance Network. These are, of course, projections on a very sensitive subject, so it is still difficult to express an accurate opinion. Solid data on antibiotic resistance is needed, not only in Europe, as examined in the report, but also in less developed countries, to take concrete action. The progress of research in the coming years will be crucial, and the drugs analyzed in this review represent only the beginning, but they are a significant step forward which, combined with individual behavior and human responsibility, can really make a difference and allow for an inversion of current estimates.
A study conducted by the American non-profit organization The Pew Charitable Trusts, published in March 2021, examines the data of about 6000 SARS-CoV-2 positive and hospitalized patients in the United States, analyzing the period from February to July 2020 . The data collected show that half of patients (52%) received antibiotic treatment in the first six months of the pandemic, increasing to 90% in March and April. On the other hand, 36% of hospital admissions led to the use of several antibiotics at the same time. Antibiotics were prescribed, in fact, to prevent bacterial infections secondary to viral infection, often even before the bacterial infection was confirmed. Another factor that contributed to worsening the situation was the difficulty, on the part of medical staff, in distinguishing bacterial pneumonia from SARS-CoV-2 viral pneumonia, evident especially in the first months of the emergency, when knowledge about SARS-CoV-2 was extremely limited. The results suggest that, most likely, there was an excessive prescription of such drugs: Many patients, in fact, did not need antibiotic treatment. Bacterial infections can actually occur in patients diagnosed with viral infections, resulting in further deterioration of the clinical condition of the patient and complicating the therapy. The 20% of SARS-CoV-2 positive patients examined were affected by bacterial pneumonia, and in particular, community-acquired pneumonia. In 96% of the COVID-19 cases, antibiotics were administered within the first 48 h of admission to hospital. Few patients received other antibiotics in the period after 48 h after admission. However, considering patients who were given at least one antibiotic, only 33% had a confirmed diagnosis of community-acquired bacterial pneumonia. This means that in the remaining cases (67%), the antibiotic was prescribed unnecessarily, helping to fuel the phenomenon of antibiotic resistance. Moreover, the study showed that the most frequently used antibiotics were macrolide azithromycin (used for more than 50% of hospital admissions), ceftriaxone (42%), vancomycin (25%), and the piperacillin-tazobactam association (23%). These antibiotics are commonly prescribed precisely for the treatment of bacterial pneumonia. Azithromycin has been administered in many subjects with interstitial pneumonia from SARS-CoV-2, as it is usually used to eradicate Legionella or Chlamydia , which can cause a similar pneumonia. It should be added that some patients (29%) have been treated with antibiotics that can increase the risk of contracting the pathogen C. difficile . The massive use of antibiotics during the pandemic, especially those with a broad antibacterial spectrum, risks hindering and slowing down the progress and results achieved by research in recent years. Some situations and particular factors can favor or prevent the transmission of MDR organisms: A study reported in the Journal of Hospital Infection from 2020 analyzes the potential impact of the SARS-CoV-2 pandemic on hospital transmission of these pathogens . It is even more evident, given the current delicate situation, that the efforts of recent years will soon have to lead to the development of more and more antibiotics effective against multidrug-resistant organisms. However, it is not only antibiotics that are being cited: Recently, numerous research groups are focusing on new therapeutic approaches, which are one more weapon in the fight against antibiotic resistance.
A possible strategy could be the destruction of the extracellular matrix that constitutes the bacterial biofilm (aggregations of microorganisms that form surface-adherent films). About 60% of microbial infections are associated with biofilm formation, as the bacteria organized in that structure are able to resist multiple antibiotics and the host’s immune system. The destruction of the biofilm leads to the release of bacteria that, therefore, regain sensitivity to the action of antibiotics. Research groups are currently studying polymeric lipid nanoparticles involving the conjugation of ramnolipids (biosurfactants secreted by the pathogen P. aeruginosa ) and polymer nanoparticles in order to combat the resistance of H. pylori bacterial biofilm to commonly used antibiotics . This system contains clarithromycin encapsulated in a polymeric core of chitosan; above all, it has antibacterial properties, also managing to prevent the formation of biofilm and bacterial adhesion. By the same principle, rhamnolipid-coated silver and iron oxide nanoparticles have been developed, which have been shown to be effective in eradicating S. aureus and P. aeruginosa biofilms . Other structures that have been evaluated for their potential as release systems for antimicrobial drugs are crystalline liquid non-lamellar nanoparticles; they are made up of several amphiphilic structures with a large surface and are able to encapsulating both hydrophilic and hydrophobic drugs . An example is the positively charged nanoparticles containing rifampicin, which showed lower MIC values respect to non-encapsulated rifampicin by inhibiting the growth of S. aureus . There are also combinations between nanoparticles and natural compounds: Rodenak-Kladniew examined the incorporation of chitosan and eugenol (a natural phenolic compound) within a lipid matrix containing the antibiotic ofloxacin . The results showed increased bactericidal action against P. aeruginosa and S. aureus . Among the new systems for the release of antibiotics is the use of polymeric materials that respond to pH and the presence of enzymes at the site of infection, which allow the release of the active ingredient precisely where the infection occurs. Other approaches under study are antimicrobial oligonucleotides and photodynamic therapy . A system has been developed that involves loading the antibiotic ciprofloxacin into photoactivable liposomes; the authors report that about 90% of the active substance was released in less than 30 s . Combined therapy is often preferred to monotherapy to treat multidrug-resistant strains because the simultaneous use of several antibiotics with synergistic action allows, in fact, avoiding antibiotic resistance, increasing the antimicrobial spectrum, and decreasing the side effects of therapy. Co-encapsulation of several antibiotics into nanosystems can offer significant benefits: Research groups have manufactured liposomes with ciprofloxacin and colistine to treat P. aeruginosa infections . In vitro results have shown that combined therapy is more effective than monotherapy. Nano-antibiotics are another promising line of research. The transformation of therapeutic agents into corresponding structures at the order of the nanoscale can modify their chemical–physical properties, increase the bioavailability of the drug, and improve its interaction and penetration into the bacterial wall and therefore its effectiveness against resistant strains. Clarithromycin formulations in nanocrystals have shown activity against multidrug-resistant H. pylori : Nanocrystals allow the drug to be directed to the desired site with a better therapeutic profile than clarithromycin suspension and powder . Some nanostructured systems containing antibiotics and antimicrobial peptides are currently in clinical trials . For example, numerous inhalation formulations of liposomal ciprofloxacin are in Phase I, II, and III, while a formulation of liposomal amikacin for the treatment of recurrent P. aeruginosa infections in patients with cystic fibrosis is already in Phase III of clinical development. In nanomedicine, the advantages of using liposomes as antibiotic carriers range from the reduction of toxicity to the improvement of pharmacokinetic parameters and in particular, of biodistribution. The fusion of liposomal vesicles with the external membrane of the bacterium allows a better release of the antibiotic and a better penetration into the bacterial cell. Although nanostructured systems are more traditionally used in oncology and cancer immunotherapy, they could also represent a revolution in antibiotic delivery, where much remains to be discovered. It is clear that translating such antibiotic-loaded nanostructured systems into clinical practice requires further investigation and efforts to combat antibiotic resistance, which today is something of a “silent” pandemic. Thanks to the use of nanoparticles, it will be possible to overcome the resistance mechanisms: These structures allow a better internalization of antibiotics, both hydrophilic and hydrophobic, that are not enzymatically inactivated and selectively reach the site of infection. As reported in numerous studies, the use of nanoparticles loaded with antibiotics or antimicrobial peptides shows significant reductions in MIC values compared to the corresponding values expected from the use of non-encapsulated active ingredients. In this way, it is possible to inhibit the development of antibiotic resistance mechanisms. Despite the promising results obtained in vitro, there are still few formulations included in clinical trials, also due to the high costs of these preparations. In 2014, Jim O’Neill published an article commissioned by the British government entitled “ Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations ”, in which there was a completely catastrophic projection: The author estimated, in fact, that by 2050, there will be approximately 10 million deaths per year caused by antibiotic resistance, even higher than the sum of deaths from cancer and diabetes . This prediction has also been mentioned in numerous other publications, including the media and health authorities. To achieve that tragic value, the model used in the report multiplied the number of bloodstream infections nationwide by national resistance rates, as reported by the European Antimicrobial Resistance Surveillance Network. These are, of course, projections on a very sensitive subject, so it is still difficult to express an accurate opinion. Solid data on antibiotic resistance is needed, not only in Europe, as examined in the report, but also in less developed countries, to take concrete action. The progress of research in the coming years will be crucial, and the drugs analyzed in this review represent only the beginning, but they are a significant step forward which, combined with individual behavior and human responsibility, can really make a difference and allow for an inversion of current estimates.
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Postmortem CT Angiography Compared with Autopsy: A Forensic Multicenter Study | b4181954-507f-4714-b00d-e3aeb738059d | 6027995 | Pathology[mh] | With the introduction of modern cross-sectional imaging techniques such as multi–detector row computed tomography (CT) and magnetic resonance (MR) imaging to postmortem investigations, forensic pathology has taken an important step forward . Relatively low maintenance costs, short examination times, and ease of operation make CT a widely used cross-sectional imaging technique in modern postmortem imaging . Compared with conventional autopsy, postmortem CT has several advantages, which can lead to important improvements in both research and postmortem investigation . The main reported weakness of postmortem CT, however, is relatively low soft-tissue contrast, especially in organ parenchyma, and poor ability to view the vascular system . Cardiovascular disease is a major cause of unexpected natural death in most developed countries , and this limitation decreases the potential of postmortem CT to help diagnose cardiovascular disease. Consequently, the reference standard for investigation of natural death, and particularly cardiovascular death, is considered to be conventional autopsy . In clinical radiology, these limitations are addressed with the use of intravenous contrast agents. Consequently, various postmortem angiographic techniques have been developed . Probably the most widespread single approach for postmortem angiography today is multiphase postmortem CT angiography , first described in 2011 . This technique uses a standardized procedure on the basis of a defined injection and a scanning protocol that uses a specific perfusion device and an oil-based contrast agent of specific viscosity. A previous study revealed that the addition of postmortem CT angiography to postmortem CT increased the sensitivity for detecting pathologic findings from 64% to approximately 81%, which is comparable to the sensitivity of conventional autopsy (∼83%). Because this study was conducted in only one center of forensic medicine and thus it involved a small number of human corpses, the technical working group for postmortem angiography methods decided in 2012 to initiate a multicenter study with the goal of validating the technique of multiphase postmortem CT angiography on a large number of cases. The purpose of this study was to determine if postmortem CT and postmortem CT angiography help to detect more lesions than autopsy in postmortem examinations, to evaluate the strengths and weaknesses of each method, and to define their indications.
Study Design The study was a prospective multicenter study. Nine European centers participated and acquired data from unenhanced postmortem CT, postmortem CT angiography, and conventional autopsy on 500 human corpses in which an autopsy was ordered. The study was conducted from February 2012 to August 2015. Inclusion criteria were as follows: subject age older than 16 years, the performance of all three examinations in accordance with the standardized protocol for every case, and a fully recorded data set. This study was partially funded by a private company (Fumedica AG, Muri, Switzerland). The funding included travel expenses, cost of tubing sets and contrast agent, rental for a Virtangio device for the duration of the study (for centers in Foggia, Italy; Krakow, Poland; Leipzig and Munich, Germany; Toulouse, France; Leicester, England; and Basel, Switzerland). Study Preparation Where required, the approval of local ethics committees was obtained (depending on the country or previously existing agreements) in accordance with local legislation. The examinations from England additionally received consent from the next of kin. Training in postmortem CT angiography was provided in a preparation phase by the principal investigator’s center to each attending center, including at least 1 week of training in the performance of postmortem CT angiography. Data Acquisition CT images were acquired according to standardized scanning protocols adapted to the equipment at each center. An overview of the CT parameters is as follows: section thickness, 0.75–3 mm; spacing interval, 0.6–2 mm; field of view, 414–500 mm; tub voltage, 110–130 mm; tube current, 100–380 mA; and standard algorithm of reconstruction. Details are provided in (online). Postmortem CT angiography was performed by using the standardized protocol described by Grabherr et al . This included the application of specific single-use sets for femoral vascular cannulation and the injection of a contrast agent mixture of an oil-based solution of defined viscosity (mixture of paraffin oil [paraffinum liquidum] with 6% oil-based contrast agent [Angiofil; Fumedica, Muri, Switzerland]) by using a special perfusion device (Virtangio; Fumedica, Muri, Switzerland) and the standardized injection protocol, including an arterial, venous, and dynamic phase of injection. Conventional forensic autopsy was performed on each body by the forensic pathologists in charge of the case in accordance with local and European requirements and standards (examination of the cranial, thoracic, and abdominal cavities) . These experts were informed of the most important radiologic findings before the autopsy was performed, which enabled them to adequately adapt their autopsy technique except in England, where autopsy was performed according to Royal College of Pathologists guidelines independent of the radiologic results. A complete autopsy report was provided by the lead forensic pathologists. Data Registration and Analysis A team from the coordinating study center evaluated the data from the included cases (S.G., a board-certified forensic pathologist with 10 years of experience interpreting radiologic data, particularly postmortem CT angiography data; J.M.G., a board-certified radiologist with 5 years of experience reading postmortem CT and postmortem CT angiography data; and P.M., K.M., and C.P., all board-certified forensic pathologists with 15–30 years of experience in the field and fluent in the local language of the visited center). The first two team members read the radiologic images of the included bodies (postmortem CT and postmortem CT angiography) and entered every pathologic finding into a spreadsheet. The second forensic pathologist extracted all pathologic findings reported by the local forensic pathologists from the autopsy report. All findings described at postmortem CT, postmortem CT angiography, and autopsy were then entered in a spreadsheet (Excel; Microsoft, Redmond, Wash) together with background case information (ie, age, sex, and circumstances of death). Regarding the circumstances of death, we grouped cases into four groups on the basis of the cause of death and the indication of the postmortem examination: natural death (eg, cardiovascular incident and sepsis); polytrauma (severe multisystem injury such as fall from great height or traffic accident); other violent death (localized injuries such as ballistic trauma, sharp force trauma, and blunt force trauma); and suspected medical error (death during or shortly after a medical intervention in which a postmortem examination was performed to prove or rule out a medical error). The different findings were jointly classified by two board-certified forensic pathologists from the coordinating study center (S.G. and either K.M., P.M., or C.P.) into three groups (essential, useful, or unimportant) on the basis of their importance to the forensic investigation of the case, such as their contribution toward identifying the cause of death, the events that led up to the death, and reconstruction of the forensic background. For example, in a cardiac death, coronary plaques, stenoses, and the associated myocardial infarction would be considered essential; other vascular pathologic analyses would be considered useful; and an old appendectomy or hip prosthesis would be considered unimportant. Findings were also categorized anatomically as bone, soft tissue (including muscles, tendons, connective and fatty tissue, and the skin), parenchyma (parenchymatous organs, intestines, and the heart), and vascular (calcification, stenosis, occlusion, aneurysm, and rupture of vessels including the coronary arteries). Findings of additional examinations and analyses before or after imaging and/or autopsy (most importantly external examination, histologic analysis, and toxicology analysis) were not considered in this study because they can be performed independently or combined with both conventional autopsy and modern imaging methods. Statistical Analysis Statistical analyses were performed by using statistical software (MedCalc version 15.6.1, MedCalc Software, Ostend, Belgium; and SPSS version 25, IBM, Armonk, NY). Postmortem CT, postmortem CT angiography, and autopsy results were compared by using Cochran Q test. The required difference between groups for pairwise comparisons was determined according to Sheskin if Cochran Q test showed a statistically significant result . Values presented in the text were additionally evaluated with McNemar test by using the simple sampling bias corrected accelerated bootstrap method with 1000 samples. A P value of less than .05 was considered to indicate statistical significance.
The study was a prospective multicenter study. Nine European centers participated and acquired data from unenhanced postmortem CT, postmortem CT angiography, and conventional autopsy on 500 human corpses in which an autopsy was ordered. The study was conducted from February 2012 to August 2015. Inclusion criteria were as follows: subject age older than 16 years, the performance of all three examinations in accordance with the standardized protocol for every case, and a fully recorded data set. This study was partially funded by a private company (Fumedica AG, Muri, Switzerland). The funding included travel expenses, cost of tubing sets and contrast agent, rental for a Virtangio device for the duration of the study (for centers in Foggia, Italy; Krakow, Poland; Leipzig and Munich, Germany; Toulouse, France; Leicester, England; and Basel, Switzerland).
Where required, the approval of local ethics committees was obtained (depending on the country or previously existing agreements) in accordance with local legislation. The examinations from England additionally received consent from the next of kin. Training in postmortem CT angiography was provided in a preparation phase by the principal investigator’s center to each attending center, including at least 1 week of training in the performance of postmortem CT angiography.
CT images were acquired according to standardized scanning protocols adapted to the equipment at each center. An overview of the CT parameters is as follows: section thickness, 0.75–3 mm; spacing interval, 0.6–2 mm; field of view, 414–500 mm; tub voltage, 110–130 mm; tube current, 100–380 mA; and standard algorithm of reconstruction. Details are provided in (online). Postmortem CT angiography was performed by using the standardized protocol described by Grabherr et al . This included the application of specific single-use sets for femoral vascular cannulation and the injection of a contrast agent mixture of an oil-based solution of defined viscosity (mixture of paraffin oil [paraffinum liquidum] with 6% oil-based contrast agent [Angiofil; Fumedica, Muri, Switzerland]) by using a special perfusion device (Virtangio; Fumedica, Muri, Switzerland) and the standardized injection protocol, including an arterial, venous, and dynamic phase of injection. Conventional forensic autopsy was performed on each body by the forensic pathologists in charge of the case in accordance with local and European requirements and standards (examination of the cranial, thoracic, and abdominal cavities) . These experts were informed of the most important radiologic findings before the autopsy was performed, which enabled them to adequately adapt their autopsy technique except in England, where autopsy was performed according to Royal College of Pathologists guidelines independent of the radiologic results. A complete autopsy report was provided by the lead forensic pathologists.
A team from the coordinating study center evaluated the data from the included cases (S.G., a board-certified forensic pathologist with 10 years of experience interpreting radiologic data, particularly postmortem CT angiography data; J.M.G., a board-certified radiologist with 5 years of experience reading postmortem CT and postmortem CT angiography data; and P.M., K.M., and C.P., all board-certified forensic pathologists with 15–30 years of experience in the field and fluent in the local language of the visited center). The first two team members read the radiologic images of the included bodies (postmortem CT and postmortem CT angiography) and entered every pathologic finding into a spreadsheet. The second forensic pathologist extracted all pathologic findings reported by the local forensic pathologists from the autopsy report. All findings described at postmortem CT, postmortem CT angiography, and autopsy were then entered in a spreadsheet (Excel; Microsoft, Redmond, Wash) together with background case information (ie, age, sex, and circumstances of death). Regarding the circumstances of death, we grouped cases into four groups on the basis of the cause of death and the indication of the postmortem examination: natural death (eg, cardiovascular incident and sepsis); polytrauma (severe multisystem injury such as fall from great height or traffic accident); other violent death (localized injuries such as ballistic trauma, sharp force trauma, and blunt force trauma); and suspected medical error (death during or shortly after a medical intervention in which a postmortem examination was performed to prove or rule out a medical error). The different findings were jointly classified by two board-certified forensic pathologists from the coordinating study center (S.G. and either K.M., P.M., or C.P.) into three groups (essential, useful, or unimportant) on the basis of their importance to the forensic investigation of the case, such as their contribution toward identifying the cause of death, the events that led up to the death, and reconstruction of the forensic background. For example, in a cardiac death, coronary plaques, stenoses, and the associated myocardial infarction would be considered essential; other vascular pathologic analyses would be considered useful; and an old appendectomy or hip prosthesis would be considered unimportant. Findings were also categorized anatomically as bone, soft tissue (including muscles, tendons, connective and fatty tissue, and the skin), parenchyma (parenchymatous organs, intestines, and the heart), and vascular (calcification, stenosis, occlusion, aneurysm, and rupture of vessels including the coronary arteries). Findings of additional examinations and analyses before or after imaging and/or autopsy (most importantly external examination, histologic analysis, and toxicology analysis) were not considered in this study because they can be performed independently or combined with both conventional autopsy and modern imaging methods.
Statistical analyses were performed by using statistical software (MedCalc version 15.6.1, MedCalc Software, Ostend, Belgium; and SPSS version 25, IBM, Armonk, NY). Postmortem CT, postmortem CT angiography, and autopsy results were compared by using Cochran Q test. The required difference between groups for pairwise comparisons was determined according to Sheskin if Cochran Q test showed a statistically significant result . Values presented in the text were additionally evaluated with McNemar test by using the simple sampling bias corrected accelerated bootstrap method with 1000 samples. A P value of less than .05 was considered to indicate statistical significance.
Autopsy was performed on the day of the postmortem CT and postmortem CT angiography examination, or the following day, in all cases. The maximum interval between death and autopsy was 5 days. After examination of 500 bodies, a total of 18 654 findings were recorded. Demographic data are in . Results of the comparisons between postmortem CT, postmortem CT angiography, and autopsy are in . Further details regarding the stratification of results across sites and for different manners of death can be found in and (online). Diagrams that display the cumulative advantage of autopsy, postmortem CT, and postmortem CT angiography are in . When viewed in conjunction, the diagrams show that about a quarter to a fifth of the essential and useful findings is missed at autopsy. When postmortem CT is performed in addition to autopsy, the proportion of missed findings is reduced to about 10%. Roughly the same amount and quality of information is delivered by combining postmortem CT and postmortem CT angiography. The best results are achieved when autopsy is combined with postmortem CT and postmortem CT angiography, especially in cases of natural death and malpractice. The greatest advantage of postmortem CT or postmortem CT angiography over autopsy was observed for the detection of bone and vascular lesions for any manner of death . For both essential findings and all findings, only 9.7% (428 of 4393) and 10.0% (1874 of 18 654), respectively ( P < .001), would have been overlooked if postmortem CT angiography had been performed without autopsy. If autopsy had been performed without postmortem CT or postmortem CT angiography, 38.7% of all findings (7221 of 18 654) and 23.4% of essential findings (1029 of 4393) would not have been reported ( P < .001). If only postmortem CT had been performed, 24.0% of all findings (4475 of 18 654) and 37.5% of essential findings (1647 of 4393) would have remained unreported ( P < .001). In cases of natural death ( [online]), only about half of the essential bone lesions (53.8%; seven of 13) and two-thirds of essential soft tissue lesions (64.0%; 32 of 50) were detected at autopsy, whereas postmortem CT angiography helped to detect 84.6% (11 of 13; P > .05) and 90.0% (45 of 50; P < .05), respectively. This superiority was less pronounced for essential vascular lesions (autopsy vs postmortem CT angiography, 79.3% [472 of 595] vs 90.8% [540 of 595], respectively; P < .001) whereas more essential parenchyma lesions were detected at autopsy (autopsy vs postmortem CT angiography, 85.9% [593 of 690] vs 81.0% [559 of 690], respectively; P < .05). In cases of violent death ( [online]), more than half of the essential vascular findings were missed at autopsy (60.9%; 95 of 156). At postmortem CT, 87.8% (137 of 156) of essential vascular findings were missed, whereas less than 1% (one of 156) were missed at postmortem CT angiography (all P < .001). However, autopsy showed a slightly higher detection rate for essential soft-tissue lesions versus postmortem CT angiography (90.2% [642 of 712] vs 88.6% [631 of 712], respectively), which was not statistically significant. In cases of polytrauma ( [online]), more than two-thirds of essential vascular lesions were not detected at autopsy (68.6%; 81 of 118). Only 5.9% of these (seven of 118) were detected at postmortem CT, whereas all were detected at postmortem CT angiography (all P < .001). Both postmortem CT and postmortem CT angiography performed better for the detection of essential bone and parenchyma findings ( P < .005); however, for essential soft-tissue lesions, postmortem CT angiography performed better than autopsy ( P < .01). The difference with postmortem CT was not statistically significant. In cases of suspected medical error ( [online]), 93.3% (125 of 134) of essential vascular lesions were detected at postmortem CT angiography. However, slightly less than a third (30.6%; 41 of 134) and two-thirds (63.4%; 85 of 134) of essential vascular lesions were detected at postmortem CT and autopsy, respectively (all P < .001).
Our results show that postmortem CT angiography is superior to autopsy for all findings except essential soft-tissue findings. The superiority of imaging over autopsy for the total and essential findings is strongly influenced by the significantly higher detection rates for bone and vascular lesions that together make up more than half of the findings. An example of a bone lesion, clearly visible at CT but not identifiable in autopsy, is shown in . Whereas the bone lesions are already visible on the CT image, the vascular findings are rendered visible at CT angiography. Many of those vascular findings were not even visible in conventional autopsy, especially if small vessels were concerned. The advantage of postmortem CT angiography over autopsy and postmortem CT is therefore most notable when vascular pathologic changes are prevalent. As expected, postmortem CT angiography resulted in additional findings compared with postmortem CT. The greatest number of additional essential findings were registered in cases of natural death and medical errors and the majority of findings were categorized as vascular and parenchyma, for which postmortem CT angiography is especially sensitive. The cases in which postmortem CT angiography showed bone lesions that were not detected at postmortem CT (eight examinations) can be explained by bone contusions or compression fractures with visible contrast agent extravasation but no morphologic fracture signs. Our results also confirm the superior detection of bone lesions with postmortem CT compared with autopsy, whereas its detection rate for essential parenchyma, soft-tissue, and vascular lesions was significantly lower. The greatest number of findings in cases of natural death and suspected medical errors were missed at postmortem CT, in which half or more of the essential findings were missed. In both of these groups, the majority of pathologic findings were categorized as vascular or parenchymal, in which unenhanced CT is less sensitive. Interestingly, the overall detection rate of soft-tissue lesions was slightly higher at postmortem CT than it was at autopsy. However, this superiority is on the basis of useful and unimportant findings, whereas for essential soft-tissue findings autopsy was superior to postmortem CT. This result may be explained by the sensitivity of postmortem CT to pathologic changes in the subcutaneous fat. These lesions are less frequently detected or described at autopsy and only rarely considered essential. Although these results should be interpreted with caution, they show that a number of essential findings are not detected at autopsy, especially bone and vascular lesions. This confirms the results of previous studies , and is also in line with our professional experience. In some cases, the interpretation of the autopsy results regarding the cause of death and events leading to death would have been incomplete or simply wrong if postmortem CT and postmortem CT angiography had not been performed. We do not know if any potential findings were missed despite the use of both postmortem CT angiography and autopsy, and if they were missed, we do not know how many. However, even though we were unable to test postmortem CT angiography and autopsy against an independent reference standard (because none exists), our results show that the combination of postmortem CT angiography and autopsy is clearly superior to autopsy alone. There has been concern in many countries about declining autopsy rates over recent decades. A high-quality postmortem examination is important not only in forensic cases, but also for the evaluation of the quality of clinical diagnosis and therapy in clinical pathologic analysis. It is thus an important instrument for both justice and medical quality control. The importance of both aspects cannot be overestimated. Postmortem CT and postmortem CT angiography might be feasible ways to increase the number of high quality postmortem examinations. An efficient postmortem examination should be performed in a stepwise manner, beginning with a thorough external examination of the body and the circumstances of death. The next step would be to perform postmortem CT, which may be sufficient to confirm a suspected cause and manner of death. Even if the results are inconclusive, it acts as a triage tool because findings from postmortem CT help to determine whether autopsy, postmortem CT angiography, targeted histologic analysis, or any combination would be best to help ascertain the cause and manner of death. If used in this way, postmortem CT would in many cases shorten the length of postmortem examinations and reduce the effort required to perform them. The more time-consuming and more expensive techniques of postmortem CT angiography, autopsy, and histologic analysis would be reserved for ambiguous postmortem CT results. The number of high-quality postmortem examinations could be increased relatively easily to counter declining autopsy rates and the overall quality of postmortem examinations would increase. Our study has limitations. In some institutes that participated in this study, postmortem CT and postmortem CT angiography were routine practice as a means of informing the autopsy. Therefore, in these examinations it was not possible to blind the forensic pathologists who performed the autopsy to the imaging results for both legal and ethical reasons. As a result, we expect that some findings were reported at autopsy that might otherwise have remained undetected, improving the apparent diagnostic ability of autopsy. Because we were unable to use a double-blind study design, we could not assess the accuracy of the final diagnosis of the cause of death. For the same reason, the sensitivity and specificity of the applied methods were not calculated because the value would not be sufficiently objective. Therefore, only the lesion detection rate was used for comparisons. This can be interpreted as sensitivity if the combination of autopsy with postmortem CT or postmortem CT angiography is considered the ground truth. The study design enabled us to evaluate organ-specific findings, which can be identified by using different techniques. It did not focus on the advantages and limitations for specific case groups. Further studies focused on particular demands (eg, cardiovascular deaths, deaths in childhood, and deaths related to gunshots) and anatomic regions (eg, cardiac and cerebral) are required. Our study leads to several important conclusions. A number of important findings remain unreported when postmortem CT, postmortem CT angiography, or autopsy are not considered in conjunction with each other. Indeed, postmortem CT angiography detects a greater number of important findings than autopsy, especially vascular and bone findings; it is therefore the method of choice for vascular findings. For parenchyma and soft-tissue findings, small differences are shown at autopsy and postmortem CT angiography, but postmortem CT alone is inferior. By combining autopsy and CT angiography, the reported number of findings can be increased, leading to a better postmortem examination. If only imaging or autopsy can be applied, the choice depends on the investigated case and the suspected findings. Summary If autopsy had been performed without postmortem CT, 39% of all findings and 23% of essential findings would not have been reported. Implications for Patient Care ■ Postmortem CT angiography is superior to autopsy and CT without angiography to help detect forensically essential findings. ■ The combination of autopsy and multiphase CT angiography helps to reveals most findings.
If autopsy had been performed without postmortem CT, 39% of all findings and 23% of essential findings would not have been reported.
■ Postmortem CT angiography is superior to autopsy and CT without angiography to help detect forensically essential findings. ■ The combination of autopsy and multiphase CT angiography helps to reveals most findings.
Tables E1–E3 (PDF)
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Information theory approaches to improve glioma diagnostic workflows in surgical neuropathology | 5ce8380a-30f0-4cd5-85e1-c4406468228d | 9425010 | Pathology[mh] | INTRODUCTION The integration of molecular subtyping in brain tumor diagnosis for patients in low‐resource settings represents a huge inequity in clinical care and is the premier leadership challenge facing the neuropathology community. Without a doubt, the integration of molecular data with histology has improved diagnosis and prognostication and assisted with neuro‐oncology and radiation oncology treatment planning . However, the deployment of these technologies globally is fraught with caveats. Per recent reports, the United States has less than 450 formally trained neuropathologists (and fewer with molecular diagnostics experience), and the number of pathologists per capita shows a negative trajectory over time . This trend is worse throughout the world. Globally, pathologist training is under‐resourced, pathology practice is poorly compensated, dedicated subspecialty training is nonexistent, and poorly implemented laboratory procedures for basic histochemical and immunohistochemical assays dampen the prospects of molecular assay deployment. Although some of the technical costs of molecular assays have declined, low‐resource healthcare ecosystems also suffer from a paucity of clinical bioinformaticists . This lack of human resource investment in clinical bioinformaticists may even be a more dire threat to molecular pathology deployment than the distribution of sequencing technologies. Furthermore, as primary brain cancers are rare, brain tumor‐specific immunohistochemical biomarker testing is not commonly available even in some academic pathology laboratories. These realities directly conflict with the diagnostic recommendations put forth in the WHO Classification of CNS Tumors. Solutions that attempt to overcome the lack of investment in reagents, physical resources, and human resources are needed. For these reasons, a strong interest within neuropathology has emerged to implement machine learning/artificial intelligence‐based workflows in routine clinical practice. Alternative solutions, including centralized diagnostic services, have been attempted. For instance, Crosier et al. reported on a centralized neuropathology review strategy for medulloblastoma in the United Kingdom which evaluated histology, RNAseq, and DNA methylation data of ~80 medulloblastoma patients per year . They reported a modified risk stratification for 29% of patients and provided a critical feasibility analysis of this model. However, such a centralized review may not be feasible in all healthcare ecosystems. For instance, in the United States, DNA methylation profiling is not widely available or clinically validated in many laboratories. Expanding testing to all brain tumors is highly problematic. Chief among these challenges is the well‐known problem that diagnostic tests lose their predictive values when disease incidence decreases in the sampling population. This phenomenon most commonly occurs when increasing the number of subjects being tested without regard to pretest probability thresholds. Furthermore, in some clinical paradigms, next‐generation sequencing (NGS) has shown to be cost‐effective only at specific pretest probability thresholds . Triaging tests based on the incorporation of clinical, histologic, and immunohistochemical findings have been proposed as a cost‐effective solution . With these concerns in mind, we focused on generating simple, easily deployable tools for assessing the most critical investment strategies for low‐resource laboratories. To achieve this, we utilized information theory to identify the most critical data required for diagnostics and posit that simple information theory calculations can aid in determining investment strategies for biomarker acquisition. MATERIALS AND METHODS 2.1 Brain cancer simulated population model Our brain cancer population simulation is composed of 51 diagnostic entities obtained from the WHO Classification of CNS Tumors . Data on these features were extracted from the scientific literature to generate a simulation of patient populations with these entities. The clinical features, the histologic features, immunoohistochemichal markers, and molecular features were delineated in , with all references (summary of each entity is available in Figures ). The importance of each feature in the dignosis of this simulation model was performed by separating all the data into a training set (70%) and a test set (30%). The randomForest algorithm was called from R’s randomForest package using the default values, and the efficiency of the model was tested by evaluation of a confusion matrix by the confusionMatrix function call in R’s caret package, which showed that this model had an accuracy of 0.95 (95% CI = [0.952, 0.9564], no information rate = 0.068, and p < 10 −12 ). We then split the dataframe by diagnosis and, for each diagnosis, passed the a random forest classifier followed on the original dataframe. For each feature of the dataframe, we scrambled each diagnostic feature by performing a random sampling of the original dataframe containing all diagnoses and saved the value of the accuracy generated by random forest classifier. These data are presented in Table . 2.2 Real‐world data sets for information theory validation Nine different data sets from different countries were used for the calculations of Bayesian probabilities and mutual information. The numbers of available cases and details were delineated in Table . 2.3 Age distribution and probability calculation Age distribution of IDH1/2 ‐mutant gliomas was presented as density plots. For the Bayesian probability plots, probabilities of 1p/19q codeletion in IDH1/2 mutant tumors given age and gender were calculated using the “naivebayes” package in R with a Laplace correction for smoothing. 2.4 Information theory calculations Information theory, initially developed by Claude E. Shannon at Bell Labs in 1948 , rests on the notion that information can be objectively quantified into units called bits (or nats, where 1 nat =log2(e)*bit). Mutual information is the exact opposite term of entropy, where entropy represents the uncertainty of the information. Maximum mutual information indicates all information quantity needed for the 1p/19q codeletion status (or diagnosis) in our example. For simplicity, the provided information with features is represented as a percentage of maximum mutual information (as in Figures , , and ). Details were delineated in , . 2.5 Image acquisition Hematoxylin–eosin (H&E)‐stained sections and p53 and Olig2 immunohistochemistry‐stained sections of IDH ‐mutant diffuse gliomas were selected for image segmentation. Details were delineated in , . 2.6 Segmentation methods (unsupervised and supervised) and evaluation of segmentation fidelity Two types of segmentation methods were used in our analysis: unsupervised (Deconvolution combined with Otsu thresholding, K‐means, and the Cut‐Cluster‐Classify) and supervised methods (Trainable Weka Segmentation and U‐Net). We made a ground truth using GIMP and R for the evaluation of segmentation fidelity and U‐Net training. Details were delineated in , . 2.7 Evaluation of segmentation fidelity For our evaluation, we only have two regions of interest: cell nuclei (stained in purple for H&E images and brown for Olig2 and p53 images) and background. Since one of the challenges of segmenting our data set comes from the various levels of noise, we also developed an image noise assay to test how robust the methods are to noise. Percentage ratios of added noise were selected as 0, 5, 10 15, 25, 50, 75, 90 to evaluate the whole pattern of noise resistance. We report accuracy and intersection over union (IoU). Details were delineated in , . 2.8 Data processing 2.8.1 Condensation of data and probability calculation of Olig2 features Eighty‐nine features were extracted from the nuclei in each channel of the images using the masks after the segmentation with the deconvolution method. After feature extraction, we excluded incomplete nuclei in the images for a better representation. Extracted features are shape, moment, pixel intensity, and Haralick's features. We had 213 features left after the elimination of repeated features in different channels. After principal component analysis (PCA) application, 8 principal components (PCs) in Olig2 features and 9 principal components (PCs) in H&E features were selected for the representation of 90% variation of features. We calculated the sample density of nuclei in a multidimensional space of PCs. Three hundred and fifty nuclei were sampled in each case for density calculation of features extracted from H&E‐ and Olig2‐stained images. The mean, median, and standard deviation values of the distances to 10th (d10), 20th (d20), 40th (d40), and 60th (d60) closest nuclei were calculated in a distance map as “density features.” The histogram parameters, such as mean, standard deviation, kurtosis, and skewness, on selected 3 shape features (major axis, eccentricity, and area) were used as “histogram features.” The mutual information of these density and histogram features of H&E‐ and Olig2‐stained slides were used for feature selection. 2.8.2 Automated p53 positivity workflow An automated positivity workflow for p53 immunoreactivity was developed for objective quantification. The detailed workflow was explained in the Results section. Briefly, we did segmentation on 1 image from positive control, 1 image from negative control, and 4 images from the tumor on the slide. After feature extraction, we labeled nuclei in the positive and negative control and used these labels as ground truth to make an RF modeling for the prediction of nuclei in the tumor images. After prediction, the positivity percentage was calculated as a percentage of positive nuclei to the total. We compared the automated positivity workflow results of p53 with human observers’ evaluations. These human observers were practicing neuropathologists at different hospitals who utilized slightly distinct interpretation workflows for p53 immunohistochemistry. Each neuropathologist interpreted the p53 immunohistochemistry as indicative of “mutant” or “wild type” in 82 cases of p53 images. After completion, the same 74 cases (8 cases do not have ATRX images) were evaluated with ATRX images to detect any bias of ATRX information on p53 positivity results. For models utilized in Figure , the continuous scale %p53 positive cells obtained from our automated workflow was utilized. Brain cancer simulated population model Our brain cancer population simulation is composed of 51 diagnostic entities obtained from the WHO Classification of CNS Tumors . Data on these features were extracted from the scientific literature to generate a simulation of patient populations with these entities. The clinical features, the histologic features, immunoohistochemichal markers, and molecular features were delineated in , with all references (summary of each entity is available in Figures ). The importance of each feature in the dignosis of this simulation model was performed by separating all the data into a training set (70%) and a test set (30%). The randomForest algorithm was called from R’s randomForest package using the default values, and the efficiency of the model was tested by evaluation of a confusion matrix by the confusionMatrix function call in R’s caret package, which showed that this model had an accuracy of 0.95 (95% CI = [0.952, 0.9564], no information rate = 0.068, and p < 10 −12 ). We then split the dataframe by diagnosis and, for each diagnosis, passed the a random forest classifier followed on the original dataframe. For each feature of the dataframe, we scrambled each diagnostic feature by performing a random sampling of the original dataframe containing all diagnoses and saved the value of the accuracy generated by random forest classifier. These data are presented in Table . Real‐world data sets for information theory validation Nine different data sets from different countries were used for the calculations of Bayesian probabilities and mutual information. The numbers of available cases and details were delineated in Table . Age distribution and probability calculation Age distribution of IDH1/2 ‐mutant gliomas was presented as density plots. For the Bayesian probability plots, probabilities of 1p/19q codeletion in IDH1/2 mutant tumors given age and gender were calculated using the “naivebayes” package in R with a Laplace correction for smoothing. Information theory calculations Information theory, initially developed by Claude E. Shannon at Bell Labs in 1948 , rests on the notion that information can be objectively quantified into units called bits (or nats, where 1 nat =log2(e)*bit). Mutual information is the exact opposite term of entropy, where entropy represents the uncertainty of the information. Maximum mutual information indicates all information quantity needed for the 1p/19q codeletion status (or diagnosis) in our example. For simplicity, the provided information with features is represented as a percentage of maximum mutual information (as in Figures , , and ). Details were delineated in , . Image acquisition Hematoxylin–eosin (H&E)‐stained sections and p53 and Olig2 immunohistochemistry‐stained sections of IDH ‐mutant diffuse gliomas were selected for image segmentation. Details were delineated in , . Segmentation methods (unsupervised and supervised) and evaluation of segmentation fidelity Two types of segmentation methods were used in our analysis: unsupervised (Deconvolution combined with Otsu thresholding, K‐means, and the Cut‐Cluster‐Classify) and supervised methods (Trainable Weka Segmentation and U‐Net). We made a ground truth using GIMP and R for the evaluation of segmentation fidelity and U‐Net training. Details were delineated in , . Evaluation of segmentation fidelity For our evaluation, we only have two regions of interest: cell nuclei (stained in purple for H&E images and brown for Olig2 and p53 images) and background. Since one of the challenges of segmenting our data set comes from the various levels of noise, we also developed an image noise assay to test how robust the methods are to noise. Percentage ratios of added noise were selected as 0, 5, 10 15, 25, 50, 75, 90 to evaluate the whole pattern of noise resistance. We report accuracy and intersection over union (IoU). Details were delineated in , . Data processing 2.8.1 Condensation of data and probability calculation of Olig2 features Eighty‐nine features were extracted from the nuclei in each channel of the images using the masks after the segmentation with the deconvolution method. After feature extraction, we excluded incomplete nuclei in the images for a better representation. Extracted features are shape, moment, pixel intensity, and Haralick's features. We had 213 features left after the elimination of repeated features in different channels. After principal component analysis (PCA) application, 8 principal components (PCs) in Olig2 features and 9 principal components (PCs) in H&E features were selected for the representation of 90% variation of features. We calculated the sample density of nuclei in a multidimensional space of PCs. Three hundred and fifty nuclei were sampled in each case for density calculation of features extracted from H&E‐ and Olig2‐stained images. The mean, median, and standard deviation values of the distances to 10th (d10), 20th (d20), 40th (d40), and 60th (d60) closest nuclei were calculated in a distance map as “density features.” The histogram parameters, such as mean, standard deviation, kurtosis, and skewness, on selected 3 shape features (major axis, eccentricity, and area) were used as “histogram features.” The mutual information of these density and histogram features of H&E‐ and Olig2‐stained slides were used for feature selection. 2.8.2 Automated p53 positivity workflow An automated positivity workflow for p53 immunoreactivity was developed for objective quantification. The detailed workflow was explained in the Results section. Briefly, we did segmentation on 1 image from positive control, 1 image from negative control, and 4 images from the tumor on the slide. After feature extraction, we labeled nuclei in the positive and negative control and used these labels as ground truth to make an RF modeling for the prediction of nuclei in the tumor images. After prediction, the positivity percentage was calculated as a percentage of positive nuclei to the total. We compared the automated positivity workflow results of p53 with human observers’ evaluations. These human observers were practicing neuropathologists at different hospitals who utilized slightly distinct interpretation workflows for p53 immunohistochemistry. Each neuropathologist interpreted the p53 immunohistochemistry as indicative of “mutant” or “wild type” in 82 cases of p53 images. After completion, the same 74 cases (8 cases do not have ATRX images) were evaluated with ATRX images to detect any bias of ATRX information on p53 positivity results. For models utilized in Figure , the continuous scale %p53 positive cells obtained from our automated workflow was utilized. Condensation of data and probability calculation of Olig2 features Eighty‐nine features were extracted from the nuclei in each channel of the images using the masks after the segmentation with the deconvolution method. After feature extraction, we excluded incomplete nuclei in the images for a better representation. Extracted features are shape, moment, pixel intensity, and Haralick's features. We had 213 features left after the elimination of repeated features in different channels. After principal component analysis (PCA) application, 8 principal components (PCs) in Olig2 features and 9 principal components (PCs) in H&E features were selected for the representation of 90% variation of features. We calculated the sample density of nuclei in a multidimensional space of PCs. Three hundred and fifty nuclei were sampled in each case for density calculation of features extracted from H&E‐ and Olig2‐stained images. The mean, median, and standard deviation values of the distances to 10th (d10), 20th (d20), 40th (d40), and 60th (d60) closest nuclei were calculated in a distance map as “density features.” The histogram parameters, such as mean, standard deviation, kurtosis, and skewness, on selected 3 shape features (major axis, eccentricity, and area) were used as “histogram features.” The mutual information of these density and histogram features of H&E‐ and Olig2‐stained slides were used for feature selection. Automated p53 positivity workflow An automated positivity workflow for p53 immunoreactivity was developed for objective quantification. The detailed workflow was explained in the Results section. Briefly, we did segmentation on 1 image from positive control, 1 image from negative control, and 4 images from the tumor on the slide. After feature extraction, we labeled nuclei in the positive and negative control and used these labels as ground truth to make an RF modeling for the prediction of nuclei in the tumor images. After prediction, the positivity percentage was calculated as a percentage of positive nuclei to the total. We compared the automated positivity workflow results of p53 with human observers’ evaluations. These human observers were practicing neuropathologists at different hospitals who utilized slightly distinct interpretation workflows for p53 immunohistochemistry. Each neuropathologist interpreted the p53 immunohistochemistry as indicative of “mutant” or “wild type” in 82 cases of p53 images. After completion, the same 74 cases (8 cases do not have ATRX images) were evaluated with ATRX images to detect any bias of ATRX information on p53 positivity results. For models utilized in Figure , the continuous scale %p53 positive cells obtained from our automated workflow was utilized. RESULTS 3.1 Information theory enhances understanding of individual feature impact in diagnostic neuropathology Our ultimate goal is to generate tools and workflows that improve pathologist decision‐making throughout the world, with a particular emphasis on resource‐poor settings. To this end, we generated a brain cancer population simulation, whereby we obtained specific features known to neuropathologists who are capable of distinguishing tumors and performed a thorough review of the scientific literature to obtain the data upon which to build this simulation (see ). We utilized three easily obtainable clinical features (age, neuroanatomical site, and gender), histologic features, and immunohistochemical stains (GFAP, ATRX, Ki67, IDH1 R132H, P53, CD34, EMA, Olig2, reticulin, and synaptophysin). We also utilized the results of basic molecular assays that are commonly reimbursed by private insurers, public aid, and Medicare, in the United States. All features for each entity simulated are delineated in the . We first performed dimensionality reduction using principal component analysis (PCA) as well as a nonlinear algorithm, the tSNE, on our simulated brain tumor population (Figure ). We note that plotting tumors only on clinical features (age, site, and gender) with tSNE and PCA results in no discernable cluster generation (Figure ) . By including histologic features from H&E‐stained sections, one is able to visibly appreciate the generation of specific clusters of entities (Figure ). The addition of immunohistochemistry further increased the clusters relative to clinical features plus immunohistochemistry. However, the addition of molecular features did not necessarily increase the clustering upon visual inspection (compared Figure with Figure ). These data are in‐line with well‐known concepts in the neuropathology community that emphasize the integration of clinical context with histology. We further conclude that this simulation provides a useful in silico framework to test workflow concepts by Monte Carlo‐like simulations prior to real‐life deployment. Then we generated a thought experiment where we imagined a scenario where the biopsy/resection procedure represented a conduit of data transmission from the patient to the pathologist. This scenario is analogous to the flow of information in information processing and amenable to information theory calculations (see ). We evaluated all the features delineated in our brain cancer simulation model and quantified the mutual information (Figure ). We note that age, neuroanatomical site, and Ki67 proliferation index were the features that carried the most amount of information in the glioma simulation model. A combination of individual features showed that histology data provided nearly 80% of the information necessary for diagnosis (Figure ). Surprisingly, basic clinical information (age, site, and gender) provided just over half of all information necessary for a diagnosis, whereas molecular data alone provided less than 20% of the information needed. Combining clinical data with histology data and immunohistochemistry increased the information to over 95%, with only minimal information gained by the molecular data. To identify the most critically important features for each diagnosis in our simulation, we generated a randomForest based workflow that identified the most critical diagnostic features in this simulation (Table ). After evaluating the information theory calculations and the results of our randomForest analysis, we note a significant asymmetry in the type of information contained in patient data, with the neuroanatomical site, age, and Ki67 labeling index being the most important features in diagnosis. This asymmetry produces a significant tendency to generate bias in machine learning‐based diagnostic algorithms. In order to illustrate how such simulations can be used for strategic planning, we subsetted the simulated brain cancer population for IDH‐mutated tumors and performed information theory calculations to determine the utility of age, gender, site, IDH status, 1p19q status, and immunohistochemical detection of Olig2, GFAP, CD34, synaptophysin, p53, and ATRX (Figure ). We noted that p53 and ATRX status provided a significant quantity of information as to 1p19q status. We further conclude that basic information theory calculations permit the rapid acquisition of which features are most impactful for diagnostic neuropathology. 3.2 Age, p53, and ATRX status have significant contribution in the diagnosis of IDH ‐mutant tumors Then we focused on IDH ‐mutant tumors to validate these information theory‐focused workflows. To achieve this, we took advantage of publicly available data sets from the Cancer Genome Atlas and the Chinese Glioma Genome Atlas as well as data from hospitals in Turkey, the United States, Argentina, and Brazil to provide a global perspective (see map in Figure ). The data sets collected included patient age (continuous numeric), gender (binary), p53 (binary), ATRX (binary), and 1p/19q codeletion status (binary), summing to 1390 total IDH ‐mutant gliomas. We noticed that IDH ‐mutant gliomas showed a different distribution of patient age. Generating a probability distribution of diagnosis as a function of age, we noticed an increase in the probability of 1p/19q codeletion with advanced age (Figure ). Confirming other studies, we found no association of gender with 1p/19q codeletion status (Figure ). We then evaluated the relationship of these biomarkers to 1p/19q codeletion status by two linear methodologies: correlation matrix and principal component analysis (PCA). To achieve this, we generated dummy variables of the features. Figure shows that ATRX and p53 mutations show a high correlation with each other and, as expected, are highly negatively correlated with 1p/19q codeletion status. Then we interrogated the data variance by PCA. As shown in Figure , PC1 and PC2 account for over 70% of the variance in the data set. By plotting the eigenvectors along PC1 and PC2, we can appreciate the significant redundancy between ATRX and p53 status in the PC1/2 graph (note the near superimposition of the ATRX and p53 eigenvectors in Figure ). Also, the eigenvector for gender shows a nearly orthogonal orientation relative to 1p/19q codeletion eigenvector, indicating no association. These evaluations raise the possibility that data for P53 and ATRX show significant redundancy, and if so, assessing both biomarkers would be unnecessary in diagnostic workflows. To test this hypothesis, we performed information theory calculations of these parameters (Figure ). We found a statistically significant increase in information when both ATRX and p53 were evaluated together, a finding in‐line with current cIMPACT‐NOW clinical practice . This means new information gain is present when combining both p53 and ATRX data points. We conclude that information theory analysis is capable of determining if biomarkers showing significant correlation and covariance add significant information when used in combination. It also provides significant additional insight into linear computational methods. 3.3 Auto‐adjustable image analysis workflows for surgical neuropathology We next sought to evaluate the information gain extractable from tissue morphology. A well‐appreciated morphologic distinction between IDH ‐mutant gliomas is the propensity of the former to show significantly greater nuclear pleomorphism. We first evaluated Olig2 expression by immunohistochemistry, a biomarker diffusely expressed in infiltrating gliomas , and which was a commonly utilized biomarker among our global collaborative group. Olig2’s nuclear localization also facilitates image segmentation. We then evaluated a variety of supervised and unsupervised segmentation algorithms, which included (1) matrix deconvolution with OTSU thresholding , (2) WEKA segmentation , (3) k‐means segmentation, (4) Cut‐Cluster‐Classify segmentation , and (5) a convolutional neural network with a UNET architecture . We also performed a separate analysis utilizing a UNET algorithm trained with experimental noise (see Figure S57 for Image Noise Assay workflow and comparison of segmentation methods). Our data were derived from the OSU patient archives. We note that matrix deconvolution with OTSU thresholding showed high accuracy up until 25% noise for both H&E staining and Olig2 immunohistochemistry (Figure , blue line), whereas UNET trained with significant noise was able to maintain high accuracy with high levels of noise. However, when evaluating IoU, both methods showed significant drop‐off with more than 25% noise (Figure ). We conclude that for simple segmentation processes where the high signal to noise exist, matrix deconvolution with OTSU thresholding is the simplest approach. We then generated image masks of all Olig2‐stained nuclei and overlaid the image masks onto the red, green, and blue image matrices to extract features of cell nuclei. These features included shape, pixel intensity, and pixel texture representing over 200 features per nucleus. To reduce the dimensions, we performed PCA which provided 90% of the variance in 8 principal components for Olig2 staining. Evaluation of morphologic features in some cases showed a tendency for oligodendroglioma cases to show higher kurtosis in histograms describing nuclear shape features (Figure ). However, the absolute values of these parameters differed on a case‐by‐case basis, and we were not able to use these raw measurements. We also felt that the pleomorphism among cells likely also included marked variance in DAB‐staining intensity and texture (e.g., compare astrocytoma staining pattern for Olig2 in Figure ). We, therefore, evaluated the principal components in multidimensional space (Figure shows the workflow). An example of our analysis is shown in Figure for a sample case of IDH ‐mutant astrocytoma and oligodendroglioma. Note that the IDH ‐mutant astrocytoma shows a wide scatter of coordinates, thus showing a lower data density. In contrast, oligodendroglioma is more heavily clustered in one area. This dense packing of the oligodendroglioma nuclei represents a reflection of the nuclear monomorphism in oligodendroglioma relative to astrocytoma. We calculated the mean Euclidean distance of each cell to its 10th, 20th, 40th, and 60th neighbor in the multidimensional PC space for each patient in our data set. An increased Euclidean distance to its n th neighbor would indicate that, on average, larger heterogeneity in morphology, Olig2 intensity, and Olig2 texture is present in tumor cell nuclei. For each distance measure, we note that IDH ‐mutant astrocytoma showed elevated Euclidean distance relative to oligodendroglioma, indicating a lower density in multidimensional space (Figure ). Data density calculations from Euclidean Distance capture the nuclear pleomorphism differences between IDH ‐mutant gliomas. We further posit that this methodology, which is based on within‐sample differences, represents a superior metric for evaluating morphology as it would be more resistant to interlaboratory methodologies. We next focused our analysis on features of p53 immunohistochemistry, an important biomarker for glioma classification. Intense p53 nuclear immunoreactivity is utilized as a surrogate for TP53 mutation but suffers significant limitations. In addition to some inactivating TP53 mutations resulting in complete negative staining , Takami, et al. demonstrated that in addition to immunoreactivity intensity, percent of immunoreactivity was also a critical feature . However, other studies have set a cutoff of positivity for p53 positivity by IHC as 10% regardless of intensity in other tumor paradigms . Generating a universal intensity and texture cutoff for p53 immunohistochemistry is challenging due to variance in laboratory practices. In our institution, we routinely perform our glioma‐related p53 immunohistochemistry with a serous carcinoma positive control and a benign lymph node as a negative control, mounted on the same slide. We utilized these internal controls as our metric for the designation of p53 positive or negative for each case (see the workflow in Figure ). The mean accuracies of random forests for each case are 0.991 (95% CI: 0.985–0.996). Once the model is trained, we passed it on to the actual data from the patient to generate the prediction. For benchmarking, we compared the performance of our model to two human neuropathologists (see Table for concordance data). We note that Lin's concordance for interobserver neuropathology recordings showed overlap in the 95% CI of neuropathology observer 1: automated workflow and neuropathology and observer 2: automated workflow. These data indicate that the automated workflow shows similar a concordance level to neuropathologists as neuropathologists concord with each other. We next tested the extent to which mean pixel intensity extracted from the nuclei were distinct between the astrocytoma and oligodendroglioma cases in the red, green, and blue channels (Figure ). Mean pixel intensity distribution showed a clear distinction between the tumor nuclei and control nuclei for the modeling. Figure demonstrates the distribution of p53 positive cells, as determined by our random forest classifier for each case. The variance between the p53 quantification is quite large in the astrocytoma cases, whereas in the oligodendroglioma, the majority of the cases showed low %p53 detection. Based on these data, we then generated a probability function using the naïve Bayes function in R (Figure ). As %p53 positivity increases, the probability of 1p/19q codeletion in our data set decreases in an exponential manner. Figure demonstrates information theory calculations for this p53 continuous‐scale data set. Automated p53 quantification using this workflow represents an objective alternative to subjective p53 interpretation. Also, the addition of ATRX information to p53 positivity percentage provides a significant information increase for diagnosis. We also used a mutual information approach to determine the significant features in the histogram and density features of H&E‐ and Olig2‐stained images (Figure ). There are 12 significant Olig2 features (Figure ), whereas 3 significant H&E features were noted (Figure ). 3.4 Informatics‐based clinical practice guidelines for the evaluation of IDH ‐mutant tumors We next trained a random forest classifier that used the significant Olig2 features of data density, p53 labeling (continuous scale quantification), and a combined ATRX/age feature on 82 cases. The ATRX/age feature was obtained from probability estimates of a random forest classifier trained on 1107 cases of the global data set after splitting the train (70%) and test (30%) sets, where 1p/19q codel status was modeled as a function of ATRX and patient age. This ATRX/age feature was calculated on the 82 cases of the OSU data. We note that in our OSU archival data set, missing (3 cases) or equivocal (2 cases, 1 on 82 cases, 1 on validation data set) ATRX status existed, and so we imputed the ATRX status based on a random forest classifier trained on the global data set, where ATRX status was modeled as a function of age and p53 status. For our 82 cases, we used an 80% threshold to convert the p53 continuous scale to a binary result (positive or negative) for this prediction. Then we tested the random forest classifier on the testing set (~30%, representing 25 cases). Our results showed an accuracy of 0.92%, p = 0.000022. We then implemented this algorithm on a series of 11 diagnostically challenging validation cases (Figure and summarized in Table ), most of which represented tumor recurrences. Detailed clinical histories of these patients are delineated in the , . Our model incorrectly classified four astrocytoma cases (Case 2, Case 3‐2, Case 4, and Case 10). All the incorrectly classified cases showed either high‐grade architectural features (necrosis and microvascular hyperplasia) or represented recurrent cases. Based on these results, we propose a clinical practice guideline to facilitate decisions related to the ordering of expensive nonreimbursable modern molecular tests (Figure ). We note that our algorithm is quite robust for low‐grade cases. In the setting of a low‐grade IDH ‐mutant tumor, if our model prediction is less than 0.5 for 1p/19q codeletion, the case can be easily diagnosed as an astrocytoma without further workup. If the low‐grade glioma shows a prediction of >0.5, 1p/19q codeletion by FISH is a sufficient diagnostic test to diagnose accurately IDH ‐mutant astrocytoma and oligodendroglioma. For IDH ‐mutant tumors that are recurrent or show high‐grade morphology, a model prediction of >0.5 should trigger a chromosomal microarray or NGS‐based 1p/19q codeletion assay so as to avoid the potential of 1p/19q FISH false positives. 1p/19q FISH false positives are the instances in which there is a discordance between 1p19q FISH and 1p19q analysis by chromosomal microarray or next‐generation sequencing as false‐positive cases with regard to the diagnosis of oligodendroglioma. Information theory enhances understanding of individual feature impact in diagnostic neuropathology Our ultimate goal is to generate tools and workflows that improve pathologist decision‐making throughout the world, with a particular emphasis on resource‐poor settings. To this end, we generated a brain cancer population simulation, whereby we obtained specific features known to neuropathologists who are capable of distinguishing tumors and performed a thorough review of the scientific literature to obtain the data upon which to build this simulation (see ). We utilized three easily obtainable clinical features (age, neuroanatomical site, and gender), histologic features, and immunohistochemical stains (GFAP, ATRX, Ki67, IDH1 R132H, P53, CD34, EMA, Olig2, reticulin, and synaptophysin). We also utilized the results of basic molecular assays that are commonly reimbursed by private insurers, public aid, and Medicare, in the United States. All features for each entity simulated are delineated in the . We first performed dimensionality reduction using principal component analysis (PCA) as well as a nonlinear algorithm, the tSNE, on our simulated brain tumor population (Figure ). We note that plotting tumors only on clinical features (age, site, and gender) with tSNE and PCA results in no discernable cluster generation (Figure ) . By including histologic features from H&E‐stained sections, one is able to visibly appreciate the generation of specific clusters of entities (Figure ). The addition of immunohistochemistry further increased the clusters relative to clinical features plus immunohistochemistry. However, the addition of molecular features did not necessarily increase the clustering upon visual inspection (compared Figure with Figure ). These data are in‐line with well‐known concepts in the neuropathology community that emphasize the integration of clinical context with histology. We further conclude that this simulation provides a useful in silico framework to test workflow concepts by Monte Carlo‐like simulations prior to real‐life deployment. Then we generated a thought experiment where we imagined a scenario where the biopsy/resection procedure represented a conduit of data transmission from the patient to the pathologist. This scenario is analogous to the flow of information in information processing and amenable to information theory calculations (see ). We evaluated all the features delineated in our brain cancer simulation model and quantified the mutual information (Figure ). We note that age, neuroanatomical site, and Ki67 proliferation index were the features that carried the most amount of information in the glioma simulation model. A combination of individual features showed that histology data provided nearly 80% of the information necessary for diagnosis (Figure ). Surprisingly, basic clinical information (age, site, and gender) provided just over half of all information necessary for a diagnosis, whereas molecular data alone provided less than 20% of the information needed. Combining clinical data with histology data and immunohistochemistry increased the information to over 95%, with only minimal information gained by the molecular data. To identify the most critically important features for each diagnosis in our simulation, we generated a randomForest based workflow that identified the most critical diagnostic features in this simulation (Table ). After evaluating the information theory calculations and the results of our randomForest analysis, we note a significant asymmetry in the type of information contained in patient data, with the neuroanatomical site, age, and Ki67 labeling index being the most important features in diagnosis. This asymmetry produces a significant tendency to generate bias in machine learning‐based diagnostic algorithms. In order to illustrate how such simulations can be used for strategic planning, we subsetted the simulated brain cancer population for IDH‐mutated tumors and performed information theory calculations to determine the utility of age, gender, site, IDH status, 1p19q status, and immunohistochemical detection of Olig2, GFAP, CD34, synaptophysin, p53, and ATRX (Figure ). We noted that p53 and ATRX status provided a significant quantity of information as to 1p19q status. We further conclude that basic information theory calculations permit the rapid acquisition of which features are most impactful for diagnostic neuropathology. Age, p53, and ATRX status have significant contribution in the diagnosis of IDH ‐mutant tumors Then we focused on IDH ‐mutant tumors to validate these information theory‐focused workflows. To achieve this, we took advantage of publicly available data sets from the Cancer Genome Atlas and the Chinese Glioma Genome Atlas as well as data from hospitals in Turkey, the United States, Argentina, and Brazil to provide a global perspective (see map in Figure ). The data sets collected included patient age (continuous numeric), gender (binary), p53 (binary), ATRX (binary), and 1p/19q codeletion status (binary), summing to 1390 total IDH ‐mutant gliomas. We noticed that IDH ‐mutant gliomas showed a different distribution of patient age. Generating a probability distribution of diagnosis as a function of age, we noticed an increase in the probability of 1p/19q codeletion with advanced age (Figure ). Confirming other studies, we found no association of gender with 1p/19q codeletion status (Figure ). We then evaluated the relationship of these biomarkers to 1p/19q codeletion status by two linear methodologies: correlation matrix and principal component analysis (PCA). To achieve this, we generated dummy variables of the features. Figure shows that ATRX and p53 mutations show a high correlation with each other and, as expected, are highly negatively correlated with 1p/19q codeletion status. Then we interrogated the data variance by PCA. As shown in Figure , PC1 and PC2 account for over 70% of the variance in the data set. By plotting the eigenvectors along PC1 and PC2, we can appreciate the significant redundancy between ATRX and p53 status in the PC1/2 graph (note the near superimposition of the ATRX and p53 eigenvectors in Figure ). Also, the eigenvector for gender shows a nearly orthogonal orientation relative to 1p/19q codeletion eigenvector, indicating no association. These evaluations raise the possibility that data for P53 and ATRX show significant redundancy, and if so, assessing both biomarkers would be unnecessary in diagnostic workflows. To test this hypothesis, we performed information theory calculations of these parameters (Figure ). We found a statistically significant increase in information when both ATRX and p53 were evaluated together, a finding in‐line with current cIMPACT‐NOW clinical practice . This means new information gain is present when combining both p53 and ATRX data points. We conclude that information theory analysis is capable of determining if biomarkers showing significant correlation and covariance add significant information when used in combination. It also provides significant additional insight into linear computational methods. Auto‐adjustable image analysis workflows for surgical neuropathology We next sought to evaluate the information gain extractable from tissue morphology. A well‐appreciated morphologic distinction between IDH ‐mutant gliomas is the propensity of the former to show significantly greater nuclear pleomorphism. We first evaluated Olig2 expression by immunohistochemistry, a biomarker diffusely expressed in infiltrating gliomas , and which was a commonly utilized biomarker among our global collaborative group. Olig2’s nuclear localization also facilitates image segmentation. We then evaluated a variety of supervised and unsupervised segmentation algorithms, which included (1) matrix deconvolution with OTSU thresholding , (2) WEKA segmentation , (3) k‐means segmentation, (4) Cut‐Cluster‐Classify segmentation , and (5) a convolutional neural network with a UNET architecture . We also performed a separate analysis utilizing a UNET algorithm trained with experimental noise (see Figure S57 for Image Noise Assay workflow and comparison of segmentation methods). Our data were derived from the OSU patient archives. We note that matrix deconvolution with OTSU thresholding showed high accuracy up until 25% noise for both H&E staining and Olig2 immunohistochemistry (Figure , blue line), whereas UNET trained with significant noise was able to maintain high accuracy with high levels of noise. However, when evaluating IoU, both methods showed significant drop‐off with more than 25% noise (Figure ). We conclude that for simple segmentation processes where the high signal to noise exist, matrix deconvolution with OTSU thresholding is the simplest approach. We then generated image masks of all Olig2‐stained nuclei and overlaid the image masks onto the red, green, and blue image matrices to extract features of cell nuclei. These features included shape, pixel intensity, and pixel texture representing over 200 features per nucleus. To reduce the dimensions, we performed PCA which provided 90% of the variance in 8 principal components for Olig2 staining. Evaluation of morphologic features in some cases showed a tendency for oligodendroglioma cases to show higher kurtosis in histograms describing nuclear shape features (Figure ). However, the absolute values of these parameters differed on a case‐by‐case basis, and we were not able to use these raw measurements. We also felt that the pleomorphism among cells likely also included marked variance in DAB‐staining intensity and texture (e.g., compare astrocytoma staining pattern for Olig2 in Figure ). We, therefore, evaluated the principal components in multidimensional space (Figure shows the workflow). An example of our analysis is shown in Figure for a sample case of IDH ‐mutant astrocytoma and oligodendroglioma. Note that the IDH ‐mutant astrocytoma shows a wide scatter of coordinates, thus showing a lower data density. In contrast, oligodendroglioma is more heavily clustered in one area. This dense packing of the oligodendroglioma nuclei represents a reflection of the nuclear monomorphism in oligodendroglioma relative to astrocytoma. We calculated the mean Euclidean distance of each cell to its 10th, 20th, 40th, and 60th neighbor in the multidimensional PC space for each patient in our data set. An increased Euclidean distance to its n th neighbor would indicate that, on average, larger heterogeneity in morphology, Olig2 intensity, and Olig2 texture is present in tumor cell nuclei. For each distance measure, we note that IDH ‐mutant astrocytoma showed elevated Euclidean distance relative to oligodendroglioma, indicating a lower density in multidimensional space (Figure ). Data density calculations from Euclidean Distance capture the nuclear pleomorphism differences between IDH ‐mutant gliomas. We further posit that this methodology, which is based on within‐sample differences, represents a superior metric for evaluating morphology as it would be more resistant to interlaboratory methodologies. We next focused our analysis on features of p53 immunohistochemistry, an important biomarker for glioma classification. Intense p53 nuclear immunoreactivity is utilized as a surrogate for TP53 mutation but suffers significant limitations. In addition to some inactivating TP53 mutations resulting in complete negative staining , Takami, et al. demonstrated that in addition to immunoreactivity intensity, percent of immunoreactivity was also a critical feature . However, other studies have set a cutoff of positivity for p53 positivity by IHC as 10% regardless of intensity in other tumor paradigms . Generating a universal intensity and texture cutoff for p53 immunohistochemistry is challenging due to variance in laboratory practices. In our institution, we routinely perform our glioma‐related p53 immunohistochemistry with a serous carcinoma positive control and a benign lymph node as a negative control, mounted on the same slide. We utilized these internal controls as our metric for the designation of p53 positive or negative for each case (see the workflow in Figure ). The mean accuracies of random forests for each case are 0.991 (95% CI: 0.985–0.996). Once the model is trained, we passed it on to the actual data from the patient to generate the prediction. For benchmarking, we compared the performance of our model to two human neuropathologists (see Table for concordance data). We note that Lin's concordance for interobserver neuropathology recordings showed overlap in the 95% CI of neuropathology observer 1: automated workflow and neuropathology and observer 2: automated workflow. These data indicate that the automated workflow shows similar a concordance level to neuropathologists as neuropathologists concord with each other. We next tested the extent to which mean pixel intensity extracted from the nuclei were distinct between the astrocytoma and oligodendroglioma cases in the red, green, and blue channels (Figure ). Mean pixel intensity distribution showed a clear distinction between the tumor nuclei and control nuclei for the modeling. Figure demonstrates the distribution of p53 positive cells, as determined by our random forest classifier for each case. The variance between the p53 quantification is quite large in the astrocytoma cases, whereas in the oligodendroglioma, the majority of the cases showed low %p53 detection. Based on these data, we then generated a probability function using the naïve Bayes function in R (Figure ). As %p53 positivity increases, the probability of 1p/19q codeletion in our data set decreases in an exponential manner. Figure demonstrates information theory calculations for this p53 continuous‐scale data set. Automated p53 quantification using this workflow represents an objective alternative to subjective p53 interpretation. Also, the addition of ATRX information to p53 positivity percentage provides a significant information increase for diagnosis. We also used a mutual information approach to determine the significant features in the histogram and density features of H&E‐ and Olig2‐stained images (Figure ). There are 12 significant Olig2 features (Figure ), whereas 3 significant H&E features were noted (Figure ). Informatics‐based clinical practice guidelines for the evaluation of IDH ‐mutant tumors We next trained a random forest classifier that used the significant Olig2 features of data density, p53 labeling (continuous scale quantification), and a combined ATRX/age feature on 82 cases. The ATRX/age feature was obtained from probability estimates of a random forest classifier trained on 1107 cases of the global data set after splitting the train (70%) and test (30%) sets, where 1p/19q codel status was modeled as a function of ATRX and patient age. This ATRX/age feature was calculated on the 82 cases of the OSU data. We note that in our OSU archival data set, missing (3 cases) or equivocal (2 cases, 1 on 82 cases, 1 on validation data set) ATRX status existed, and so we imputed the ATRX status based on a random forest classifier trained on the global data set, where ATRX status was modeled as a function of age and p53 status. For our 82 cases, we used an 80% threshold to convert the p53 continuous scale to a binary result (positive or negative) for this prediction. Then we tested the random forest classifier on the testing set (~30%, representing 25 cases). Our results showed an accuracy of 0.92%, p = 0.000022. We then implemented this algorithm on a series of 11 diagnostically challenging validation cases (Figure and summarized in Table ), most of which represented tumor recurrences. Detailed clinical histories of these patients are delineated in the , . Our model incorrectly classified four astrocytoma cases (Case 2, Case 3‐2, Case 4, and Case 10). All the incorrectly classified cases showed either high‐grade architectural features (necrosis and microvascular hyperplasia) or represented recurrent cases. Based on these results, we propose a clinical practice guideline to facilitate decisions related to the ordering of expensive nonreimbursable modern molecular tests (Figure ). We note that our algorithm is quite robust for low‐grade cases. In the setting of a low‐grade IDH ‐mutant tumor, if our model prediction is less than 0.5 for 1p/19q codeletion, the case can be easily diagnosed as an astrocytoma without further workup. If the low‐grade glioma shows a prediction of >0.5, 1p/19q codeletion by FISH is a sufficient diagnostic test to diagnose accurately IDH ‐mutant astrocytoma and oligodendroglioma. For IDH ‐mutant tumors that are recurrent or show high‐grade morphology, a model prediction of >0.5 should trigger a chromosomal microarray or NGS‐based 1p/19q codeletion assay so as to avoid the potential of 1p/19q FISH false positives. 1p/19q FISH false positives are the instances in which there is a discordance between 1p19q FISH and 1p19q analysis by chromosomal microarray or next‐generation sequencing as false‐positive cases with regard to the diagnosis of oligodendroglioma. DISCUSSION In this study, we implemented, for the first time, information theory approaches as a conceptual and practical framework upon which to determine the features that offer the most information to reach an integrated neuropathologic diagnosis. We tested this hypothesis first with a highly comprehensive simulation of brain cancer patients and validated this approach using real‐world data. We developed, validated, and deployed a simple image analysis workflow that could be adopted in resource‐poor settings using open‐source software as well as translated to a variety of diagnostic applications and proposed a clinical practice guideline recommendation in the evaluation of IDH ‐mutant tumors. We note that few emerging concepts in modern pathology have experienced as much hype as machine learning and AI (for instance, see Ref. and related its editorial Ref. ). We propose that our information theory approach represents a simple way for resource‐challenged healthcare ecosystems to make informed decisions on reagent and testing platform investments. We further propose that in silico simulation, such as performed by our glioma population simulation, provides an inexpensive framework amenable to Monte Carlo‐like simulation and permits the testing of how specific datapoints may affect the differential diagnosis. We posit that generating such Monte Carlo‐like simulations can provide multiple insights to other surgical pathology paradigms. The workflow presented in the , can easily be substituted by other tumor types and/or other types of information by creating an m × n matrix in Excel with each row corresponding to a specific tumor entity, each column a feature/biomarker, and each cell a proportion of cases showing positivity for the designated feature/biomarker. Such a table would then be imported into Rstudio or Python to generate the simulation using a sampling function as delineated in the methods. 4.1 Implications of information theory on diagnostic neuropathology The glioma diagnosis simulation and our information theory analysis raise some very interesting points regarding the future development and deployment of clinical diagnostic aids for neuropathology. At the forefront of our findings is the identification that simple information provides the majority of information. Specifically, basic clinical history, radiographic documentation of tumor site, and histologic findings carried the most information. This is rather intuitive, but these findings are of utmost importance for low‐resource healthcare ecosystems. These findings are in‐line with recommendations for low‐to‐middle‐income countries (LMIC) that have emphasized local healthcare ecosystems to embrace a culture of quality in pathology laboratories, in particular, by adopting appropriate standard operating procedures, quality control, and quality assurance practices . Underscoring this point, a comprehensive study by Fleming et al. generated a recommended capabilities list for Pathology Laboratories in LMICs. Chief among these recommendations included high‐quality histologic/cytologic services as well as training for laboratory staff and physician‐pathologists . Our findings from the brain cancer population simulation underscore the key importance of quality histology in diagnostic neuropathology. Building upon these recommendations, we would further advocate that evaluation of clinical, histologic, and biomarker features by information theory could provide a quantitative framework from which to make data‐driven decisions on diagnostic services. Our work raises significant concerns with ML‐/AI‐based clinical diagnostic aids in neuropathology. First, most brain cancer data set structures include high‐resolution genomic data on very few samples. Pooling data sets from various centers results in missing data elements that are often beyond the capabilities of imputation for clinical outcomes research . In addition, as demonstrated in our glioma simulation, the quantity of information present is highly asymmetrical, and many brain tumor entities are exceedingly rare. This results in ML‐/AI‐based algorithms, nearly all of which utilize some version of information theory, to focus on age, neuroanatomical site, and proliferative index for diagnosis. As a result, predictive algorithms may generate adequate differential diagnoses, but these would be of minimal utility in routine practice. Our approach to overcoming this problem is to identify specific, granular diagnostic conundrums as candidate branch points in clinical decision‐making, where ML/AI can make an impact. This permits the focused evaluation of biomarker information with several advantages over traditional analyses. PCA and correlation plots indicated such a high correlation and covariance between p53 and ATRX status that it raised the possibility that these two biomarkers were fully redundant, legitimately questioning employing both antibodies. In contrast, using an information theory approach, we found that the incorporation of both biomarkers simultaneously resulted in a statistically significant information gain. This synergy in information gain was masked using traditional linear analyses. Similar evaluations could be employed to evaluate additional frequently paired biomarkers. 4.2 Simple image analysis workflows deployable throughout the world The advent of CNN networks and image recognition has revolutionized the modern world, and it will continue to provide significant impacts to diagnostic pathology. However, its deployment in the practice of pathology globally has several limitations. First and foremost, although pathologists worldwide have access to brightfield microscopes equipped with cameras, few laboratories contain whole slide imaging (WSI)‐capable scanners. In addition to capturing all of the tissue examined, WSI has the added benefit of imposing a standard operating procedure (SOP) in image acquisition, whereas traditional photomicroscopy is nearly impossible to be replicated from one session to another. In our study, we utilized tiles of image patches taken from WSI images to mimic pathologists capturing images on their desktop microscopes. A second challenge is that the lack of adherence to SOPs for histopathology across the globe results in highly variable interlaboratory fixation and staining protocols . Although several attempts to correct this have been published (e.g., Ref. ), the nonlinearity in histologic color maps has posed a significant challenge when controlling for fixation and staining conditions across labs and may be unsurmountable in labs with high batch variance in staining. For this reason, we focused on incorporating self‐normalizing workflows when dealing with color intensity and color texture features. One simple approach is to use internal known positive controls, such as p53 immunohistochemistry in ovarian serous carcinoma with a known negative control (in our case, a benign lymph node), and train within each case an algorithm based on pixel intensity and texture features. This internal slide control provides an unbiased methodology. A second approach we took was using the concept of data density within multidimensional space for the evaluation of nuclear pleomorphism in glioma. These analyses can be performed across histopathology labs as they would only require image acquisition using the same objective magnification and numerical aperture. 4.3 Machine learning‐workflow improvement to WHO and cIMPACT‐NOW clinical guidelines Our test‐case scenario dealt with the need to confirm 1p/19q codeletion status in IDH ‐mutant gliomas. In many healthcare ecosystems, even in the United States, no reimbursement for chromosomal microarray to confirm 1p/19q codeletion exists. In contrast, 1p/19q codeletion testing by FISH, although suffering in positive predictive value relative to more advanced techniques, reimburses routinely. This leads to a situation in which pathologists must make a decision to perform unreimbursed reflex testing in a serial fashion to confirm 1p/19q codeletion, further delaying treatment planning by neuro‐oncology/radiation oncology. The cIMPACT guidelines, which are anticipated to be incorporated into the 2021 WHO classification, represent a significant improvement to prior recommendations . Specific to our test case at hand is that current cIMPACT guidelines dictate that documentation of TP53 mutation and concurrent ATRX mutation within an IDH ‐mutant tumor excludes the diagnosis of oligodendroglioma. A specific problem arises when pathologists identify an IDH ‐mutant tumor, and either ATRX immunohistochemistry is unavailable, or ATRX immunohistochemistry is equivocal (as was shown in Case 4 of Figure ) or negative due to copy number loss involving the ATRX locus in a tumor with 1p/19q codeletion. Furthermore, the designation of TP53 mutation subjectively by immunohistochemistry is fraught with caveats . We developed a predictive model as a clinical diagnostic aid that incorporates Olig2 features, % of cells with strong p53 immunoreactivity, and the probability based on age and ATRX status. The goal of this model is to provide guidance in the event that ATRX immunohistochemistry is either unavailable or equivocal. Our validation set (Figure ) included 5 false‐positive 1p/19q FISH results, only two of which could have been prevented by adherence to cIMPACT‐NOW guidelines, whereas our improved guidelines handle all cases in the validation set and would prevent all false‐positive 1p/19q in the assays we analyzed. Implications of information theory on diagnostic neuropathology The glioma diagnosis simulation and our information theory analysis raise some very interesting points regarding the future development and deployment of clinical diagnostic aids for neuropathology. At the forefront of our findings is the identification that simple information provides the majority of information. Specifically, basic clinical history, radiographic documentation of tumor site, and histologic findings carried the most information. This is rather intuitive, but these findings are of utmost importance for low‐resource healthcare ecosystems. These findings are in‐line with recommendations for low‐to‐middle‐income countries (LMIC) that have emphasized local healthcare ecosystems to embrace a culture of quality in pathology laboratories, in particular, by adopting appropriate standard operating procedures, quality control, and quality assurance practices . Underscoring this point, a comprehensive study by Fleming et al. generated a recommended capabilities list for Pathology Laboratories in LMICs. Chief among these recommendations included high‐quality histologic/cytologic services as well as training for laboratory staff and physician‐pathologists . Our findings from the brain cancer population simulation underscore the key importance of quality histology in diagnostic neuropathology. Building upon these recommendations, we would further advocate that evaluation of clinical, histologic, and biomarker features by information theory could provide a quantitative framework from which to make data‐driven decisions on diagnostic services. Our work raises significant concerns with ML‐/AI‐based clinical diagnostic aids in neuropathology. First, most brain cancer data set structures include high‐resolution genomic data on very few samples. Pooling data sets from various centers results in missing data elements that are often beyond the capabilities of imputation for clinical outcomes research . In addition, as demonstrated in our glioma simulation, the quantity of information present is highly asymmetrical, and many brain tumor entities are exceedingly rare. This results in ML‐/AI‐based algorithms, nearly all of which utilize some version of information theory, to focus on age, neuroanatomical site, and proliferative index for diagnosis. As a result, predictive algorithms may generate adequate differential diagnoses, but these would be of minimal utility in routine practice. Our approach to overcoming this problem is to identify specific, granular diagnostic conundrums as candidate branch points in clinical decision‐making, where ML/AI can make an impact. This permits the focused evaluation of biomarker information with several advantages over traditional analyses. PCA and correlation plots indicated such a high correlation and covariance between p53 and ATRX status that it raised the possibility that these two biomarkers were fully redundant, legitimately questioning employing both antibodies. In contrast, using an information theory approach, we found that the incorporation of both biomarkers simultaneously resulted in a statistically significant information gain. This synergy in information gain was masked using traditional linear analyses. Similar evaluations could be employed to evaluate additional frequently paired biomarkers. Simple image analysis workflows deployable throughout the world The advent of CNN networks and image recognition has revolutionized the modern world, and it will continue to provide significant impacts to diagnostic pathology. However, its deployment in the practice of pathology globally has several limitations. First and foremost, although pathologists worldwide have access to brightfield microscopes equipped with cameras, few laboratories contain whole slide imaging (WSI)‐capable scanners. In addition to capturing all of the tissue examined, WSI has the added benefit of imposing a standard operating procedure (SOP) in image acquisition, whereas traditional photomicroscopy is nearly impossible to be replicated from one session to another. In our study, we utilized tiles of image patches taken from WSI images to mimic pathologists capturing images on their desktop microscopes. A second challenge is that the lack of adherence to SOPs for histopathology across the globe results in highly variable interlaboratory fixation and staining protocols . Although several attempts to correct this have been published (e.g., Ref. ), the nonlinearity in histologic color maps has posed a significant challenge when controlling for fixation and staining conditions across labs and may be unsurmountable in labs with high batch variance in staining. For this reason, we focused on incorporating self‐normalizing workflows when dealing with color intensity and color texture features. One simple approach is to use internal known positive controls, such as p53 immunohistochemistry in ovarian serous carcinoma with a known negative control (in our case, a benign lymph node), and train within each case an algorithm based on pixel intensity and texture features. This internal slide control provides an unbiased methodology. A second approach we took was using the concept of data density within multidimensional space for the evaluation of nuclear pleomorphism in glioma. These analyses can be performed across histopathology labs as they would only require image acquisition using the same objective magnification and numerical aperture. Machine learning‐workflow improvement to WHO and cIMPACT‐NOW clinical guidelines Our test‐case scenario dealt with the need to confirm 1p/19q codeletion status in IDH ‐mutant gliomas. In many healthcare ecosystems, even in the United States, no reimbursement for chromosomal microarray to confirm 1p/19q codeletion exists. In contrast, 1p/19q codeletion testing by FISH, although suffering in positive predictive value relative to more advanced techniques, reimburses routinely. This leads to a situation in which pathologists must make a decision to perform unreimbursed reflex testing in a serial fashion to confirm 1p/19q codeletion, further delaying treatment planning by neuro‐oncology/radiation oncology. The cIMPACT guidelines, which are anticipated to be incorporated into the 2021 WHO classification, represent a significant improvement to prior recommendations . Specific to our test case at hand is that current cIMPACT guidelines dictate that documentation of TP53 mutation and concurrent ATRX mutation within an IDH ‐mutant tumor excludes the diagnosis of oligodendroglioma. A specific problem arises when pathologists identify an IDH ‐mutant tumor, and either ATRX immunohistochemistry is unavailable, or ATRX immunohistochemistry is equivocal (as was shown in Case 4 of Figure ) or negative due to copy number loss involving the ATRX locus in a tumor with 1p/19q codeletion. Furthermore, the designation of TP53 mutation subjectively by immunohistochemistry is fraught with caveats . We developed a predictive model as a clinical diagnostic aid that incorporates Olig2 features, % of cells with strong p53 immunoreactivity, and the probability based on age and ATRX status. The goal of this model is to provide guidance in the event that ATRX immunohistochemistry is either unavailable or equivocal. Our validation set (Figure ) included 5 false‐positive 1p/19q FISH results, only two of which could have been prevented by adherence to cIMPACT‐NOW guidelines, whereas our improved guidelines handle all cases in the validation set and would prevent all false‐positive 1p/19q in the assays we analyzed. The authors have no conflicts of interest to declare that are relevant to the content of this article. Clinical context and clinical informatics: Diana L. Thomas, José Javier Otero, Lokman Cevik, Mehmet Tahir Aslan, Wesley Wang; Data analysis and data visualization: Catherine Czeisler, José Javier Otero, Lokman Cevik, Mehmet Tahir Aslan, Wesley Wang; Data acquisition: Francisco Jose Garagorry Guerra, Yolanda Cabello‐Izquierdo, Wesley Wang, Jing Zhao, Aline Paixao Becker, Catherine Czeisler, Anne Costa Rendeiro, Lucas Luis Sousa Véras, Maicon Fernando Zanon, Rui Manuel Reis, Marcus de Medeiros Matsushita, Koray Ozduman, M. Necmettin Pamir, Ayca Ersen Danyeli, Thomas Pearce, Michelle Felicella, Jennifer Eschbacher, Naomi Arakaki, Horacio Martinetto, Anil Parwani; Writing—original draft preparation: Diana L. Thomas, José Javier Otero, Lokman Cevik, Marilyn Vazquez Landrove, Wesley Wang; Writing—review and editing: all authors; Mathematical and statistical modeling: Marilyn Vazquez Landrove, Jing Zhao. This study involved an analysis of retrospectively collected individual patient information and digital images in patients with IDH ‐mutant gliomas. This study was approved by the ethics board of the Ohio State University and all the local ethics boards. Fig S1‐S58 FIGURE S1 Anaplastic astrocytoma, WHO grade 2, IDH−mutated FIGURE S2 Anaplastic astrocytoma, WHO grade 2, IDH−wild type FIGURE S3 Anaplastic ependymoma, posterior fossa A, WHO grade 3 FIGURE S4 Anaplastic ependymoma, posterior Fossa B, WHO grade 3 FIGURE S5 Anaplastic ependymoma, spine, WHO grade 3 FIGURE S6 Anaplastic ependymoma, supratentorial−RELA, WHO grade 3 FIGURE S7 Anaplastic ependymoma, supratentorial−YAP, WHO grade 3 FIGURE S8 Anaplastic ganglioglioma FIGURE S9 Anaplastic oligodendroglioma FIGURE S10 Anaplastic pleomorphic xanthoastrocytoma FIGURE S11 Angiocentric glioma FIGURE S12 Astroblastoma FIGURE S13 Astrocytoma, WHO grade 2, IDH−mutated FIGURE S14 Astrocytoma, WHO grade 2, IDH−wild type FIGURE S15 Atypical choroid plexus papilloma, WHO grade 2 FIGURE S16 Atypical rhabdoid tumour FIGURE S17 Central neurocytoma FIGURE S18 Cerebellar liponeurocytoma, WHO grade 2 FIGURE S19 Choroid glioma of the third ventricle FIGURE S20 Choroid plexus carcinoma FIGURE S21 Choroid plexus papilloma FIGURE S22 Desmoplastic infantile astrocytoma, WHO grade 1 FIGURE S23 Desmoplastic infantile ganglioglioma FIGURE S24 Diffuse leptomeningeal glioneuronal tumor FIGURE S25 Diffuse midline glioma, WHO grade 4 FIGURE S26 Diffuse oligodendroglioma, WHO grade 2 FIGURE S27 Dysembryoplastic neuroepithelial Tumor FIGURE S28 Dysplastic cerebellar gangliocytoma, WHO grade 1 FIGURE S29 Ependymoma, posterior fossa A, WHO grade 2 FIGURE S30 Ependymoma, posterior fossa B, WHO grade 2 FIGURE S31 Ependymoma, spine, WHO grade 2 FIGURE S32 Ependymoma, supratentorial‐YAP, WHO grade 2 FIGURE S33 Ependymoma, supratentorial, RELA, WHO grade 2 FIGURE S34 Extraventricular neurocytoma FIGURE S35 Gangliocytoma, WHO grade 1 FIGURE S36 Glioblastoma FIGURE S37 Astrocytoma, IDH−mutant, WHO grade 4 FIGURE S38 Glioblastoma, IDH−wild type, WHO grade 4 FIGURE S39 Medulloblastoma, non−WNT/non−SHH, WHO grade 4 FIGURE S40 Medulloblastoma, SHH class, WHO grade 4 FIGURE S41 Medulloblastoma, WNT group, WHO grade 4 FIGURE S42 Meningioma, WHO grade 1 FIGURE S43 Paraganglioma FIGURE S44 Pilocytic astrocytoma FIGURE S45 Pilomyxoid astrocytoma FIGURE S46 Pleomorphic xanthoastrocytoma, WHO grade 2 FIGURE S47 Rossete‐forming glioneuronl tumour, WHO grade 1 FIGURE S48 Schwannoma, WHO grade 1 FIGURE S49 Solitary Fibrous tumour/hemangiopericytoma grade 1 FIGURE S50 Subependymoma FIGURE S51 Subependymal Giant cell astrocytoma, WHO grade 1 FIGURES S1–51 Summary of each entity in simulation model. (A) Age distributions, (B) Neuroanatomical distributions, (C) Ki67 distributions, (D) Distribution of histological features, (E) Distribution of immunohistochemical features, (F) Distribution of molecular pathology features. Data represented in panels D, E, and F are violin plots of the distribution of the histological, immunohistochemical, and molecular biomarkers. Each biomarker is listed as a binary readout of 0 = negative, 1 = positive. The violin shape provides an indication of the relative proportion showing positive or negative results for this biomarker. In cases where the literature does not support a distribution, (for instance, necrosis in astrocytoma, a notch is placed at the level of the 0) FIGURE S52 Distribution of cases in the global IDH mutant (Grade 2 and 3) dataset. World map shows the distribution of the cases in the global dataset. TCGA study (purple circles) has cases from different areas of the world such as Italy, Germany, Australia, but mostly the United States. This overall distribution of global dataset shows a good variety of ethnicity and genetic background to represent a real‐world data FIGURE S53 Deconvolution with Otsu’s thresholding and Watershed. The images were splitted into R, G and B channels. Then R (red), G (green) and B (blue) channels were converted to H (hematoxylin), D (DAB) and X channels. D channel was selected for brown color in OLIG2 staining. After application of median filtering, Otsu's thresholding was applied to the D channel. The holes in the nuclei were filled out and then the nuclei on the border were eliminated for a better representation of nuclei. As a last step, watershed function was applied for overlapping or contacting nuclei FIGURE S54 K‐means clustering segmentation. The images were converted to a data frame using R, G and B pixel intensity values. All pixels were clustered into 3 clusters using k‐means clustering method. Each pixel was labeled as cluster 1, cluster 2, or cluster 3. The cluster that contain specifically the nuclei was chosen as foreground and the other 2 clusters were accepted as background FIGURE S55 Cut‐Cluster‐Classify (CCC) segmentation method. (A) All patches in the image are viewed as high dimensional points and a threshold is applied to the sample density (Cut) to get clusters. After clustering (Cluster), the patches below the threshold were classified based on the clusters (Classify). (B) Then all pixels were voted for clusters based on cluster result on overlapping patches. The cluster that contains nuclei was accepted as foreground and the other one was accepted as background. 5x5 patches were used for the segmentation FIGURE S56 Relationships of R channel pixel intensity values with G and B pixel intensity values for color of noise. Mean pixel intensity values of R channel are correlated to mean pixel intensity values of G and B channels. To better represent these spectrums, we used support vector machine (SVM) modeling to predict G and B values from R. The colors of noise are purple for H&E staining and brown for OLIG2 staining FIGURE S57 Image noise assay workflow and comparison of supervised and unsupervised image segmentation methods for H&E and OLIG2 immunostaining. (A) An image noise assay was created for the fidelity evaluation of image segmentation methods to determine the best one for our purpose. We extracted the purple color for H&E staining and brown color for OLIG2 staining. A support vector model (SVM) was made to generate a noise similar to colors of the image. After generation of noise for a full image, the pixels were randomly sampled with certain percentages to implement the noise to the images. This workflow provided us to compare the performance of segmentation methods with different amount of noise. (B and C) We plotted segmentation performance by accuracy and intersection‐over‐union (IoU). Additionally, we added the U‐Net trained with random noise generation as “U‐Net with noise” to the comparison. In U‐Net with noise, we trained a new model every time when we added noise to the image. (B) In H&E staining, U‐Net models show highest accuracies independent from the noise percentage. On the other hand, U‐Net method shows low IoU, when U‐Net with noise has a better IoU values. Overall deconvolution method has the better performance for accuracy and IoU. (C) In OLIG2 staining, similar to the H&E, deconvolution and U‐Net with noise methods have the highest performance regarding accuracy and IoU. Despite deconvolution method has a sharp decrease after 25% noise, U‐Net with noise maintained high IoU even after 25% noise FIGURE S58 (A) Gini importance of RF model for the probability of 1p19q codeletion using ATRX and age. (B) Gini importance of final RF model used for the 1p19q codeletion in validation data set. (C) Simulation of the model with different p53 percentages showing 1p19q codeletion probability in different scenarios Click here for additional data file. Table S1 TABLE S1 Impact of scrambling features on accuracy of random forest model Click here for additional data file. Supplementary Material TABLE S2 Validation set data Click here for additional data file. Supplementary Material Click here for additional data file. |
A Novel Custom Bent 27-Gauge Needle–Guided Suture Snare Technique for Scleral Fixation of Posterior Chamber Intraocular Lenses | d55a61cd-00c0-4fcf-afb7-859a9bb83c22 | 11753450 | Surgical Procedures, Operative[mh] | The study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Institutional Review Board of Liyang Hospital of Chinese Medicine. All participants undergoing the procedure provided written informed consent after possible benefits and risks of the procedure were discussed. All patients underwent comprehensive preoperative and postoperative ophthalmologic examination, and age, sex, best-corrected visual acuity, postoperative IOL position, and other data were recorded. Surgical Procedure The procedure was performed by one of the authors (C.C.) and is demonstrated in the Supplemental Digital Content (see Video , http://links.lww.com/IAE/C254 and Figures and ). The procedure was performed under retrobulbar anesthesia, and a three-haptic IOL with a haptic loop was selected. First, the IOL haptic fixation point was positioned, the exit position of the needle from the limbus was planned, and a 27-gauge needle was bent into an arc that made it easy to hold with the fingers and pass the suture through the needle (Figure ). Finally, an 8-0 polypropylene suture was threaded through the needle lumen. The 2-mm incisions were made in the ocular conjunctiva at the three fixation sites, exposing the sclera. The IOL was implanted in the anterior chamber. A hand-held needle was threaded into the eye 2 mm outside the limbus and passed through the IOL haptic loop and out the limbal incision with the assistance of a blunt, round, and hollow needle (viscoelastic needle). The suture was pulled out, and the needle was advanced into the eye again (without withdrawing the needle) by the same limbal incision, bypassing the haptic loop. The suture was threaded through the needle lumen once again, and the needle was withdrawn from the eye, thereby completing fixation of the IOL haptic loop. The same procedure was used for fixation of the other two IOL haptic loops. External scleral fixation was performed using an “overhand friction knot” technique to bury the knot in the stroma of the sclera. The tightness of the sutures at the three fixation points was adjusted at the time of securing the knot to keep the optical site of the IOL centered on the pupil. The small conjunctival incision was either not sutured or it was closed with a single 10-0 nylon suture.
The procedure was performed by one of the authors (C.C.) and is demonstrated in the Supplemental Digital Content (see Video , http://links.lww.com/IAE/C254 and Figures and ). The procedure was performed under retrobulbar anesthesia, and a three-haptic IOL with a haptic loop was selected. First, the IOL haptic fixation point was positioned, the exit position of the needle from the limbus was planned, and a 27-gauge needle was bent into an arc that made it easy to hold with the fingers and pass the suture through the needle (Figure ). Finally, an 8-0 polypropylene suture was threaded through the needle lumen. The 2-mm incisions were made in the ocular conjunctiva at the three fixation sites, exposing the sclera. The IOL was implanted in the anterior chamber. A hand-held needle was threaded into the eye 2 mm outside the limbus and passed through the IOL haptic loop and out the limbal incision with the assistance of a blunt, round, and hollow needle (viscoelastic needle). The suture was pulled out, and the needle was advanced into the eye again (without withdrawing the needle) by the same limbal incision, bypassing the haptic loop. The suture was threaded through the needle lumen once again, and the needle was withdrawn from the eye, thereby completing fixation of the IOL haptic loop. The same procedure was used for fixation of the other two IOL haptic loops. External scleral fixation was performed using an “overhand friction knot” technique to bury the knot in the stroma of the sclera. The tightness of the sutures at the three fixation points was adjusted at the time of securing the knot to keep the optical site of the IOL centered on the pupil. The small conjunctival incision was either not sutured or it was closed with a single 10-0 nylon suture.
A total of 10 eyes in 10 patients (8 males and two females) were included. The mean age of the patients was 61.5 ± 7.6 years (range: 52–76 years). Indications for surgery were as follows: 6 cases of traumatic lens dislocation, 1 case of IOL dislocation, and 3 cases of aphakia after vitrectomy. The IOL used was the Zeiss CT ASPHINA 603P PCIOL（Carl Zeiss Inc, Perigny, France). There were two cases of transient minor ciliary body hemorrhage during needle puncture. No other intraoperative complications were observed. There was one case of postoperative complications in an eye with transiently elevated intraocular pressure, which was treated with topical antiglaucoma medication. The mean follow-up period was 8.5 ± 5.6 months (range: 3–16 months). The logarithm of the minimum angle of resolution (logMAR) corrected visual acuity was 0.73 ± 0.55 (Snellen 20/107). The logMAR corrected visual acuity at the final follow-up was 0.24 ± 0.27 (Snellen 20/35); the t -test showed that the difference was statistically significant ( P < 0.05). The IOL was well-positioned in all patients (Figure ), and the mean postoperative PCIOL tilt was 2.78 ± 1.32°. There were no cases of pupillary capture, suture loosening, suture erosion, scleral atrophy, or retinal detachment at the time of the final follow-up.
Implantation of PCIOL without capsular support is generally performed in two ways: sutureless intrascleral fixation using the Yamane technique, which has become popular in recent years, or scleral suture fixation. However, the only IOL that is suitable for this technique is the three-piece IOL, which is difficult to learn using a double-needle technique, especially for IOL haptics that withdraw into the needle lumen. Many types of IOLs can be selected for scleral suture fixation, including two-haptic (including three-piece IOLs), three-haptic, and four-haptic IOLs. The bent needle-guided suture snare technique is particularly appropriate for IOLs with a haptic loop, as the snare and suture can form a “cross-buckle” fixation that remains in place. In particular, stable trocar fixation can be created for subluxated IOLs and capsular bag complexes. The bent needle-guided suture snare technique requires a 27-gauge needle with appropriate hardness, a needle body length of 38 mm, which is much longer than the diameter of the ciliary sulcus, and a puncture opening that is self-closing, with the scleral puncture entrance and the limbal exit planned first. The needle is bent into the corresponding arc shape, which facilitates stable hand-held operation, enables more direct control of the needle tip, and facilitates changing the angle and direction of the needle. The use of blunt, round, and hollow needles (viscoelastic needles) pushes away the iris tissue or pivots the IOL to expose the IOL haptic loops. This facilitates passage of the 27-gauge needle from inside and outside the IOL haptic loop and serves as a “trocar” to guide the needle out of the limbal incision, enabling the IOL haptic suture snare to be completed easily even in cases of small pupils. In some special cases where IOL haptic fixation is required at the 6-o'clock position, the length of the bent portion of the needle can be shortened to avoid interfering with the eyelid distractor or lower eyelid. Fixation of the IOL haptic is a major step in this procedure. Our technique requires only one scleral puncture, and the needle is passed through the limbal incision twice to complete fixation of the IOL haptic loop. Alternatively, in the case of a two-haptic IOL, a cow-hitch suture knot can be used for fixation. , This maintains the integrity of the sutures and reduces the damage caused by clamping of the sutures with forceps. The conventional technique is to use a needle holder to clamp a long straight needle for puncture. , However, this can lead to kinking and twisting of the needle, resulting in potential tissue damage, especially when operating in directions other than 3 o'clock and 9 o'clock (because of interference with the eyelids or eyelid distractor). The sutures are knotted on the IOL haptic with a free end, which can potentially abrade the uvea and cause glaucoma, recurrent uveitis, cystoid macular edema, and other complications. , Some researchers have used trocars to create ports to deliver sutures into the anterior chamber before IOL implantation, using retinal forceps to enter the eye to pull out sutures and even widen the incision and manually fold the IOL for implantation. , In addition to increasing the complexity of the operation, there is potential to cause leakage from the ports and hypotony. Reliable external scleral fixation is necessary for fixation of the IOL. We used the “overhand friction knot” technique to bury the knot in the stroma of the sclera, which eliminates the need for a conjunctival or scleral flap. The conventional method is burial of suture knots in a scleral flap, which may abrade and irritate local tissues leading to complications such as scleral atrophy and exposure of suture knots. Meanwhile, suture breakage and secondary IOL dislocation remain significant problems that cannot be ignored. The long-term stability of the 10-0 polypropylene suture in common use is controversial. , To reduce the risk of suture breakage because of its material properties, some researchers have experimented with a relatively thicker suture for IOL fixation, and 8-0 polypropylene suture is currently being used more and achieving good results in clinical practice. , The follow-up period of the present study was 8.5 ± 5.6 months, and no suture-related complications were observed. In summary, the custom bent 27-gauge needle–guided suture snare technique for scleral fixation of PCIOL facilitates smooth operation. The use of the normal surgical incision for IOL implantation for cataract surgery eliminates the need for additional conjunctival and scleral flaps, thereby allowing for a minimally invasive operation. In addition, it is suitable for scleral suture fixation of multiple types of PCIOLs, subluxated IOLs, and capsular bag complexes. Our experience demonstrates that this technique is safe and results in satisfactory patient outcomes. The limitations of the present study include the few cases, the short observation period, and the lack of observation of long-term effects and comparison with other methods.
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LncRNA linc01194 promotes the progress of endometrial carcinoma by up-regulating SOX2 through binding to IGF2BP1 | 70592528-d86d-4810-9ea5-dd017419520e | 10948988 | Gynaecology[mh] | Endometrial carcinoma (EC) ranks second in the incidence of female malignant tumors . The metastatic and invasive capacity of EC is strong, which is the main factor affecting the long-term survival rate of EC patients . Due to the increase in risk factors such as diabetes and obesity, the incidence of EC has gradually increased . Notably, the incidence of EC will increase by 50% by 2040 . Currently, surgery is the main therapeutic approach for EC treatment, with radiotherapy and chemotherapy applied as adjusted treatments when necessary. Although progress in EC treatment has been made, those with advanced EC have the poor prognosis . A study conducted in 2018 showed that 80% of EC patients were diagnosed as well differentiated and moderately differentiated EC, with a good prognosis. Nevertheless, those with poorly differentiated EC have low survival rate . Therefore, the discovery and development of effective early prognostic indicators are of considerable clinical significance. Previous studies have shown that less than 2% of genes in the human genome encode proteins . Non-coding RNAs (ncRNAs), as the name implies, are not involved in coding proteins. Increasingly more studies on ncRNAs have revealed that they are the key regulators in many cellular processes, such as transcription, post-transcriptional modification, cell growth, cell proliferation, apoptosis and metabolism . As a type of RNAs without coding capability, long non-coding RNAs (lncRNAs), have a length of more than 200 nucleotides . Accumulating studies had revealed that lncRNAs are related to several pathological and physiological processes, especially in cancer development . The advances in RNA sequencing, gene microarray analysis, and high-throughput sequencing have enabled the recognition of the lncRNA potential as a tumor-suppressor or an oncogene gene that can regulate cell proliferation, migration, invasion, apoptosis, and immune response has been recognized . Therefore, lncRNA can be used as a molecular marker for early diagnosis of tumors and a new target for tumor therapy . In recent years, increasingly more studies have revealed that a large number of lncRNAs, such as lncRNA-ZXF1 , lncRNA DSCAM-AS1 , and lncRNA SNHG12 and so on, have an abnormal expression in EC and are involved in its occurrence and development. The Cancer Genome Atlas (TCGA) database shows that linc01194 is upregulated in EC. We speculated that linc01194 may be involved in the development and occurrence of EC. 1. Bioinformatics analysis The expression levels of linc01194 in normal endometrial tissues and EC tissues was analyzed in the starBase database ( https://starbase.sysu.edu.cn/ ) and the TCGA database ( https://portal.gdc.cancer.gov/ ). Also, the downstream target protein of linc01194 was predicted in the starBase database. The relationship between insulin-like growth factor 2 binding protein 1 (IGF2BP1) and the overall survival rate of EC patients was revealed in the Kaplan-Meier Plotter database ( http://kmplot.com/analysis/ ). 2. Endometrial specimens We collected and analyzed the endometrial tissue of 127 gynecological patients (including 99 endometrial cancer tissues and 28 normal endometrial tissues) from the Third Affiliated Hospital of Guangzhou Medical University. All of them had not received preoperative radiotherapy and chemotherapy. Two pathologists confirmed the endometrial specimens independently. Then these endometrial specimens were stored at −80°C until they were used for further studies. Obtained the informed consent of the subjects before obtaining the specimen. The experiment was approved by the Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University (No. 2020066). All endometrial specimens were treated in accordance with ethical and legal standards and treated anonymously. 3. Cell culture The expression of linc01194 in 4 human EC cells lines (Ishikawa, HEC-1B, HEC-1A, and KLE), purchased from Jennio Biotech, was detected. Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone Laboratories Inc., Logan, UT, USA) was utilized to culture Ishikawa cells, while Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) was utilized to culture HEC-1B cells, McCoy’s 5A medium was utilized to culture HEC-1A cells and Ham’s F-12 treatment medium was utilized to culture KLE cells. All culture media contained 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were cultured in an incubator containing 5% CO 2 at 37°C. All cells were identified by short tandem repeat (STR) analysis and subjected to mycoplasma contamination tests. 4. Transfection of small interfering RNA (siRNA) and plasmid targeting linc01194 The siRNA targeting linc01194 (si-linc01194) and the negative control (si-NC) were synthesized by RiboBio Co., Ltd. (Guangzhou, China). The plasmid targeting linc01194 was used to upregulate the expression of linc01194. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was utilizes to transfect siRNA and plasmid. The concentration of si-linc01194 and control si-NC was 100 nM. The siRNA sequence targeting linc01194 was GCTTGATGGTGTAACTTCA. The si-NC sequence was provided by RiboBio Co., Ltd. The plasmid sequence targeting linc01194 was as listed in . 5. Cell proliferation detection with cell counting kit-8 (CCK-8) We followed the instructions of CCK-8 kit (Yeasen, Shanghai, China) to detect cell proliferation. First, inoculated cells into a 96-well plate containing 100 µL of medium until they adhered to the wall. The concentration is 3,000 cells/well. The samples were divided into an experimental and a negative control group, which were transfected with si-linc01194 and si-NC, respectively. At 0, 24, 48 and 72 hours, add 10 uL CCK-8 solution to each well. Then, incubate for 2 hours in an incubator with 5% CO 2 at 37°C. The absorbance was then determined at 450 nm with a microplate reader. 6. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from EC cell lines with TRIzol reagent (TaKaRa, Shiga, Japan), and then Hifair ® III 1st Strand cDNA Synthesis SuperMix was used for qPCR, and then total RNA reversed to cDNA. Next, Hieff ® qPCR SYBR ® Green Master Mix was utilized to amplify the target gene by real-time qPCR and measure its expression level. Further, 18sRNA and the U6 gene were acted as an internal reference gene. The related primer sequences were listed in the . 7. Wound-healing assay Cells were inoculated in a 6-well plate containing 2 mL of medium containing 10% FBS and cultured until fusion into a monolayer was achieved. Make vertical scratches by a 200-µL pipette in the Petri dish, and then washed with phosphate-buffered saline (PBS) twice to three times, following by adding 2 mL of serum-free medium. At 0, 24 and 48 hours, took photo under a microscope, to compare the wound sizes in the experimental and negative control groups. The software ImageJ (National Institutes of Health, Bethesda, MD, USA) was utilized to calculate the cell migration rate. All experiment were repeated 3 times. 8. Cell invasion test The diluted matrix binder was evenly placed in the small chamber of a 24-well plate Transwell chamber (Corning, Inc., Corning, NY, USA), following by incubation in an incubator containing 5% CO 2 at 37 °C for 4 hours. A total volume of 600 uL of medium containing 10% FBS, streptomycin and penicillin and the configured transfection liquid was added to the lower chamber. After 48 hours, small chamber was taken out and washed with PBS. Fix with 4% paraformaldehyde for 30 minutes, and dye with 0.1% crystal violet after washed with PBS. Remove cells on the surface of matrix binder. Cut it down and make it into a slide. Calculate cells in a field of vision. 9. Apoptosis - Flow cytometry analysis The cells were cultured into a 6-well plate until they adhered to the wall. The experimental and the negative control groups were transfected with si-linc01194 and si-NC, respectively. Then, the supernatant was collected after 48 hours. After washing with PBS twice, the cells were digested with trypsin without ethylenediaminetetraacetic acid, collected, and centrifuged. The precipitate was washed with PBS twice, centrifuged, and retained. Following the instructions of the manufacturer of the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining reagent, flow cytometry was utilized to quantitatively analyze and compare the apoptosis between the experimental and the negative control groups. 10. RNA-binding protein immunoprecipitation (RIP) Cells were washed with cold PBS twice to three times, scraped off, and transferred into a clean EP tube. Then the cell samples were centrifuged. The precipitate was retained, and RIPA buffer containing phenylmethylsulfonyl fluoride was added, followed by overnight separation at 4°C, and then centrifugation 12,000 rpm for 20 minutes at 4°C. Retain the supernatant, and take 20 uL as the input group. The remaining supernatant was divided into 2 equal portions, in one of which, we added 4 ug immunoglobulin G (IgG), while in the other, we added 4 ug IGF2BP1 antibody. The samples were then subjected to overnight shaking at 4°C. Next, 100 uL of magnetic bead suspension was added on the next day, followed by shaking at a constant speed of 4°C for 4 hours. Then, a magnetic rack was used to adsorb the magnetic beads and separate the reaction mixture. We further removed the supernatant, and utilizes PBS for 3-fold washing of the magnetic beads. TRIzol reagent was added to all samples for RNA extraction, followed by its reverse transcription into cDNA. And then compare the expression levels of linc01194 in the IgG and IGF2BP1 groups through qRT-PCR. 11. Protein nucleo-cytoplasmic separation experiment We utilized the Cytoplasm and Nuclear Protein Extraction Kit (Beyotime Biotech Inc., Haimen, China) to extract the cytoplasmic and nuclear proteins of cells from the experimental and negative control groups following to the manufacturer’s instructions. 12. Western blotting IGF2BP1 antibody (Proteintech, Rosemont, IL, USA), SOX2 antibody (Proteintech), GAPDH antibody (Proteintech) and α-tubulin antibody (Proteintech) were used in our western blotting analyses. We determined the protein concentration with the bicinchoninic acid protein concentration determination kit (Beyotime Biotech Inc.), and then PBS and 5 × loading buffer were added for quantification of the protein concentration to 2 ug/uL. After denaturation at 100°C for 15 minutes, separate the protein with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred protein to a polyvinylidene difluoride membrane. Next, seal the membrane with 5% skimmed milk, and primary antibodies were added, and then incubation of the membrane overnight at 4°C. The membrane was washed with Tris buffered saline with Tween 20 (TBST) for 10 minutes 3 times, and added the secondary antibody (1:8000, Proteintech) and incubated the membrane for 2 hours at room temperature, on the next day. And then wash the membrane with TBST for 10 minutes 3 times. Analyze the protein bands through the hypersensitive ECL chemiluminescence kit (NcmECL UItra; Sliding Biotech, Shanghai, China). ImageJ software was utilized to protein quantification. 13. Statistical analysis All data are presented as the mean ± standard error of mean (SEM). GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) was applied. One-way analysis of variance and unpaired Student’s t-test were utilized to analyze the difference. The p-value is less than 0.05 was considered statistically significant. The expression levels of linc01194 in normal endometrial tissues and EC tissues was analyzed in the starBase database ( https://starbase.sysu.edu.cn/ ) and the TCGA database ( https://portal.gdc.cancer.gov/ ). Also, the downstream target protein of linc01194 was predicted in the starBase database. The relationship between insulin-like growth factor 2 binding protein 1 (IGF2BP1) and the overall survival rate of EC patients was revealed in the Kaplan-Meier Plotter database ( http://kmplot.com/analysis/ ). We collected and analyzed the endometrial tissue of 127 gynecological patients (including 99 endometrial cancer tissues and 28 normal endometrial tissues) from the Third Affiliated Hospital of Guangzhou Medical University. All of them had not received preoperative radiotherapy and chemotherapy. Two pathologists confirmed the endometrial specimens independently. Then these endometrial specimens were stored at −80°C until they were used for further studies. Obtained the informed consent of the subjects before obtaining the specimen. The experiment was approved by the Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University (No. 2020066). All endometrial specimens were treated in accordance with ethical and legal standards and treated anonymously. The expression of linc01194 in 4 human EC cells lines (Ishikawa, HEC-1B, HEC-1A, and KLE), purchased from Jennio Biotech, was detected. Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone Laboratories Inc., Logan, UT, USA) was utilized to culture Ishikawa cells, while Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) was utilized to culture HEC-1B cells, McCoy’s 5A medium was utilized to culture HEC-1A cells and Ham’s F-12 treatment medium was utilized to culture KLE cells. All culture media contained 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were cultured in an incubator containing 5% CO 2 at 37°C. All cells were identified by short tandem repeat (STR) analysis and subjected to mycoplasma contamination tests. The siRNA targeting linc01194 (si-linc01194) and the negative control (si-NC) were synthesized by RiboBio Co., Ltd. (Guangzhou, China). The plasmid targeting linc01194 was used to upregulate the expression of linc01194. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was utilizes to transfect siRNA and plasmid. The concentration of si-linc01194 and control si-NC was 100 nM. The siRNA sequence targeting linc01194 was GCTTGATGGTGTAACTTCA. The si-NC sequence was provided by RiboBio Co., Ltd. The plasmid sequence targeting linc01194 was as listed in . We followed the instructions of CCK-8 kit (Yeasen, Shanghai, China) to detect cell proliferation. First, inoculated cells into a 96-well plate containing 100 µL of medium until they adhered to the wall. The concentration is 3,000 cells/well. The samples were divided into an experimental and a negative control group, which were transfected with si-linc01194 and si-NC, respectively. At 0, 24, 48 and 72 hours, add 10 uL CCK-8 solution to each well. Then, incubate for 2 hours in an incubator with 5% CO 2 at 37°C. The absorbance was then determined at 450 nm with a microplate reader. Total RNA was extracted from EC cell lines with TRIzol reagent (TaKaRa, Shiga, Japan), and then Hifair ® III 1st Strand cDNA Synthesis SuperMix was used for qPCR, and then total RNA reversed to cDNA. Next, Hieff ® qPCR SYBR ® Green Master Mix was utilized to amplify the target gene by real-time qPCR and measure its expression level. Further, 18sRNA and the U6 gene were acted as an internal reference gene. The related primer sequences were listed in the . Cells were inoculated in a 6-well plate containing 2 mL of medium containing 10% FBS and cultured until fusion into a monolayer was achieved. Make vertical scratches by a 200-µL pipette in the Petri dish, and then washed with phosphate-buffered saline (PBS) twice to three times, following by adding 2 mL of serum-free medium. At 0, 24 and 48 hours, took photo under a microscope, to compare the wound sizes in the experimental and negative control groups. The software ImageJ (National Institutes of Health, Bethesda, MD, USA) was utilized to calculate the cell migration rate. All experiment were repeated 3 times. The diluted matrix binder was evenly placed in the small chamber of a 24-well plate Transwell chamber (Corning, Inc., Corning, NY, USA), following by incubation in an incubator containing 5% CO 2 at 37 °C for 4 hours. A total volume of 600 uL of medium containing 10% FBS, streptomycin and penicillin and the configured transfection liquid was added to the lower chamber. After 48 hours, small chamber was taken out and washed with PBS. Fix with 4% paraformaldehyde for 30 minutes, and dye with 0.1% crystal violet after washed with PBS. Remove cells on the surface of matrix binder. Cut it down and make it into a slide. Calculate cells in a field of vision. The cells were cultured into a 6-well plate until they adhered to the wall. The experimental and the negative control groups were transfected with si-linc01194 and si-NC, respectively. Then, the supernatant was collected after 48 hours. After washing with PBS twice, the cells were digested with trypsin without ethylenediaminetetraacetic acid, collected, and centrifuged. The precipitate was washed with PBS twice, centrifuged, and retained. Following the instructions of the manufacturer of the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining reagent, flow cytometry was utilized to quantitatively analyze and compare the apoptosis between the experimental and the negative control groups. Cells were washed with cold PBS twice to three times, scraped off, and transferred into a clean EP tube. Then the cell samples were centrifuged. The precipitate was retained, and RIPA buffer containing phenylmethylsulfonyl fluoride was added, followed by overnight separation at 4°C, and then centrifugation 12,000 rpm for 20 minutes at 4°C. Retain the supernatant, and take 20 uL as the input group. The remaining supernatant was divided into 2 equal portions, in one of which, we added 4 ug immunoglobulin G (IgG), while in the other, we added 4 ug IGF2BP1 antibody. The samples were then subjected to overnight shaking at 4°C. Next, 100 uL of magnetic bead suspension was added on the next day, followed by shaking at a constant speed of 4°C for 4 hours. Then, a magnetic rack was used to adsorb the magnetic beads and separate the reaction mixture. We further removed the supernatant, and utilizes PBS for 3-fold washing of the magnetic beads. TRIzol reagent was added to all samples for RNA extraction, followed by its reverse transcription into cDNA. And then compare the expression levels of linc01194 in the IgG and IGF2BP1 groups through qRT-PCR. We utilized the Cytoplasm and Nuclear Protein Extraction Kit (Beyotime Biotech Inc., Haimen, China) to extract the cytoplasmic and nuclear proteins of cells from the experimental and negative control groups following to the manufacturer’s instructions. IGF2BP1 antibody (Proteintech, Rosemont, IL, USA), SOX2 antibody (Proteintech), GAPDH antibody (Proteintech) and α-tubulin antibody (Proteintech) were used in our western blotting analyses. We determined the protein concentration with the bicinchoninic acid protein concentration determination kit (Beyotime Biotech Inc.), and then PBS and 5 × loading buffer were added for quantification of the protein concentration to 2 ug/uL. After denaturation at 100°C for 15 minutes, separate the protein with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred protein to a polyvinylidene difluoride membrane. Next, seal the membrane with 5% skimmed milk, and primary antibodies were added, and then incubation of the membrane overnight at 4°C. The membrane was washed with Tris buffered saline with Tween 20 (TBST) for 10 minutes 3 times, and added the secondary antibody (1:8000, Proteintech) and incubated the membrane for 2 hours at room temperature, on the next day. And then wash the membrane with TBST for 10 minutes 3 times. Analyze the protein bands through the hypersensitive ECL chemiluminescence kit (NcmECL UItra; Sliding Biotech, Shanghai, China). ImageJ software was utilized to protein quantification. All data are presented as the mean ± standard error of mean (SEM). GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) was applied. One-way analysis of variance and unpaired Student’s t-test were utilized to analyze the difference. The p-value is less than 0.05 was considered statistically significant. 1. Linc01194 was overexpressed in EC Our search in starBase database and the TCGA database revealed that the level of linc01194 in EC tissues is higher than that in normal endometrial tissues (p<0.05; ). To determine the level of linc01194 in EC, through qRT-PCR, we detected the expression of linc01194 in endometrial tissues of 127 patients from the Third Affiliated Hospital of Guangzhou Medical University (including 99 patients with EC and 28 patients without EC). The results indicated that the level of linc01194 in the endometrial tissues of patients with EC was higher than that in those without EC (p<0.05; ). Surprisingly, the level of linc01194 was higher in EC with lymphovascular invasion than that in EC without lymphovascular invasion (p<0.05; ). Subsequently, we detected the expression of linc01194 in EC cell lines (Ishikawa, KLE, HEC-1B, and HEC-1A cells). The results revealed that linc01194 had higher expression level in Ishikawa cells and KLE cells, while the expression level was lowest in HEC-1B (p<0.05; ). Therefore, we chose Ishikawa cells and KLE cells for the transfection of siRNA, and HEC-1B cells for the transfection of overexpression-plasmid. 2. The downregulation of linc01194 inhibits the malignant behaviors of EC cells The expression of linc01194 decreased after the transfection of si-linc01194. To explore the effect between linc01194 and EC, we conducted CCK-8 assay, wound-healing assay, cell invasion assay and apoptosis experiments. We found that the low level of linc01194 was related to the decrease of cell proliferation, cell migration, cell invasion, and the increase of apoptosis (p<0.05; and ). 3. The upregulation of linc01194 promotes the malignant behavior of EC cells After transfection with plasmid targeting linc01194, the expression of linc01194 was increased (p<0.05; ), and cell proliferation (p<0.05; ), cell migration (p<0.05; ) and cell invasion (p<0.05; ) were promoted, while cell apoptosis was inhibited (p<0.05; ). 4. Linc01194 regulates the expression of IGF2BP1 and SOX2 in EC To identify the downstream target protein that binds to linc01194, we used the starBase database to predict the target protein of linc01194. The data obtained suggested that IGF2BP1 may be the downstream target protein of linc01194 . The relationship between the levels of IGF2BP1 and the overall survival rate of EC patients was searched in Kaplan-Meier Plotter database, which showed that high levels of IGF2BP1 was related to poor survival rate of EC patients (p<0.05; ). Additionally, we established that linc01194 binds to IGF2BP1 through RIP experiments (p<0.05; ). Moreover, western blots experiment indicated that the knockdown of linc01194 decreased the level of IGF2BP1 in the cytoplasm of EC cells (p<0.05; ). A related study found that the functions of IGF2BPs are exerted mainly in the cytoplasm . Meanwhile, we found that the knockdown of linc01194 reduced the expression level of SOX2 (p<0.05; ). Conversely, the upregulation of linc01194 increased IGF2BP1 expression in the cytoplasm (p<0.05; ). Similarly, the upregulation of linc01194 promoted the expression of SOX2 (p<0.05; ). Our search in starBase database and the TCGA database revealed that the level of linc01194 in EC tissues is higher than that in normal endometrial tissues (p<0.05; ). To determine the level of linc01194 in EC, through qRT-PCR, we detected the expression of linc01194 in endometrial tissues of 127 patients from the Third Affiliated Hospital of Guangzhou Medical University (including 99 patients with EC and 28 patients without EC). The results indicated that the level of linc01194 in the endometrial tissues of patients with EC was higher than that in those without EC (p<0.05; ). Surprisingly, the level of linc01194 was higher in EC with lymphovascular invasion than that in EC without lymphovascular invasion (p<0.05; ). Subsequently, we detected the expression of linc01194 in EC cell lines (Ishikawa, KLE, HEC-1B, and HEC-1A cells). The results revealed that linc01194 had higher expression level in Ishikawa cells and KLE cells, while the expression level was lowest in HEC-1B (p<0.05; ). Therefore, we chose Ishikawa cells and KLE cells for the transfection of siRNA, and HEC-1B cells for the transfection of overexpression-plasmid. The expression of linc01194 decreased after the transfection of si-linc01194. To explore the effect between linc01194 and EC, we conducted CCK-8 assay, wound-healing assay, cell invasion assay and apoptosis experiments. We found that the low level of linc01194 was related to the decrease of cell proliferation, cell migration, cell invasion, and the increase of apoptosis (p<0.05; and ). After transfection with plasmid targeting linc01194, the expression of linc01194 was increased (p<0.05; ), and cell proliferation (p<0.05; ), cell migration (p<0.05; ) and cell invasion (p<0.05; ) were promoted, while cell apoptosis was inhibited (p<0.05; ). To identify the downstream target protein that binds to linc01194, we used the starBase database to predict the target protein of linc01194. The data obtained suggested that IGF2BP1 may be the downstream target protein of linc01194 . The relationship between the levels of IGF2BP1 and the overall survival rate of EC patients was searched in Kaplan-Meier Plotter database, which showed that high levels of IGF2BP1 was related to poor survival rate of EC patients (p<0.05; ). Additionally, we established that linc01194 binds to IGF2BP1 through RIP experiments (p<0.05; ). Moreover, western blots experiment indicated that the knockdown of linc01194 decreased the level of IGF2BP1 in the cytoplasm of EC cells (p<0.05; ). A related study found that the functions of IGF2BPs are exerted mainly in the cytoplasm . Meanwhile, we found that the knockdown of linc01194 reduced the expression level of SOX2 (p<0.05; ). Conversely, the upregulation of linc01194 increased IGF2BP1 expression in the cytoplasm (p<0.05; ). Similarly, the upregulation of linc01194 promoted the expression of SOX2 (p<0.05; ). Accumulating evidence on lncRNA has indicated that they are key regulators in cancer pathways. They affect signal pathways and regulate gene expression at the epigenetic level. Also, they can interact with chromatin, and proteins to affect RNA splicing, and transcriptional and post-transcriptional levels, thereby regulating several cellular biological processes . Slack et al summarized the relevant mechanisms of action of lncRNAs. First, they can act by directly binding to protein complexes. For example, they direct chromatin modification complexes to target gene promoters, which affects the processes of transcriptional inhibition or activation. Additionally, they combined with transcription factors, which have extensive downstream effects on the cell transcription process. Furthermore, they directly interact with RNA-binding proteins (RBPs), which regulates mRNA processing and stability and serve as a scaffold for regulatory molecules found in nuclear speckles and paraspeckles. Second, lncRNAs also bind to nucleic acids to regulate their molecular mechanism of action. Besides, lncRNA as a competitive endogenous RNA (ceRNA) or a “sponge” for miRNA, regulated gene expression. Data in TCGA database revealed that the expression levels of linc01194 were higher in EC. In our study, we confirmed that linc01194 was overexpressed in EC by analyzing the endometrial tissues of 127 patients. Relevant research shows that linc01194 expression level in hepatocellular carcinoma , PCa , lung cancer , laryngeal squamous cell carcinoma , and colorectal cancer is increased, which promotes their occurrence and development. However, linc01194 expression in EC has not been studied. To clarify the function of linc01194 in EC, we silenced linc01194 in EC cells. We found that the cell proliferation, migration and invasion of EC cells were inhibited, whereas apoptosis was promoted. On the contrary, when we upregulated the level of linc01194, cell proliferation, migration and invasion of EC cells were promoted, while apoptosis was inhibited. LncRNA can exert these effects by binding to a target protein. Therefore, through the starbase database, we predicted that linc01194 might bind to IGF2BP1. The results of RIP and WB experiments showed that linc01194 could combine with IGF2BP1, and the expression level of linc01194 is positively correlated with the expression level of IGF2BP1 in the cytoplasm. In addition, an earlier study showed that the activities of IGF2BPs are exerted mainly in the cytoplasm . The insulin-like growth factor (IGF) system is related to organism development and cell function maintenance. This system includes IGF family ligands, binding proteins and receptors . IGF2BPs are members of this system, which are highly expressed in human cancer tissues. Meanwhile, a high level of IGF2BPs is associated with a poor prognosis in cancer patients . IGF2BPs are involved in cell growth, stem cell maintenance and differentiation during cell development . IGF2BP1 is a member of the IGF2BPs. IGF2BPs consists of 6 typical RNA-binding domains, including 4 K homology (KH) domains and 2 RNA recognition motif (RRM) domains. Of them, KH-1/2 is known to stabilize IGF2BP-RNA complexes. IGF2BPs preferentially recognize mRNAs modified by m6A and promote an m6A-dependent mode, thereby affecting gene expression. In tumors, IGF2BPs may play a carcinogenic role by stabilizing m6A-modified mRNAs . In their study, Xue et al. established that IGF2BP1 enhanced the stability of the sex-determining Y-box 2 (SOX2) mRNA in EC in an m6A-dependent manner, thereby promoting the expression of SOX2. On the other hand, SOX2 is a key transcription factor of embryonic development and the maintenance stem cell characteristics . As a carcinogenic transcription factor, SOX2 is related to cell stemness in tumors . In cancer, SOX2 promotes cell proliferation, inhibits apoptosis, and regulates cell invasion, migration and metastasis . In addition, SOX2 was upregulated in many human cancers, such as colorectal cancer , breast cancer , esophageal cancer , glioblastoma , lung cancer , hepatocellular carcinoma , and to promote the occurrence and progression of tumors. In our study, we found that the knockdown of linc01194 downregulated the expression of IGF2BP1 and SOX2. Combined with the research results of Xue et al. , our present findings suggest that linc01194 may promote the expression of SOX2 in an m6A-dependent manner through the interaction with IGF2BP1, promoting the progress of EC. Our study demonstrated for the first time that linc01194 can promote the expression of downstream protein SOX2 through binding to IGF2BP1, thus promoting the occurrence and development of EC, which may provide a new approach for the diagnosis and treatment of EC. However, further research on the mechanism and function of linc01194 is needed, including the design of diagnostic kits, research on specific inhibitors, and, if possible, preclinical transformation research based on organoids. |
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